Abstract
Bony defects in the craniofacial skeleton remain a major and challenging health concern. Recent studies have shown that baculovirus-mediated gene therapy combined with bone marrow-derived mesenchymal stem cell (BMSC) therapy can improve the calvarial bone healing. Aside from BMSC, adipose-derived stem cells (ASC) have proven to be an abundant source of multipotent stem cells. However, ASC are less superior to BMSC in their osteogenesis potential. Therefore, hereby we aimed to combine baculovirus-mediated microRNA (miRNA) expression with ASC therapy, hoping that the miRNA can assist the regulation of osteogenesis and improve bone healing. We constructed multiple baculoviruses harboring miRNAs putatively associated with osteogenesis, and discovered that miR-148b can up-regulate osteogenic marker gene expression in osteogenic medium. Transduction of human ASC (hASC) with miR-148b and BMP-2, which not only extended the BMP2 expression beyond 14 days but augmented the ASC osteogenesis. Furthermore, the gelatin-coated poly(lactic-co-glycolic acid) (PLGA) scaffold seeded with engineered hASC was transplanted into mice critical size calvarial defect. After transplantation, μCT and histological analyses demonstrated that miR-148b remarkably ameliorated the regeneration of critical size calvarial defect, when compared with the group implanted with hASC only expressing BMP2. The hASC modulated with miR-148b accelerated the bone remodeling and regenerated the bone through the intramembranous pathway, filling ≈90% of the area and volume in 12 weeks. Furthermore, target prediction analysis tools and experimental validation by luciferase 3’ UTR reporter assay identified NOG (Noggin, a BMP antagonist) as a direct target of miR-148b. It was seen that over-expression of miR-148b and BMP-2 leads promotion of BMP-2/p-Smad1/5 signaling than that only expression of BMP-2. This strongly suggests that miR-148b promotes osteogenic differentiation by repressing NOG and its downstream signaling. Our study provides evidence that miRNA-based therapy can be a valuable tool to promote bone regeneration.