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.

中文翻译：

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含有表达 miR20a 的质粒的基因激活基质促进大鼠颅骨增强

基因激活基质 (GAM) 在骨工程中具有潜在用途，可作为持久释放成骨蛋白的替代策略，但产生非病毒 GAM 的有效方法仍有待建立。在这项研究中，我们研究了基于去端胶原的 GAM 是否含有编码 microRNA (miR) 20a 的裸质粒 (p) DNA，这可能通过与间充质干细胞/祖细胞 (MSCs) 的成骨分化相关的多种途径促进体内成骨。 ，有利于大鼠颅骨增大。首先，我们在体外证实了生成的编码 miR20a (pmiR20a) 的 pDNA 的成骨细胞分化功能，其转染调节了大鼠骨髓间充质干细胞中 Bambi1 和 PPARγ 等几个靶基因的表达，并诱导 BMP4 的表达增加。然后，当将 100 μl 2% 牛去端胶原、20 mg β-TCP 颗粒和 0.5 mg (3.3 μg/μl) 编码 miR20a 的 AcGFP 质粒载体混合制成的 GAM 被移植到大鼠颅骨表面时，可以清楚地认识到促进的垂直骨增强移植后长达 8 周，移植早期阶段 VEGF 和 BMP4 表达的上调也是如此。因此，基于 GAM 的 miR 递送可以通过可调节多种途径的小序列提高转基因功效，从而提供一种替代的非病毒方法。移植早期阶段 VEGF 和 BMP4 表达的上调也是如此。因此，基于 GAM 的 miR 递送可以通过可调节多种途径的小序列提高转基因功效，从而提供一种替代的非病毒方法。移植早期阶段 VEGF 和 BMP4 表达的上调也是如此。因此，基于 GAM 的 miR 递送可以通过可调节多种途径的小序列提高转基因功效，从而提供一种替代的非病毒方法。