Gene-activated matrix (GAM) has a potential usefulness in bone engineering as an alternate strategy for the lasting release of osteogenic proteins but efficient methods to generate non-viral GAM remain to be established. In this study, we investigated whether an atelocollagen-based GAM containing naked-plasmid (p) DNAs encoding microRNA (miR) 20a, which may promote osteogenesis in vivo via multiple pathways associated with the osteogenic differentiation of mesenchymal stem/progenitor cells (MSCs), facilitates rat cranial bone augmentation. First, we confirmed the osteoblastic differentiation functions of generated pDNA encoding miR20a (pmiR20a) in vitro, and its transfection regulated the expression of several of target genes, such as Bambi1 and PPARγ, in rat bone marrow MSCs and induced the increased expression of BMP4. Then, when GAMs fabricated by mixing 100 μl of 2% bovine atelocollagen, 20 mg β-TCP granules and 0.5 mg (3.3 μg/μl) AcGFP plasmid-vectors encoding miR20a were transplanted to rat cranial bone surface, the promoted vertical bone augmentation was clearly recognized up to 8 weeks after transplantation, as were upregulation of VEGFs and BMP4 expressions at the early stages of transplantation. Thus, GAM-based miR delivery may provide an alternative non-viral approach by improving transgene efficacy via a small sequence that can regulate the multiple pathways.

Bone morphogenetic protein-2 (BMP-2) has diverse functions and is especially important in bone and cartilage development. Recombinant human BMP-2 (rhBMP-2) is an osteoinductive growth factor that has been clinically applied as a bone graft substitute. However, high-dose rhBMP-2 can cause complications such as induction of significant swelling that can endanger the patient's life. Atelocollagen sponge (ACS) is the commercially provided standard carrier of rhBMP-2 in clinical applications. However, a large concentration of rhBMP-2 is required to be clinically effective with ACS as the carrier. Octacalcium phosphate/collagen (OCP/Col) has been shown to be an excellent bone substitute compared with other bone substitute materials such as hydroxyapatite or β-tricalcium phosphate due to its biological properties. In this study, we evaluated the use of OCP/Col as a carrier to minimize the effective dose of rhBMP-2. ACS or OCP/Col discs impregnated with different rhBMP-2 concentrations were implanted in mice calvarial bone defects. Morphological analysis with micro-CT both at 4 and 6 weeks post-implantation showed homogenous hard tissue formation in the defects of the OCP/Col group at all rhBMP-2 concentrations tested (0, 0.25, 0.50, or 1.00 μg). In contrast, ACS alone or with 0.25 μg of rhBMP-2 showed almost no bone formation. However, bone mineral density in all groups of ACS and OCP/Col was not dependent on rhB-MP-2 concentration. Histological evaluation indicated that bone formation progressed depending on rhBMP-2 concentration in the defects of both the ACS and OCP/Col groups, although the newly formed bone area was significantly higher in the OCP/Col group than in the ACS group. These results indicate that OCP/Col could be an effective carrier of rhBMP-2, minimizing the application dose of rhBMP-2 in clinical settings and avoiding the complications caused by high-dose rhB-MP-2.

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The aim of this study was to clarify whether hydroxyapatite/ collagen composite material (HAp/Col) could be useful as a graft material for maxillary sinus floor augmentation (MSFA). MSFA and implant placement were performed simultaneously. When the lateral approach was employed, 3 out of 19 implants failed in 3 maxillary sinuses (success rate; 84.2%), and in these cases the alveolar bone heights, cortical bone thicknesses and values of the implant stability quotient were smaller. If alveolar the bone height, cortical bone thickness, and healing period are optimized, HAp/Col can be a useful graft material for MSFA.