Shido Rena, Sumita Yoshinori, Hara Masahito, Iwatake Mayumi, Narahara Shun, Umebayashi Mayumi, Miura Kei-Ichiro, Kodama Yukinobu, Asahina Izumi
Department of Regenerative Oral Surgery, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
Basic & Translational Research Center for Hard Tissue Disease, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
Regen Biomater. 2021 Mar 13;8(2):rbaa060. doi: 10.1093/rb/rbaa060. eCollection 2021 Mar.
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 () DNAs encoding microRNA (miR) 20a, which may promote osteogenesis 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 DNA encoding miR20a (miR20a) , 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.
基因激活基质（GAM）作为一种使成骨蛋白持续释放的替代策略，在骨工程中具有潜在的应用价值，但生成非病毒GAM的有效方法仍有待确立。在本研究中，我们探究了一种基于去端胶原蛋白的GAM，其包含编码微小RNA（miR）20a的裸质粒DNA，该微小RNA可能通过与间充质干/祖细胞（MSC）成骨分化相关的多种途径促进骨生成，是否有助于大鼠颅骨增大。首先，我们证实了所生成的编码miR20a（miR20a）的DNA的成骨细胞分化功能，并且其转染调节了大鼠骨髓间充质干细胞中几个靶基因（如Bambi1和PPARγ）的表达，并诱导了BMP4表达的增加。然后，当将通过混合100μl 2%牛去端胶原蛋白、20mg β - TCP颗粒和0.5mg（3.3μg/μl）编码miR20a的AcGFP质粒载体制备的GAM移植到大鼠颅骨表面时，在移植后长达8周时明显观察到垂直骨增大得到促进，在移植早期VEGFs和BMP4表达也上调。因此，基于GAM的miR递送可能通过一个能够调节多种途径的小序列提高转基因效率，从而提供一种替代性的非病毒方法。
