New craniofacial bone engineering through miR-23-27-24 cluster mediated osteogenic angiogenic coupling

血管影响骨骼生理学的各个方面，包括矿物质生长 在新骨形成过程中。将骨骼改造成血管组织 专注于microRNA作为分子工程工具，因为它们能够 可逆地靶向多个调节网络。最近的研究从我们和其他 实验室已经证明，miRNA促进骨谱系分化， 通过WNT、BMP和Notch信号通路形成新骨， 骨吸收在我们寻求新的治疗方法，促进耦合骨 重塑和血管生成来治疗大面积骨缺损 发现miR-27~23~24 microRNA簇是一个非常有希望的候选miRNA 试剂.我们的初步分析揭示了多个miR-23和miR-27结合位点， Wnt拮抗剂Sfrp 1和Nlk 3 '的非翻译区（UTR）， 与血管生成抑制剂BIM的UTR上的miR-24结合位点结合 和SEMA 4A证明了miR-27-23-24 microRNA簇对于 结合成骨和血管生成分子工程策略。 在初步研究中，我们已经证明miR-23-27-24簇成员 在炎性组织中表达大大降低。相反，miR-27模拟物 治疗具有严重垂直骨缺损的患病牙周组织， 80%完全恢复证明了意外广泛的新骨形成 垂直骨高度和新的矿化组织沉积在大颅骨 miR-27应用8周后的缺陷。含有PEG-PLGA-PLL-miR- 27纳米颗粒促进基质重塑，新血管形成， 在临界尺寸缺损中的大量颅骨再生。在细胞 水平，miR-27过表达增加碱性磷酸酶活性和骨标志物 基因表达，而抑制miR-27则导致细胞凋亡显著增加。 破骨细胞生成相关基因的表达与骨吸收。miR-24和 miR-27促进内皮细胞增殖和血管管形成。 总之，这些令人兴奋的初步数据促使我们提出了一种新的分子 工程策略，将利用成骨和促血管生成的质量， miR-23/27/24簇成员结合合适的支架和再平衡 牙槽骨和颅骨骨的动态平衡，以促进新的颅面骨形成。

Blood vessels affect all aspects of bone physiology, including mineral growth during new bone formation. To engineer bone as a vascularized tissue we have now focused on microRNAs as molecular engineering tools because of their ability to reversibly target multiple regulatory networks. Recent studies from our and other laboratories have demonstrated that miRNAs promote bone lineage differentiation and new bone formation through WNT, BMP, and Notch signaling pathways and also control bone resorption. In our quest for novel therapeutics that promote coupled bone remodeling and angiogenesis for the treatment of large bone defects we have here identified the miR-27~23~24 microRNA cluster as a highly promising miRNA candidate reagent. Our preliminary analysis revealed multiple miR-23 and miR-27 binding sites on the untranslated region (UTR) of the Wnt antagonist Sfrp1 and Nlk 3’, which in conjunction with miR-24 binding sites on the UTR of the angiogenesis inhibitors BIM and SEMA4A demonstrate the potential of the miR-27-23-24 microRNA cluster for coupled osteogenic and angiogenic molecular engineering strategies. In preliminary studies, we have demonstrated that miR-23-27-24 cluster member expression was greatly reduced in inflammatory tissues. Conversely, miR-27 mimic treatment of diseased periodontal tissues with severe vertical bone defects resulted in unexpectedly extensive new bone formation as evidenced by 80% complete restoration of vertical bone height and new deposition of mineralized tissue in large calvarial bone defects after 8 weeks of miR-27 application. Implants containing PEG-PLGA-PLL-miR- 27 nanoparticles promoted matrix remodeling, new blood vessel formation, and substantial amounts of calvarial bone regeneration in critical size defects. On a cellular level, miR-27 overexpression increased alkaline phosphatase activity and bone marker gene expression, while inhibition of miR-27 resulted in a dramatic increase in the expression of osteoclastogenesis related genes and bone resorption. Both miR-24 and miR-27 promoted endothelial cell proliferation and blood vessel tube formation. Together, these exciting preliminary data prompted us to propose a novel molecular engineering strategy that will harness the osteogenic and pro-angiogenic qualities of miR-23/27/24 cluster members in conjunction with suitable scaffolds and re-balance alveolar and calvarial bone homeostasis for new craniofacial bone formation.