Construction And Observation Of Tissue Engineering Bone With Recombinant Adenovirus Vectors Co-expressing HBMP2 And HVEGF165 Infected BMSCs And Combined N-HA/PA66

Background The critical bone defect caused by trauma and tumor can be surgically treated with various procedures. In such cases, the use of autologous bone is still regarded as the golden standard.The transplation of autologous bone, however, is limited by its quantitative availability, loss of vascularity, invasion of normal tissue, and risk of infection during harvesting. Recent research has been focused on the bone regeneration favored by a rapid development of MSCs-based therapies. Bone marrow mesenchymal stem cells (BMSCs) are characterized by its self-renewal capacity, immunosuppressive property, and the pluripotency to differentiate into several lineages of cells including osteocytic, chondrocytic, muslecytic and adipocytic cells. Accordingly, BMSCs are considered as a potential therapeutic target of stem cells in tissue engineering filed. In addition, several growth factors have been proved to increase bone regeneration facilitated by a rapid development of gene therapy technology, especially as the progression of application of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP) in vacularization and bone regeneration. As a member of the transforming growth facor-β(TGF-β) super family, BMP2 are the only signal molecules that are individually sufficient for the induction of bone formation at orthotopic and heterotopic sites. The VEGF family is one of the most important angiogenesis-regulating that is characterized by its ability to induce angiopoiesis and promote endothelial proliferation. VEGF 165 plays a vital role in regulating bone formation and repair by attracting endothelial cells and osteoclasts, and enhancing the osteoblasts differentiation, especially for large and critical bone defects. Some reports concluded that the n-HA/PA66 has good biocompatibility and safely, which acting as a scaffold to repair bone defect were applied in clinical. However, single application of n-HA/PA66 to repair bone defects are having some defects and deficiencies. In order to overcome the exogenous growth factors in the body for easier filling and lower titer, short-time effect, choosing the vectors that express some cell growth factors infected BMSCs have sustainable and efficient expression.The adenoviral vector in our study was produced by the AdMax packaging system and consisted of a type 5 replication defective adenovirus with double loss of El and E3. The framework plasmids containing most of the adenovirus genome with the Cre/loxP recombinase system and shuttle plasmids inserted the exogenous genes, and they were assembled in HEK293 cellswhich thereby avoided the recombination in bacteria, as well as the corresponding shuttle plasmid linearization and improved recombination efficiency. Additionally, the T2A-like (Thosea asigna virus 2A like) sequence was selected as the multicistronic connection in the vector, ensuring separate but coordinated expression of several genesPreliminary experiments have shown good therapeutic effect that BMP2 protein and VEGF165 protein to repair rabbit bone defect. Based on the previous research, we select the adenovirus expression vector which co-expressing hBMP2 and hVEGF165 infected BMSCs compound n-HA/PA66 to construct tissue engineering bone. We dedicated to provide a technical support and theoretical basis of molecular biology to peers which the BMSCs growth characteristic and its osteoblast differentiation in constructed tissue engineering bone observed in vitro.Objective1. Isolation, culture and identification of bone marrow mesenchymal stem cells, we hope to obtain seed cells of bone tissue engineering.2. Construction and identification of recombinant adenovirus vector co-expressing hBMP2 and hVEGF165 and observation of its transfecting rabbit BMSCs, we hope to obtain the efficient exogenous growth factor expression vector.3. Construction and observation of tissue engineering bone with recombinant adeno virus vectors co-expressing hBMP2 and hVEGF165 infected BMSCs and combined n-HA/PA66. We dedicated to provide a technical support and theoretical basis of molecular biology to peersMethods1.Using the method of density gradient centrifugation to separate from bone marrow mesenchymal stem cells and identifing the cell phenotype of third generation of cells; Osteoblast induction training after 21 d and alizarin red staining conformed to observe intracellular calcium nodules formation; The third generation of BMSCs were slected as subjects.2. BMP2 and VEGF165 genes were amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2-VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ (delta) E1,3Cre. The recombinant adenovirus was purified and virus titer was determined, and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMSCs. In our study, the hBMP2 and hVEGF165 genes were inserted into the opposite side of the T2A to construct a bicistronic frame, and secreted in supernatant of BM-MSCsThe result revealed that the T2A sequence may be a superiorstrategy for multiple genes co-expression with adenovirus vector.3. Selecting the third generation of BMSCs as subjects, vectors of Ad-BMP2-VEGF165 and Ad-GFP infected BMSCs were regarded as experimental group and control group, respectively. The infected cells were inoculated into the n-HA/PA66 and observed the growth conditions state by scanning electron microscope after 7 days. Meanswhile, after 7 days of coulping with n-HA/PA66, BMSCs were collected and protein of osteocalcin and osteopontin were detected by Western Blot, and at 3 and 7 days later, Alkaline phosphatase activity were also determinated.Results1. Using the method of density gradient centrifugation to separate from bone marrow mesenchymal stem cells and identifing the cell phenotype of third generation of cells. Some cells attach plastic wall after 24 h and have polygon or small spindle sharp and the nucleus is clear. The swirling or herringbone pattern cells with the extension of incubation time is gradually increasing colony after 48 h; The cells can reach 80% fusion after 6-8 days and alizarin red staining conformed to observe intracellular calcium nodule formation.The results of cell surface antigen detected by flow cytometry after 3rd passage showed that BMSCs were hypso-expression CD29 (99.82%) and CD44 (94.14%), and then the CD 14 (3.11%) andCD34 (0.34%) were hypo-expression.2. The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the motheds of gene analyzing,colony PCR,Western blot and observing GFP expression, and the titer of the adenovirus was 1 x 1010PFU/ml. The Ad-BMP2-VEGF165 group expression of hBMP2 and hVEGF165 were markedly higher than the control (either 3 day or 28 day). Notably, the gene expression levels have been gradually enhencing from 3 to 5 day.3. The protein levels of BMP2 and VEGF165 in the cells and cultured medium after infection was detected. In addition, SEM examination revealed extensive cellular attachment and growth on the n-HA/PA66. Detection of ALP activity at 3 and 7 days and protein expression of OC and OPN in experimental group at 7 days were shown higher expression than control group (P<0.05)Conclusion We used Adenovirus as a gene delivery system to co-express BMP2 and VEGF165 genes in BMSCs,which was verified by direct sequencing, visual detection of GFP expression, RT-PCR and ELISA assays. The synergistic biological effects of BMP2 and VEGF165 facilitated by the present method might be able to provide theory support and experimental basis for gene therapy of critical bone defect.1. Successfully isolation, culture and identification of BMSCs and the earlier generation of cells (P1-P3) having strong proliferation ability could be used as a good seed cells for subsequent experiments.2. Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained.3. Ad-BMP2-VEGF165 could infect BMSC with high efficiency and interesting proteins are higher expression than control groups. The cells after infection grew well on the n-HA/PA66 scaffold and tissue engineering bone is constructed successfully.