The Effects And Mechanism Of VEGF And BMP2Genetic Modification On Vascularized Tissue Engineered Bone

Bone regeneration is required to repair mandible defects arising from trauma, inflammation or tumor. Bone tissue engineering which based on seed cells, grow factors and scaffold material are currently being combined to assess their potential to develop effective concepts for the treatment of extensive loss of osseous tissue. Bone seed cells which share the collective goal of creating osteoconductive and osteoinductive bone graft substitutes is the primary and basic element of bone tissue engineering. Bone marrow stem cells (BMSCs) are pluripotent cells having the potential to differentiate into bone, cartilage, muscle, and ligament have been implicated in the enhancement of bone repair. Although bone morphogenetic protein2(BMP2) can produce orthotopic bone in the hindlimbs of animal model, the short half-life and dispersion limit their effection of directly application. However, the strategy of gene modification can efficiently solve the deficience of directly application and adenovirus vectors have been successfully used for transient gene expression avoiding side effect of gene overexpression. BMP2gene modification of BMSCs provides a new prospect for bone tissue engineering.Osteogenic stem cells with over-expressed osteogenic factors were less effective in treating large bone defects due to inefficient blood supply in the entire bone graft. The vascularization has been involved in bone tissue engineering with the seed cells, grow factors and scaffold. Implanting the tissue valve embedment with vessel pedicle or blood vessel bundle to reconstruct bone graft blood circulation is an efficient way to vascularization. This method can offer rich blood supply to the construction by an independent vascular system, but multiple surgeries, trauma of vascular area and long treatment cycle limit to a small area and the repair of soft tissue trauma. Administration of angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and angiopoietin (Ang) together with biological materials to promote the vascularization. However, the angiogenic factors have short half life in vivo, which cannot reach the effective entration. Angiogenic cells combined with osteogenic cells are a more promising approach to vascularize tissue engineered bone. Endothelial Progenitor Cells (EPCs) with strong ability of promoting the angiogenesis after born updates the traditional concept of angiogenesis, and provides a new thought for ischemic disease. EPCs are a kind of immature cells, which can differentiate into endothelial cells and directly involved in angiogenesis. The treatment of animal model of hindlimb ischemia, myocardial ischemia and injuried cornea by EPCs has achieved some success. But the insufficient number of EPCs limits application in larger bone defect area, VEGF-modified EPCs can compensate for the insufficient number of shortcomings for VEGF can directly induce angiogenesis and induce EPCs recruitment. Thus in this experiment, we adopt the approach of angiogenic cells combined with osteogenic cells to promote vascularize bone graft. In vitro co-culture VEGF-EPC with BMP2-BMSC in osteogenic differentiation medium, then detected ALP activity, expression of osteogenic genes such as ALP, OC and Col I and alizarin red staining observed the number of mineralized nodules. Alginate gel mixed with cells was implanted into lateral femoral muscle, after implantation for4and6weeks immunohistochemistry stainning observed blood vessels and bone formation in order to detect the effction of angiogenesis and osteogenesis after combination of VEGF-EPC and BMP2-BMSC. The main experimental methods and results are as follows:1. Mononuclear cells from autogenous animal bone marrow had strong proliferative ability when cultured in plastic culture flask in vitro and would amplified in short period, which finally were induced to BMSCs with high purity and quantity. Under some specific conditions, BMSCs could differentiate into osteoblast and adipocyte. So BMSCs were ideal seed cells of bone tissue engineering.2. Mononuclear cells were separated and purified from the rat bone marrow by density gradient centrifugation. After amplification and culture under special inductive conditions, high purity EPCs could be obtained in vitro, which were identified by cell morphology, cell phenotype and cell function tests including cellular immune fluorescence and tube formation experiment. Mixed EPCs and BMSCs with different proportions and tested cell proliferation by MTT method, the result shows BMSCs:EPCs best mixing ratio of1:1.3. BMP2and VEGF gene were constructed into recombinant adenovirus expression vector by AdMax vector system (Ad-BMP2,Ad-VEGF). Purified recombinant adenovirus and determined for virus titer:BMSCs and EPCs were transfected by Ad-BMP2and Ad-VEGF in gradient MOI, according to the expression of green fluorescent observation to determine the best MOI viral transfection is40. Growth curve shown that cells proliferative capacity was not affected after adenovirus transfer at MOI=40. Extract RNA of recombinant adenovirus transfection group and blank virus transfection