The Research On Repairing Bone Defect With New Composite Tissue Engineering Bone Constructed By 3D Printing Technology

Objective:A new PCL scaffold constructed by 3D printing technology with high porosity and controllable pore size was combined with Platelet-rich gelatin of bone marrow mesenchymal stem cells(BMSCs)carrying BMP-7 gene.It is called a new type of composite tissue engineering bone.To explore the osteogenesis effect of new composite tissue engineered bone in rabbit tibia bone defect,and to provide a theoretical basis for the repair and reconstruction of large bone defect.Methods:Bone marrow mesenchymal stem cells(BMSCs)from New Zealand white rabbits were isolated and cultured in vitro.After subcultures,BMSCs were divided into groups A,B and C.Group A was BMSCs transfected with control adenovirus,group B was BMSCs transfected with BMP-7 gene over-expressing adenovirus,and group C was BMSCs transfected with BMP-7 gene silencing adenovirus.Realtime-PCR,Western blot and Alzarin-Red S staining were used to detect OPN-mRNA,BMP-7-mRNA,BMP-7,OPN and calcified nodules in normal BMSCs and group A,B,C.Platelet rich plasma(PRP)was prepared by Landersberg method.After mixing with BMSCs,BMSCs was injected into the double cavity catheter to obtain PRP gel containing transfected BMSCs.A new PCL scaffold constructed by 3D printing technology with high porosity and controllable pore size was combined with Platelet-rich gelatin of transfected BMSCs.Twenty-four rabbits weighing approximately three kg were randomly divided into four groups,six in each group.The tibia bone defect model of about one cm in length was established by surgery.The structural data of individual rabbit tibial defect were obtained by CT three-dimensional reconstruction.According to the data obtained,the molten polycaprolactone(PCL)was printed out by a 3D printer.The PRP gel tissue containing transfected BMSCs was perfused into a PCL scaffold through a 3D printer to construct a new composite tissue engineered bone.Each group was implanted with different materials into the bone defect.Group A: blank control group;Group B: autogenous bone reconstruction;Group C: scaffold + normal BMSCS reconstruction;Group D: scaffold + transfected BMSCs reconstruction,and four groups were fixed with small tibial plate.Three months after operation,all rabbits in four groups were sacrificed and the whole tibia was taken out.The bone fusion was observed by micro-CT scanning.Results:(1)Rabbit BMSCs cells were obtained by Percoll density gradient centrifugation,and proved to be rabbit BMSCs cells by experiments.(2)Real-time fluorescence quantitative PCR detection: BMP-7-mRNA values in group A and B were higher than group C(P<0.05),BMP-7-mRNA values in group B were higher than group A(P <0.05).OPN-mRNA values in group B were higher than group A and C(P<0.05),and there was no significant difference between group A and C(P>0.05).(3)Western blot detection: BMP-7 and OPN values in group A and B were higher than group C(P<0.05).BMP-7 and OPN values in group B were higher than group A(P<0.05).(4)Alzarin-Red S: Under the microscope,the staining depth and the number of calcium nodules in group B were higher than group A and C,the staining depth and number of calcium nodules in group A were higher than group C.The results of semi-quantitative determination of calcium content showed that the calcium content in group A and B was higher than in group C(P<0.05),while the calcium content in group B was slightly higher than group A(P>0.5).(5)Micro-CT results of rabbit tibia after 3 months showed the amount of callus formation in D and B groups was significantly better than the other two groups,and there was no significant difference in callus formation between group D and group B.Conclusion:(1)Adenovirus carrying BMP-7 gene can effectively infect BMSCs.After transfection,BMSCs can express OPN and BMP-7 protein in large quantities.(2)The PCL scaffold constructed by 3D printing has high porosity and controllable pore size,which can ensure that the scaffold can obtain good oxygen,nutrients and facilitate the discharge of metabolic substances.(3)The new composite tissue engineered bone constructed by 3D printing has good osteoinductive ability and can replace autologous bone transplantation.