The Dynamic Change Of Endothelial Progenitor Cells In Distraction Osteogenesis And Involveld Mechanism

Purpose We established a rat tibia DO model,to study the mobilization of EPCs through analysing the cell expression of EPCs in peripheral blood;Using fracture healing as control group,the main research areas contained function of EPCs in DO,mobilization mechanism of EPCs,and important roles of SDF-1?,/CXCR4 reaction axis,MAPK signaling pathways in the process,by Microfil perfusion,HE staining observation,gene and protein expression detection of SDF-1?,CXCR4,and MAPK signaling pathways;To investigate the migration and mechanical stress reaction of EPCs in response to SDF-1? and CXCR4 in a Transwell chamber assay and mechanical stretching assay.Methods The first part: 2 months male SD rats were divided into fracture control group and DO group.5 animals each group were killed every 3 days before and after cutting bone.Endothelial progenitor cells in peripheral blood were detection by flow cytometry for identification and expression level analysis.The second part: 2 months male SD rats were divided into fracture control group and DO group.The rat tibia DO and fracture models were established.(1)In 13 days and 16 days after the osteoectomy,rats from fracture and DO groups were anaesthetized for a Microfil perfusion experiment operation,and Micro-CT scans for Micro-CT images.After the scanning,the software is used to calculate number of vessels,thickness of blood vessels,blood vessel volume/total volume and vascular spaces.(2)Blood flow from the animals on days 10,13,and 16 after surgery was measured with laser Doppler flowmeter.(3)The animals from fracture and DO groups were killed through anesthesia on the days 10,13,16,19,and 20 after surgery.Tissue extracts from both DO and fracture callus were prepared.Immunohistochemistry were used for SDF-1? detection according to the manufacturer's protocol.(4)Total RNAs in callus were extracted and reverse transcribed into c DNA.SDF-1? and CXCR4 expression level was detected by quantitative PCR according to the manufacturer's protocol.(5)Total proteins were extracted from callus tissue of rats according to themanufacturer's protocol for Western blot detection p38,JNK,ERK and their respective phosphorylation products p-p38,p-JNK,p-ERK,CXCR4 and beta actin expression level.The third part: Rat bone-marrow derived EPCs were isolated from the tibias and femurs of SD rats and characterized by immunohistochemical staining,and immunofluorescence double-staining.(1)In a Transwell chamber assay with 8-?m pores,the upper chambers were loaded with 1�105 EPCs in 500 ?L EBM-2,and dilutions of 0,100,200,300 and 400 ng/m L of recombinant SDF-1? in 1.5 m L of media were placed to the lower side of the membrane.For neutralization studies cells were pretreated with a potent and specific antagonist of CXCR4(AMD3100,5?g/m L)for 30 minutes at 37�C.After 24 hours of incubation at 37�C in 5% CO2,the migrated cells were stained and counted under a light microscope.(2)EPCs were seeded in silicon-based plates pre-coated with fbrinogen for 24 hours.Mechanical stretching of EPCs by 5% and 10% was performed.Differentiation ability was checked.Total protein and RNA were extracted from the cells with or without mechanical stretching for 6h,and the expression of CXCR4,SDF-1? was detected.Results Part I:(1)In fracture control model,the number of endothelial progenitor cells in peripheral blood gave a temporary increase after osteoectomy.Compared with none osteoectomy control,the number of endothelial progenitor cells in peripheral blood increased to the peak after 3 days of osteoectomy,then gradually reduced.(2)Compared with fracture group,the EPCs(including mature and immature)increased in the number,the difference was statistically significant(P<0.05).In the consolidation phase difference between the two groups has no statistical significance.(3)After osteoectomy CD133 positive cells number continues to decline,indicating that number of immature EPCs conversion to mature cells increased.But immature EPCs increased in the DO group.(4)After osteoectomy CD34 positive cells number had a rising trend,which indicated that number of stem cells increased in a single nuclear cell groups.And compared with fracture group CD34 positive cell number increased in the DO group.Part II:(1)Microfil results show that,compared with controlled fracture group,more blood vessels were formed on the days 13 and 16 after osteoectomy.The results were characterized by the concentrated distribution of blood vessels in the osteoectomy point.According to the results of Micro-CTscanning,on the day 16 after osteoectomy vascular volume of DO group was higher than fracture group,the difference was statistically significant,and the number of blood vessels on Microfil perfusion was no statistical significance.