Tissue Engineering Vascularized Bone Regulated Bone Repair And Vascular Endothelial Growth Factor Release In Critical Size Defect Model On The Femur In Rabbit

ObjectiveThe long history of orthopedic practice has confirmed the efficacy of autogenous bone grafting,but significant problems such as donor site morbidity and the limited supply associated with autogenous bone harvesting remain.The Advances in bone tissue engineering have enabled this technique to hold promise for the eventual application.The use of tissue engineering vascularized bone for reconstructive surgery is in harmony with the bionics perspective and may be an ideal solution to large segmental bone defects.Tissue engineering vascularized bone can significantly shortens the time for bone healing.In another way,angiogenesis is an essential component of skeletal development and repair.Blood supply and angiogenesis might also be important for bone remodeling.Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair.Of these,vascular endothelial growth factor(VEGF) is of particular interest because of its ability to induce neovascularization(angiogenesis).Meanwhile,VEGF is a major actor regulating not only angiogenesis but also osteoblast function.The aim of the present study was to evaluate whether tissue engineering vascularized bone regulated bone repair and vascular endothelial growth factor release in critical size defect model on the femur in rabbit.Samples were evaluated by means of histology to determine the extent of new bone formation,by means of immunohistochemistry,real-time PCR and western blot analysis to determine the level of VEGF expression.MethodsThirty-two New Zealand rabbits(male or female) were weighing between 2 and 2.5kg were purchased from the Experimental Center of the Southern Hospital and randomly divided into two groups of 16 rabbits each.In both groups(n=16),a segmental and critical bone defect 15ram in length was made at the left femur,a tissue engineering bone was inserted into the bone defect.In experimental group, femur vascular bundle was implanted into tissue engineering bone.In control group, there was no vascular implanted.At 2,4,8,and 12 weeks after implantation,samples were evaluated by means of histology to determine the extent of new bone formation, by means of immunohistochemistry,real-time PCR and western blot analysis to determine the level of VEGF expression.Autologous bone marrow was isolated from the rabbits and bone marrow mesenchymal stem cells were proliferated in vitro.At the fourth passage,10~7/ml cells were seeded onto 8 mm diameter porousβ-tricalcium phosphate(β-TCP) scaffolds and cultured in vitro for seven days prior to implantation.A 6-hole reconstruction common steel plate was placed in front of the right femur and a 15mm length of femur was intercepted between the second and the fifth hole of the plate.Tissue engineering bone was imbedded into the defect at this location.A length of femoral vascular bundle was implanted to the side groove of tissue engineering bone and fixed with thread(Experimental group,n=16).Neither the vascular bundle graft nor the nerve graft was implanted into control group(n=16).Four animals in each group were sacrificed a t 2,4,8,and 12 weeks after implantation.The expressions of VEGF were determined.The integrated optical density(IOD) of each blot was measured. Statistical software was used for statistical analysis.2×3 factorial design ANOVA was used to analyze the mean of each group.When differences were statistically significant,the one-way ANOVA was used to analyze the mean of each group and the SNK method was applied for multiple comparisons.Results1.Histological observation and results analysis:With the time prolong,new bone formation were gradually increased,the difference has statistical significance(F= 471.55,P＜0.05).The effects of vascular bundle implantation in osteogenesis have statistical significance(F = 224.64,P＜0.05 ).Factorial design ANOVA showed time and vascular bundle implantation have interaction(F= 34.51,P＜0.05 ).The formation of new bone in experimental group was superior to in control group at each time point.2.Immunohistochemistry stain results of VEGF:With the time prolong,the expression of VEGF in each group were gradually changed,the difference has statistical significance(F= 155.95,P＜0.05 ).The effects of vascular bundle implantation in increasing the expression of VEGF have statistical significance(F= 681.78,P＜0.05).Factorial design ANOVA showed time,vascular bundle implantation have interaction(F= 59.51,P＜0.05).The integrated optical density of VEGF in experimental group was superior to in control group at each time point.At 4 weeks,the level of VEGF expression was peaked at 4 weeks.3.Real-time PCR results of VEGF mRNA:With the time prolong,the expression of VEGF mRNA in each group were gradually changed,the difference has statistical significance(F=71.66,P＜0.05).The effects of vascular bundle implantation in increasing the expression of VEGF have statistical significance(F= 1121.07,P＜0.05). Factorial design ANOVA showed time,vascular bundle implantation have interaction (F=17.38,P＜0.05).The ratio of VEGF mRNA in experimental group was superior than in control group at each time point.At 4 weeks,the level of VEGF mRNA expression was peaked at 4 weeks.4.Western blot results of VEGF:With the time prolong,the expression of VEGF in each group were gradually changed,the difference has statistical significance(F= 110.78,P＜0.05).The effects of vascular bundle implantation in increasing the expression of VEGF have statistical significance(F=2641.51,P＜0.05).Factorial design ANOVA showed time,vascular bundle implantation have interaction(F= 75.33,P＜0.05).The integrated optical density of VEGF in experimental group was superior to in control group at each time point.At 4 weeks,the level of VEGF expression was peaked at 4 weeks.Conclusion1.Compared with control group,the extent of new bone formation in the group of tissue engineering vascularized bone was increased with the time prolong.At 2 weeks after implantation,there is no significantly between the control group and the experimental group.In the other time point,the extent of new bone formation in the experimental group was significantly higher than the control group.The way of bone repair majored in enchondral bone formation.2.At 12 weeks,the new bone expressed the fiberⅡtype in the experimental group,and the control group expressed the fiberⅠtype.3.At 2 weeks,vascular bundle implantation group can be seen larger lumen neovascularization in the pores of scaffold,neovascularization of the control group is relatively small,and mainly fibrous tissue.At 12 weeks,the vascular diameter was narrowed,and vascular structures were more mature.4.At the group of tissue engineering vascularized bone,the level of VEGF expression changed with time and the expression of VEGF was significantly higher than the control group at each time point,which means vascular implantation can significantly promote the expression of endogenous VEGF.5.The expression of VEGF in the experimental group began increasing in 2 weeks,and peaked at 4 weeks,and started to decline in 8,12 weeks,so it's considered VEGF are essential to stimulate angiogenesis in the early time.6.VEGF can promote endochondral ossification,expecially in the experimental group.Tissue engineering vascularized bone accelerated bone repair and increased VEGF release in critical size defect model on the femur in rabbit.