Expression Of Vascular Endothelial Growth Factor In Bone Morphogenetic Protein-2 Induced Osteogenesis

Part â…  : Analysis of Expression of Vascular Endothelial Growth Factor in Bone Morphogenetic Protein-2 Induced OsteogenesisObjective Through observing the expression patterns of vascular endothelial growth factor (VEGF) in bone morphogenetic protein-2 (BMP-2) induced osteogenesis, to deepen our knowledge of the inducing mechanism of BMP-2, so to search the theoretical foundation for BMP-2's further clinical application.Methods An experimental model of femoral muscular pouch in 20 mice was adopted. The left legs of the mouse were treated as experimental group and the right control group. A composite of gelatin and hydroxyapatite was adopted as carrier of rhBMP-2. RhBMP-2(0.25mg)/carrier composite and carrier only were implanted into the femoral muscular pouch of experimental group and control group, respectively. Tissues in the muscular pouch were extracted on the 3^rd, 7^th, 14^th and 21^st day, respectively. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method.Results The expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenchymal cells, but their expression levels were low. As the mesenchymal cells differentiated into precartilage, the expression signals disappeared in mesenchymal cells, but appeared in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14^th day, accompanied by numerous hypertrophic chondrocytes. When mature cartilage calcified and new bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. Signals of VEGF mRNA and VEGF can not be detected in the control group.Conclusion The expression of VEGF mRNA and VEGF was increased significantly, compared to the control group, in BMP-2 induced osteogenesis and they were expressed most strongly in mature chondrocytes. BMP-2 has the effect of promoting the synthesis and secretion of VEGF. Although the role of VEGF in the process is still unknown, this new finding will throw some light on the inducing mechanism of BMPs and establish the theoretical foundation for BMP-2's further clinical application. Part â…¡: Primary culture and identification of fetal mouse osteoblastsObjective To investigate an economical and efficient method for primary osteoblasts culture, with the aim to establish a foundation for further experimental study. Methods The calvaria which harvested from 19 day fetal mouse was stripped off all the soft tissues including the periosteum, rinsed and cut to trivial bone block under sterile condition. Then, the bone block was subcultured in culture flask after digested with 0.25% trypsin for 10 min, the acquired cells were identified with inverted microscope and transmission electron microscope observation, alkaline phosphorase stain, immunohistochemistry stain of osteocalcin and alizarin bordeaux stain of calcified nodules.Results The cells obtained from fetal mouse calvaria were identified to be osteoblasts by inverted microscope and transmission electron microscope observation, alkaline phosphorase stain, immunohistochemistry stain of osteocalcin and alizarin bordeaux stain of calcified nodule.Conclusions The modified tissue mess method is an ideal method to obtain and culture osteoblasts which having typical characteristics. This method saves money and time and is easy to manipulate.Part â…¢: Dose-dependent Effect of rhBMP-2 on the VEGF Expression in Fetal Mouse OsteoblastsObjective To investigate the effect of different dose of recombinant human bone morphogenetic protein-2(rhBMP-2) on vascular endothelial growth factor (VEGF) expression in fetal mouse osteoblasts.Methods Calvaria osteoblasts of 19 day's fetal mouse were cultured, the effect of different dose of rhBMP-2 on VEGF mRNA and protein expression in fetal mouse osteoblasts were observed with reverse transcription-polymerase chain raction (RT-PCR) and western blot respectively.Results In the present study, the steady-state level of VEGF in control confluent fetal mouse osteoblasts was measured by RT-PCR and western blot. The levels of VEGF increased in an apparent biphasic manner with a maximum stimulation (about 2-fold above the control, P<0.05) of both mRNA species observed at 300 ng/ml of rhBMP-2.After 48 h of OP-1 treatment, the VEGF mRNA and protein levels approached that of control.Conclusions A steady-state level of VEGF mRNA and protein in control confluent fetal mouse osteoblasts could be measured by RT-PCR and western blot; RhBMP-2 can promote the expression of VEGF in a dose-dependent manner, the levels of VEGF increased in an apparent biphasic manner with a maximum stimulation at 300 ng/ml of rhBMP-2.Part â…£: Time-dependent Effect of rhBMP-2 promoting the VEGF Expression in Fetal Mouse OsteoblastsObjective To investigate the time