Oral and Craniofacial Bone Regeneration using MicroRNA Modulation

项目摘要 自体移植和同种异体移植是目前口腔和颅面缺损骨修复的标准策略。 然而，每一种方法都有局限性，例如使用自体移植时供体部位的发病率以及发生移植失败的风险。 通过使用同种异体移植物来缓解传播。基于组织工程策略的人工骨移植替代物 Egy代表了一种克服这些固有局限性的替代方法。MicroRNAs(MiR)是 小的非编码的RNA已经成为重要的转录调节因子，在生理和 病理生理条件。我们之前已经证明，miR-200家族的成员积极地调节 成骨分化、骨发育和炎症。我们的长期目标是开发基于mir的 可有效促进成骨分化和骨再生的基因治疗方案 用于修复口腔和颅面部的骨缺损。我们最近的研究表明，miR-200C负载胶原 海绵在体内促进了骨的形成。我们还观察到miR-200C上调了Wnt sig-1的活性。 降低SOx2和Klf4。与之相关的是，miR-200A已被证明直接抑制Wnt和Wnt 骨形态发生蛋白/转化生长因子信号通路。在我们的初步研究中，我们已经表明，使用我们的Plas-2抑制miR-200A- 基于MID的miR抑制系统(PMIs)可以有效地促进成骨分化和骨形成 在活体内。我们在这项应用中的目标是了解和优化miR-200C的分子功能 和PMIS-200A(miR-200A抑制剂)对成骨分化和骨形成的影响，并验证其 局部给药可以用来促进骨再生。这个项目的中心假设是 MIR-200C联合miR-200A抑制通过上调Wnt和TGF-β促进成骨分化 /BMP信令。可以优化miR-200C和PMIS-200A载体以促进骨再生 口腔和颅面部缺陷。在本项目中，我们将确定miR-200C在Wnt sig-1上的分子功能。 以及Sox2和Klf4在miR-200C介导的成骨分化中的作用(目标1)。我们会 通过了解PMIS-200A在成骨分化和骨形成中的作用 PMIS-200A对Wnt和BMP/TGF信号的调节(目标2)。我们将提高工作的有效性。 MIR-200C和PMIS-200A通过使用可生物降解的纳米颗粒优化基因传递系统。在目标中 3我们将使用临界大小的颅骨缺损和牙周炎诱导的骨缺损模型来研究 MiR-200C联合PMIS-200A能否在体内诱导明显的骨再生。在复杂的情况下- 随着这个项目的完成，我们将大大扩展我们对miR-200C分子功能的理解 和miR-200A抑制，并证明了这些miRs对口腔和颅面的翻译能力 骨骼再生。

Project Summary Autografts and allografts are current standard strategies for bone repair of oral and craniofacial defects. However, each possesses limitations, such as donor-site morbidity with the use of autograft and the risk of dis- ease transmission with the use of allograft. Synthetic bone-graft substitute based on a tissue engineering strat- egy has represented an alternative approach to overcome these inherent limitations. MicroRNAs (miRs) are small non-coding RNAs that have emerged as important transcriptional regulators in both physiologic and pathophysiological conditions. We have previously shown that members of the miR-200 family actively regulate osteogenic differentiation, bone development, and inflammation. Our long-term goal is to develop a miR-based gene therapy program that can be used to effectively promote osteogenic differentiation and bone regeneration for restoring oral and craniofacial bone defects. Our recent studies showed that miR-200c loaded in collagen sponge promoted bone formation in vivo. We also observed that miR-200c up-regulated the activity of Wnt sig- naling and reduced Sox2 and Klf4. Relatedly, miR-200a has been demonstrated to directly suppress Wnt and BMP/TGF signal pathways. In our preliminary studies we have shown that inhibiting miR-200a using our Plas- mid-based miR Inhibitor System (PMIS) can effectively improve osteogenic differentiation and bone formation in vivo. Our objectives in this application are to understand and optimize the molecular function of miR-200c and PMIS-200a (miR-200a inhibitor) on osteogenic differentiation and bone formation and to validate that their local administration can be used to improve bone regeneration. The central hypothesis of this project is that miR-200c combined with miR-200a inhibition improve osteogenic differentiation by up-regulating Wnt and TGF- /BMP signaling. Plasmid miR-200c and PMIS-200a delivery can be optimized to promote bone regeneration for oral and craniofacial defects. In this project, we will determine the molecular function of miR-200c on Wnt sig- naling and the roles of Sox2 and Klf4 in the osteogenic differentiation mediated by miR-200c (Aim 1). We will also determine the function of PMIS-200a on osteogenic differentiation and bone formation by understanding the regulation of PMIS-200a on Wnt and BMP/TGF signaling (Aim 2). We will improve the effectiveness of miR-200c and PMIS-200a by optimizing a gene delivery system using biodegradable nanoparticles. In the Aim 3 we will use critical-sized calvarial defects and periodontitis-induced bone defects in rat models to investigate whether miR-200c combined with PMIS-200a can induce significant bone regeneration in vivo. At the comple- tion of this project, we will have significantly expanded our understanding of the molecular function of miR-200c and miR-200a inhibition and demonstrated the translational capabilities of these miRs for oral and craniofacial bone regeneration.