(2)Blood flow measurement results show that the blood flow both in the fracture and the DO groups animals were higher than the none osteoectomy control;The blood flow of DO group animals obviously was higher than that of the fracture groups animals.(3)Blood vessels measurement results show that the blood vessel number both in the fracture and the DO groups animals decreased gradually.Blood vessels area and number in the DO group was obviously higher than that of fracture group.Blood vessels number in the distraction stage was higher than that in the consolidation stage.The number of blood vessels in the DO group were higher than the fracture group.The difference on the day 16 had statistical significance(P < 0.05);The vascular area in the DO group has slowly rising trend over time,fracture group vascular area decreased.And the vascular area in the DO group was higher than the fracture group,the difference was statistically significant(P < 0.05).(4)As the extension of fracture time,SDF-1? expression in callus gradually reduced.SDF-1? expression in the DO group were higher than that in the fracture group,but it showed a trend of decline in consolidation phase.(5)SDF-1? and CXCR4 expression level in the callus of animals of experiment group and the fracture group at the different time points are analyzed through RT-PCR.It showed that after the osteoectomy SDF-1? expression increased,but there is no statistical significance for the expression differences on the day 1,3,and 5(P > 0.05).But in the distraction stage,SDF-1? expression was higher than that in the fracture group.SDF-1? expression in the distraction stage increased to the peak on the day 13 after osteoectomy,then decreased to the level as the same as that in the fracture group on the day 21 after osteoectomy.Compared to the none osteoectomy control,CXCR4 expression increased both in the DO group and the fracture group.But there was no significant difference between the two groups excepted on the day 19 after osteoectomy.(6)The p-p38 expression levels in the fracture healing gradually decreased,but p-38 level increased through the protein Western blot test.Compared with fracture group,the expression level of phosphorylation products of p38,JNK and ERK increased in the DO group.Part III:(1)The migration of EPCsincreased according to the different doses of SDF-1? in a dose-dependent manner(P<0.05).Blocking of CXCR4 using 5?g/m L AMD3100 reduced the migrated cell number markedly(P<0.05).(2)The formation of vessel networks increased according the mechanical stress both in vessel networks area and longth(P<0.05).Compared with control cells,the SDF-1? and CXCR4 expression levels in cells subjected to mechanical stress increased through the protein Western blot test and RT-PCR test(P<0.05).Conclusions(1)Experimental results demonstrated that the number of endothelial progenitor cells in peripheral blood in the fracture process increased,but relative to this,the number of endothelial progenitor cells in peripheral blood in DO process has a higher number of increases.It showed that the DO produced the mobilization of EPCs from to the marrow to the peripheral blood with a special mechanism.(2)Compared to the fracture group,the number and area of blood vessels and blood flow increased to some extent in the DO model.It showed that in the process of DO,there were more EPCs mobilization and angiogenesis.And the way of mobilization and angiogenesis was different from the fracture model.Even in the different stages of DO,the number and area of blood vessels were also different.In detail,the number and area of blood vessels were higher in the distraction stage than the that in the consolidation stage.It may caused by more lesion in DO.(3)Both in the process of fracture healing and DO,the expression level of SDF-1? and CXCR4 showed a downward trend after rising.Rising mainly appeared in the early stages of bone cutting,and mainly was caused by osteoectomy lesion.The change way was the same as that in the EPCs mobilization,it showed that there must be some relationship between the SDF-1?,CXCR4 and the EPCs.Compared to the fracture model,the expression level of SDF-1? and CXCR4 and the EPCs mobilization both increase in the DO model.It showed that in the DO model,SDF-1? and CXCR4 must play important roles in EPCs mobilization.(4)Compared with fracture group,the expression level of phosphorylation products of p38,JNK and ERK increased in the DO group.It remained that EPCs mobilization in the DO process may be regulated through MAPK signal pathway.(5)SDF-1? facilitated the migration of EPCs in vitro,and this can be inhibited by treatment of EPCs with CXCR4 antagonistAMD3100.(6)We also found that the angiogenic functions of EPCs can be regulated by factor SDF-1? and CXCR4.Accordingly,the formation of vessel networks on Matrigel was significantly increased in cells subjected to mechanical stress.SDF-1? and CXCR4 expression was significantly increased by mechanical stress.