To Explore The Platelet-derived Growth Factor-BB In Bone Reconstruction

Objective:a.To explore the effect of PDGF-BB on vascular endothelial cells.b.To investigate the osteogenesis of PDGF-BB;c.To explore the biocompatibility of hydroxyapatite-chitosan-PDGF-BB.Methods:a.New Zealand white rabbit renalvas cular endothelial cells and identified vascular endothelial cells in vitro.Take a 4th generation cells according to the 2×10~4/mL vaccination in 96 well plates diluted with dissolved PDGF-BB proteins for 5ng/mL,10ng/mL,15ng/mL,20ng/mL,25ng/mL,30ng/mL concentration gradient PDGF-BB proteins intervention in vascular end othelial cells 24h.PDGF-BB is measured by CCK 8 for vascular endothelial cell toxicity.Take a 4th generation cells according to the 2×10~4/mL vaccination in petridishes,randomly divided into 4 groups(n=10):the control group(without factor training group),PDGF-BB factor to cultivate 24h group,PDGF BB factor to cultivate 48 group;PDGF-BB factor to cultivate 72 group.The expression of VEGF mRNA and protein in EPCs of each group was detected by fluorescence real-time quantitative polysaccharide chain reaction(Q-PCR)and Western Blot.b.In vitro cul ture of New Zealand white rabbit skull osteoblasts and identification of osteoblasts.Take a 4th generation cells according to the 2×10~4mL vaccination in 96 well pla tes,diluted with dissolved PDGF-BB proteins for 5ng/mL,10ng/mL,15ng/mL,20n g/mL,25ng/mL,30ng/mL,30ng/mL concentration gradient PDGF-BB 24h BB prote ins intervention in vascular endothelial cells,PDGF-BB is measured by CCK-8 protein on vascular endothelial cell toxicity.Take a 4th generation cells according to the 2×10~4mL vaccination in petri dishes,randomly divided into 4 groups(n=10):the control group(without factor training group),and PDGF-BB factor,PDGF-BB factor to cultivate 24h group,PDGF-BB factor to cultivate 48h group.PDGf-BB f actor to cultivate 72h group The expression of OPG mRNA and protein in EPCs of each group was detected by fluorescence real-time quantitative polysaccharide chain reaction(Q-PCR)and Western Blot.c.The precipitation to obtain chitosan coating on titanium plate.oxide coating of pure titanium plate,remove oxide coating of pure titanium plate and precipitation of coating of pure titanium steel were observed by scanning electron microscopy.Painted hydroxyapatite chitosan-slow-release coating on titanium plate PDGF-BB factor of slow release curve.The toxicity of hydroxyapatite-chitosan–PDGF-BB was detected by CCK-8.The coated steel plates and osteoblasts were cultured to observe the compatibility of the finished products.The hydroxyapatite-chitosan-PDGF-BB factor slow-release coating on titanium plate im planted into New Zealand white rabbit femoral,Testing the New zealand white rabbit of the main internal organs toxicity after 8 weeks.Results:a.The gray value of VEGF/GAPDH in control group,group with PDGF-BB cultured for 24h,group with PDGF-BB cultured for 48d and group with PDGF-BB culture for 72h was 0.375±0.004,0.613±0.004,0.661±0.005,0.845±0.002,which showed a significant difference between different groups(P<0.05).The results of Q-PCR showed that lowest expression of VEGF in control group(0.340±0.004)and highest VEGF expression in group with PDGF-BB culture for 72h(3.646±0.023)(P<0.05),which showed significant difference between different groups.b.The gray value of VEGF/GAPDH in control group,group with PDGF-BB cultured for 24h,group with PDGF-BB cult ured for 48 d and group with PDGF-BB culture for 72h was 0.365±0.003,0.516±0.003,0.761±0.002,0.795±0.0044,which showed a significant difference between different groups(P<0.05).The results of Q-PCR showed that lowest expression of VEGF in control group(0.240±0.006)and highest VEGF expression in group with PDGF-BB culture for 72h(3.556±0.003)(P<0.05),which showed significant difference between different groups.c.The releasing curve of PDGF-BB,PDGF-BB is gradually increasing during 0~10h,PDGF-BB release rate is relatively high in 10~40h and reached the maximum after 40h.The different types of titanium plate electron microscope observation morphology showed that pure titanium plate has a good cell compatibility.But the compatibility of the noneoxide coating,the co-culture of osteoblasts,and the cells is less than the pure titanium plate.Conclusion:PDGF-BB can promote the vascular endothelial cells to express the mRNA and protein of VEGF Gene during vascularization.PDGF-BB can promote the secretion of OPG factor in osteoblast cells.The hydroxyapatite-chitosan-PDGF-BB titanium plate has good mechanical efficiency and biocompatibility.Hydroxyapatite-chitosan-PDGF-BB has good mechanical performance,biocompatibility has a prosperous of application in orthopedic surgery and can be well applied to in clinical research.