Construction And Osteogenesis Of Biomimetic Bone Complex Of ?-TCP Optimized With Sirt3 Transfected BMSC

Objective:Bone defect caused by traumas,infections,tumors and other diseases are now facing a huge clinical need.As a new type of bone repair material,tissue engineering bone(the bone material generated via tissue engineering)obtains a great attention.However,there are two major challenges for its application.Firstly,the bone marrow mesenchymal stem cells(rBMSCs)isolated from autologous bone marrow has both low proliferation rate and low differentiation efficiency,and are difficult to use as the seed cells for tissue engineering.Secondly,although the manufacturing strategy of bone tissue engineering scaffolds advocate to simulate the shape and structure of a natural bone tissue,there is not any theoretical model reported for biomimetic scaffold structure.The purpose of this study was to provide a new method on tissue engineering concept for repairing bone defects,by providing a biomimetic scaffold model suitable for in vitro cell enrichment and bone reconstruction,and constructing a bone tissue engineering seed cell line with rapid proliferation and differentiation potential on the engineering bone.Methods:(1)A biomimetic scaffold model was designed by parametric modeling process,as a template for tissue engineering scaffold.After that,the ?-tricalcium phosphate(?-TCP)material was prepared using hydrothermal method as the raw material for scaffold preparation.The biomimetic scaffold and 0°/90° woven-type scaffold were printed out by 3D-printing using ?-TCP.Besides,the natural cancellous bone scaffold was prepared using ammonium dihydrogen phosphate calcination method.The physicochemical properties of the three types of scaffold were analyzed by fourier transform infrared spectroscopy(FT-IR)and X-ray diffraction(XRD).The structural characterizations were detected by Micro-CT scan reconstruction,scanning electron microscopy(SEM)and compression experiments.(2)The rBMSCs of SD rat were isolated and identified by Giemsa staining and flow cytometry,followed by cell/scaffold co-culture.The proliferation and adhesion potential of rBMSCs were evaluated by live/dead cell staining and DNA content assay.After switching to osteogenic inducing medium,alkaline phosphatase activity assay and RT-PCR were used to detect the expression level of osteogenic related genes,and to evaluate the effects of three scaffold structures on rBMSCs adhesion,proliferation and differentiation.Finally we chosen one scaffold with the best vitro biocompatibility,the biomimetic scaffold,for the following experiment.(3)The eukaryotic expression vector pcDNA3.1-Sirt3 was constructed and the recombinant plasmid was transfected into rBMSCs(Sirt3,rBMSCs).The Sirt3 protein expression was assessed by an analysis under fluorescence microscopy and Western Blot.Then cellular oxygen consumption,ATP production and key enzyme activities were also measured.After culturing rBMSCs on the scaffold,MTT and RT-PCR were used to analyze the changes of cell proliferation and expression level of the genes bone related to osteogenesis and bone formation,(4)Animal models were constructed by surgical drilling for a 6.0 mm diameter circular defects of SD rat skull.The Sirt3-rBMSCs and the control rBMSCs were compounded respectively on the ?-TCP-based biomimetic scaffold,and implanted into the bone defects.Micro-CT scan reconstruction and immunohistochemical staining were performed at the time of 4 weeks and 8 weeks after implantation to evaluate the effect of osteogenesis and bone formation in vivo.Results:In this study,we designed and fabricated three different types of P-TCP-based biomimetic scaffolds and studied their characteristics,including in vitro biocompatibility analysis.Then the gene Sirt3 was transfected to rBMSCs with liposome,which promoted the oxidative phosphorylation and eventually improved cell proliferation and differentiation.Applying an in vivo experiment,we used rat cell/scaffold complexes to repair the cranial defect of SD rats,and evaluated the potential of osteogenesis.The material prepared by the hydrothermal method and the calcinated bovine femur material were detected as(?-TCP.The biomimetic scaffold prepared by 3D-printing gave a relatively higher compressive modulus,porosity and surface area.Additionally,the co-culture of rBMSCs with biomimetic scaffold shows excellent biocompatibility at in vitro experiments.The Sirt3-rBMSCs was successfully generated,and constantly expressed Sirt3 protein,which improved the feature of rBMSCs for bone repairing.The oxygen consumption and ATP production of rBMSCs were increased in Sirt3-rBMSCs;their related glucose metabolism enzyme activity was vigorous;and the proliferation and differentiation rates of rBMSCs were also significantly increased.After 8 weeks of implantation of Sirt3-transfected rBMSCs/?-TCP-based biomimetic scaffold complexes into the cranial defects of the SD rats,a large amount of osteogenesis and new bone formation were identified by Micro-CT and immunohistochemistry analysis.Conclusion:With this study we built a promising new system for tissue engineering scaffold potentially useful in bone defect repairing.In this system,we optimized a new construct of bone with ?-TCP-based biomimetic scaffolds,and confirmed their characteristics for bone re-construct particularly its in vitro biocompatibility.We also engineered a new type of seed cells for tissue engineering,with active Sirt3 gene transfected in the rBMSC,which when were seeded in the construct,improved the proliferating and differentiation of the osteoblast from autologous BMSCs,and promoted the bone formation.We further showed the practical repairing effect of this system in an in vivo model of SD rats with defect cranial.In summary,this study provides a promising new system for repairing a defect bone,including an optimized construction of ?-TCP-based biomimetic scaffolds,and a type of useful seed cells generated with Sirt3-transfected BMSC for the tissue engineering.