To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.
The purpose of this study was to evaluate the effect of bone marrow mesenchymal stem cells (MSCs) transfected with the basic fibroblast growth factor (bFGF)-expressing recombinant adeno-associated virus vector (rAAV2-bFGF), on early angiogenesis of calvarial defects in rats. The MSCs were cultured and transfected with rAAV2-bFGF after differential adherence isolation. The transfection efficiency was detected by RT-PCR and Western blotting. The transfected MSCs were compounded with poly-DL-lactide/hydroxyapatite (PDLLA/HA) in vitro. The cranial defect models in 36 male SD rats were created. Nothing (group A), PDLLA/HA alone (group B), PDLLA/HA combined with MSCs (group C), and PDLLA/HA combined with rAAV2-bFGF transfected MSCs (group D) were implanted in rat calvarial defects. The specimens were harvested for hematoxylin-eosin staining on the day 1, 3 and 7 after implantation. Factor Ⅷ immunohistochemical staining and histomorphometric analysis were carried out to evaluate neovascularization around the implantation. The results indicated that MSCs could indeed be successfully transfected with the rAAV2-bFGF vector. Histological and histomorphometric analysis revealed that the angiogenesis in group D was significantly enhanced as compared with the rest groups (P<0.05). These results strongly suggest that MSCs transfected with rAAV2-bFGF in combination with PDLLA/HA can effectively promote the early angiogenesis of calvarial defects in rats, which played an important role in creating an environment suitable for the survival and activity of transplanted cells for further applications in cranio-maxillofacial bone regeneration.