patterns of bone morphogenetic protein-2(rhBMP-2)-stimulated vascular endothelial growth factor (VEGF) expression in fetal mouse osteoblasts.Methods Calvaria osteoblasts of 19 day's fetal mouse were cultured, the effect of rhBMP-2 on VEGF mRNA and protein expression at different times in fetal mouse osteoblasts were observed with reverse transcription-polymerase chain raction (RT-PCR) and western blot respectively.Results The levels of VEGF mRNA increased in an apparent biphasic manner with a maximum stimulation (about 2-fold above the control, P<0.05) of both mRNA species observed at 300 ng/ml of rhBMP-2. After 48 h of OP-1 treatment, the VEGF mRNA levels approached that of control. When compared with the individual control value at each time point, the stimulation in VEGF mRNA level by rhBMP-2 was also time-dependent. An apparent maximum change (about 2.7-fold above the control, P<0.05) occurred at 6h. The apparent switch in peak time from 3 to 6 h is mainly due to a continuous drop in the control value at 6h. The second peak induction is about 2-fold above the control and occurred at about 36h. The relative level remained elevated at 48h following rhBMP-2 treatment.Conclusions RhBMP-2 can promote the expression of VEGF in a time-dependent manner. The VEGF mRNA levels appeared to be biphasic in rhBMP-2-treated cultures, showing peak induction at 3 and 24h. The level remained elevated at 48h. When compared with the individual control value at each time point, an apparent maximum change occurred at 6h. The second peak induction is about 2-fold above the control and occurred at about 36h. The relative level remained elevated at 48h following rhBMP-2 treatment.Partâ…¤: Mechanism of VEGF expression in fetal mouse osteoblasts induced by rhBMP-2Objective To investigate the mechanism involved in the bone morphogenetic protein-2(rhBMP-2)-stimulated vascular endothelial growth factor (VEGF) expression in fetal mouse osteoblasts.Methods Calvaria osteoblasts of 19 day's fetal mouse were cultured, effects of actinomycin D, cycloheximide, and hydroxyurea on VEGF mRNA expression level in fetal mouse osteoblasts in the presence or absence of rhBMP-2 were observed with reverse transcription-polymerase chain raction (RT-PCR).Results Inhibition of transcription by actinomycin D reduced VEGF transcription in control cells and almost completely prevented the stimulation of VEGF expression by rhBMP-2. Cycloheximide increased VEGF expression in control cells but it did not abolish the rhBMP-2 effect. Inhibition of DNA synthesis blocked the stimulatory effects of rhBMP-2 on VEGF gene expression.Conclusions The observation suggests that the observed enhancement in VEGF expression induced by rhBMP-2 is dependent on new transcription and cell replication (the mitogenic activity of rhBMP-2). The results indicate that de novo protein synthesis might not be essential for the increase in VEGF mRNA. Partâ…¥: Roles of vascular endothelial growth factor play in theosteogenesis induced by rhBMP-2Objective To investigate the roles of vascular endothelial growth factor (VEGF) play in the osteogenesis induced recombinant human bone morphogenetic protein-2(rhBMP-2). Methods (1) Calvaria osteoblasts of 19 day's fetal mouse were cultured, gene expression of VEGF receptors were examined with reverse transcription-polymerase chain raction (RT-PCR). (2) To measure alkaline phosphatase (AP) activity and bone nodule formation of osteoblasts after the inhibition of VEGF synthesis and function by VEGF antisense oligodeoxynucleotides (VEGF ASODNs) and suramin respectively. (3) Bone nodule formation was examined after 14 day's treatment with varying dosages of VEGF in the presence or absence of rhBMP-2.Results (1) The mRNA levels for both types of VEGF receptors, Flk-1 and Flt-1 were low but detectable in osteoblasts cells by RT-PCR and were not changed by rhBMP-2. (2) Inhibition of VEGF synthesis and function by antisense oligonucleotide and by suramin, respectively arrested the rhBMP-2-induced alkaline phosphatase activity and mineralized bone nodule formation. (3)VEGF alone did not have any effect on nodule formation. VEGF, in the concentration range tested, did not affect the extent of rhBMP-2-induced bone nodule formation.Conclusions The present studies demonstrated that VEGF acts in an autocrine manner on the osteoblastic cells and that VEGF is involved, at least partly, in the differentiation activity of rhBMP-2 in primary cultures of osteoblastic cells. Together with published data on the angiogenic activity of VEGF on endothelial cells, the present finding, suggests that VEGF can act as an autocrine and a paracrine. Future studies will be necessary to elucidate the role of VEGF in the OP-1-induced osteoblast cell differentiation.