group and blank cell group, Real-TimePCR detected the BMP2and VEGF level. The expression of BMP2in Ad-BMP2-BMSC group and the expression of VEGF in Ad-VEGF-EPC was significantly higher than their respective control group (p<0.05). Cell supernatant of each groups were collected, Elisa detected the expression of BMP2and VEGF in protein level. The expression of BMP2in Ad-BMP2-BMSCs group and the expression of VEGF in Ad-VEGF-EPC was significantly higher than their respective control group (p<0.05). These results proved the effectiveness of Ad-of BMP2and Ad-VEGF.4. The co-cultured cells designated as four groups including BMSC+EPC, Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC, and Ad-BMP2-BMSC+Ad-VEGF-EPC groups and cultured in osteogenic differentiation medium. Osteoblastic gene expression of cultured cells in vitro was evaluated after transplantation for7,14,21, and28days. ALP gene in Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC, and Ad-BMP2-BMSC+Ad-VEGF-EPC groups reached a peak level at the21st day. However, Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups showed higher ALP expression than other groups (p<0.05). All groups showed slow increase in the expression of OC. Interestingly, the Col I expression of all groups increased time dependently, and the Ad-BMP2-BMSC+Ad-VEGF-EPC group had the highest Col I level at21st day (P<0.05).5. Four groups including BMSC+EPC, Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups were seeded on an alginate gel and then implanted in rat intramuscularly to evaluate the effects on angiogenesis and osteogenesis. After implantation for4weeks, decalcified sections stained with H&E and sirius red from all groups exhibited ectopic bone formation and Col I formation. The volume of the alginate gel became smaller and smaller due to the degradation and absorption by host tissues. Long-round or shuttle-like cells were observed along the alginate gel. Many neo-collagen tissues and irregular premature bone-like matrix were also observed on the interface between alginate gel and tissues. Newly formed bone began to cover the surface of scaffolds in all groups after4weeks. At week6, the alginate scaffold still remained in vivo, and more collagen tissues were observed. The quantitative analysis showed that Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups had higher bone area than others at week4and6, and there was significant difference between these two groups at week6(P<0.05). The amount of Col I formation in Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups was higher than that in the BMSCs+EPC and BMSC+Ad-VEGF-EPC groups at week4and6, but there was no significant difference between Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups (P<0.05). The occurrence of angiogenesis at4th and6th week was confirmed by the expression of CD31, a special marker of endothelial cells. The BMSC+Ad-VEGF-EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups displayed the higher density of blood vessels than other groups at week4and6, and there was significant difference between these two groups at week6(P<0.05), which was also verified by the quantitative analysis for blood vessel density.6. The RealTime-PCR revealed that VEGF induced7-fold increases in the expression of Id1gene. The expression of Idl gene was found to be up-regulated more than5-fold in Ad-BMP2-BMSC group.Conclusions:1. Bone marrow mononuclear cells were separated by density gradient centrifugation which can be induced to BMSCs and EPCs under certain conditions. Angiogenic ability of EPCs and osteogenic differentiation capabilityof BMSCs can be identified by the cell function test, respectively. This method can obtain the high quality BMSCs and EPCs.2. BMSCs and EPCs could be infected with Ad-BMP2and Ad-VEGF efficiently in vitro. The BMP2was successfully produced in transfected BMSCs and the VEGF can be efficient secreted in EPCs. Then BMP2can enhance osteogenic differentiation of BMSCs and VEGF induce EPCs into the capillary-like structures in vitro.3. VEGF-modified EPCs co-cultured with BMP2-modified BMSCs can promote the vascularization of transplant tissues.4. Id1protein might promote proliferation of early osteoblast progenitor cells and. In this regard, we proposed the overexpression of Id1can be induced by BMP2and VEGF, which down-regulated the terminal differentiation of committed osteoblasts and promote angiogenesis. VEGF-modified EPCs co-cultured with BMP2-modified BMSCs can accelerate osteogenesis and bone formation through promoting the vascularization of transplant tissues. Although this approach did not exhibit perfect bone formation, the relationship between Idl gene and osteogenesis has the potential to provide a new vision for the engineered bone tissues.Originality1. We demonstrate, for the first time, that BMP modified BMSCs and VEGF-modified EPCs together as seed cells for bone tissue engineering. The vascularization of VEGF-EPC can promote the repair of bone defects and provide a solution and strategie to a large area of bone defect repair.2. This study was to explore the role of the Idl gene in osteogenesis and angiogenesis, provides a new idea for the study of bone tissue engineering.