背景:骨缺损今天仍然是一个挑战。除了成骨激活,血管生成的关键作用也受到关注。特别是,血管内皮生长因子(VEGF)可能在骨再生中起重要作用,不仅可以恢复血液供应,还可以直接参与间充质干细胞的成骨分化。在这项研究中,在骨再生过程中产生附加的血管生成-成骨作用,VEGF和Runt相关转录因子2(Runx2),成骨分化必需的转录因子,与信使RNA(mRNA)共同给予大鼠下颌骨的骨缺损。
方法:通过体外转录(IVT)制备编码VEGF或Runx2的mRNA。使用原代成骨细胞样细胞评估mRNA转染后的成骨分化,然后评估成骨标志物的基因表达水平。然后使用我们的原始阳离子聚合物载体将mRNA施用到大鼠下颌骨中制备的骨缺损中,复合物纳米胶束。通过显微计算机断层扫描(μCT)成像评估骨再生,和组织学分析。
结果:mRNA转染后,成骨标志物如骨钙蛋白(Ocn)和骨桥蛋白(Opn)显著上调。VEGFmRNA显示具有与Runx2mRNA相似的独特成骨细胞功能,并且两种mRNA的组合使用导致标记的进一步上调。体内给药后进入骨缺损,随着骨矿化的增加,两种mRNA诱导骨再生的显著增强。使用针对分化簇31蛋白(CD31)的抗体进行组织学分析,碱性磷酸酶(ALP),或OCN揭示了mRNA诱导缺损中成骨标志物的上调,随着血管形成的增加,导致快速的骨形成。
结论:这些结果证明了使用mRNA药物引入各种治疗因子的可行性,包括转录因子,进入目标站点。这项研究为组织工程mRNA疗法的开发提供了有价值的信息。
BACKGROUND: Bone defects remain a challenge today. In addition to osteogenic activation, the crucial role of angiogenesis has also gained attention. In particular, vascular endothelial growth factor (VEGF) is likely to play a significant role in bone regeneration, not only to restore blood supply but also to be directly involved in the osteogenic differentiation of mesenchymal stem cells. In this study, to produce additive angiogenic-osteogenic effects in the process of bone regeneration, VEGF and Runt-related transcription factor 2 (Runx2), an essential transcription factor for osteogenic differentiation, were coadministered with messenger RNAs (mRNAs) to bone defects in the rat mandible.
METHODS: The mRNAs encoding VEGF or Runx2 were prepared via in vitro transcription (IVT). Osteogenic differentiation after mRNA transfection was evaluated using primary osteoblast-like cells, followed by an evaluation of the gene expression levels of osteogenic markers. The mRNAs were then administered to a bone defect prepared in the rat mandible using our original cationic polymer-based carrier, the polyplex nanomicelle. The bone regeneration was evaluated by micro-computerized tomography (μCT) imaging, and histologic analyses.
RESULTS: Osteogenic markers such as osteocalcin (Ocn) and osteopontin (Opn) were significantly upregulated after mRNA transfection. VEGF mRNA was revealed to have a distinct osteoblastic function similar to that of Runx2 mRNA, and the combined use of the two mRNAs resulted in further upregulation of the markers. After in vivo administration into the bone defect, the two mRNAs induced significant enhancement of bone regeneration with increased bone mineralization. Histological analyses using antibodies against the Cluster of Differentiation 31 protein (CD31), alkaline phosphatase (ALP), or OCN revealed that the mRNAs induced the upregulation of osteogenic markers in the defect, together with increased vessel formation, leading to rapid bone formation.
CONCLUSIONS: These results demonstrate the feasibility of using mRNA medicines to introduce various therapeutic factors, including transcription factors, into target sites. This study provides valuable information for the development of mRNA therapeutics for tissue engineering.