The Experimental Research On The Construction Of Double Gene-modified Tissue-engineered Bone

Background: It still remains as a clinical problem for orthopaedic surgeons to repair large bone defect. Tissue engineered bone has been advancing rapidly in recent years and has become a research focus in the treatment of bone defect. But the vascularization process is a major bottleneck for its clinical application. The osteogenic ability of bone morphogenetic protein 2 (BMP2) has been widely admitted and vascular endothelial growth factors (VEGF) is special mitogen in vascular endothelial cell, accelerating the growth of new vessels. Researches show that the expression of BMP and VEGF exists mutual influence relations, leading to a synergetic effect on bone regeneration. United reconstruction of bone formation and bone supply can be realized by combined application of both genes, becoming a novel method to repair bone defect. It has become a research focus to transfer endogenous gene to seed cell of tissue-engineered bone through recombinant virus vector system and then combined with scaffolds to form gene-modified tissue engineered bone. Lentiviral vector can infect most none-dividing cells and owns the capacity to express exogenous genes efficiently and stably. It has been widely applied in researches on gene therapy.Objectives: (1) Promote the angiogenesis and osteogenesis processes of tissue-engineered bone by virtue of the synergetic effect of BMP2 and VEGF genes. (2) Master the technique of recombinant lentivirus production to apply in future researches, leading to a stable and high expression of relating exogenous genes.Materials and methods: (1) Human VEGF165 and BMP2 cDNAs were obtained from human osteosarcoma cell line MG63. The expression lentivirus vectors carrying VEGF165 or BMP2 were constructed respectively. Recombinant lentivirus carrying VEGF165 (Lv-VEGF) or BMP2 (Lv-BMP) were packaged and produced respectively. (2) Rat bone marrow mesenchymal stem cells (MSCs) were isolated and expanded in vitro. (3) MSCs were co-transfected with Lv-VEGF and Lv-BMP (BMP+VEGF group), or each alone (BMP group and VEGF group), or with no virus (control group). (4) The mRNA expression of human VEGF165 and BMP2 genes in each group was detected by real-time PCR at 7 days after transfection. And the protein expression of human VEGF165 and BMP2 genes in each group was detected by enzyme linked immunosorbent assay (ELISA) at 1,4,8 weeks after transfection.(5) Alkaline phosphatase activity (ALP) staining as well as ALP activity was performed in each group at 14 days after transfection. (6) MSCs of each group were collected and seeded on theβ-tricalcium phosphate (β-TCP) scaffolds. Theβ-TCP scaffolds combining MSCs of BMP+VEGF group were randomly chosen for scanning electron microscopy (SEM) observation at 14 days after cell seeding. Cell proliferation of each group was determined every day by a hoechst assay for up to 2 weeks after cell seeding. The ALP activity assay of cell-seeded scaffolds in each group were analyzed 14 days after cell seeding.(7)Engineered constructs of each group were implanted subcutaneously to nude mice. The implants were harvested and used for histological analysis at 4 and 8 weeks after implantation. Angiogenesis was also observed at 4 and 8 weeks after implantation by CD31 staining.Results: (1) Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed. (2) VEGF165 and BMP2 genes were co-expressed in BMP+VEGF group. No significant difference of BMP2 expression was detected between BMP+VEGF and BMP groups (P>0.05), similarly no significant difference of VEGF165 expression between BMP+VEGF and VEGF groups (P>0.05). And both genes were successfully co-expressed in the co-transfection group for up to 8 weeks confirmed by ELISA. (3) The ALP activity of MSCs was significantly augmented by the co-transfection with both genes than any single gene transfection (p<0.01).(4) After seeding MSCs onto the scaffolds, SEM observation showed that MSCs grew and proliferated well in co-transfection group at 14 days. There was no significant difference among all the groups in hoechst DNA assay for cell proliferation for 14 days after cell seeding (P>0.05), but the highest ALP activity was observed in the co-transfection group at 14 days after cell seeding (p<0.01). In nude mice, ectopic osteogenesis was observed in BMP group and BMP+VEGF group, and no ectopic osteogenesis was found in the other groups. At 4 and 8 weeks post-operation, BMP+VEGF group has better bone formation than BMP group in the HE staining sections (p<0.01). At 4 weeks post-operation, more CD31 positive cells were observed in BMP+VEGF group than in BMP group.Conclusions:In our study, we constructed tissue-engineered bone usingβ-TCP combined with MSCs lentivirally co-transfected with BMP2 and VEGF165 and implant these engineered constructs subcutaneously to nude mice to observe the bone formation in vivo. We can conclude: (1) A stable and relatively long term co-expression of both genes can be achieved through lentivirus mediated co-transfection of both genes. (2) Combined use of BMP2 and VEGF gene can produce better osteogenesis and angiogenesis than each single gene alone in virtue of the synergistic effect of both genes.