Dual Delivery of Engineered EVs and Growth Factor for Bone Regeneration

项目概要/摘要 与慢性免疫相关的炎症状态和复杂的生理变化， 2型糖尿病（T2 DM）对伤口愈合的反应挑战了骨治疗的结果， 再生与骨愈合失败相关的发病率和成本是相当大的， 增加。最近的发现表明，免疫系统与骨骼生理学密切相关 并且MSC衍生的细胞外囊泡（EV）在骨修复的免疫调节中起显著作用。 而重组人骨形态发生蛋白2（rhBMP 2）介导的骨再生效果良好， 建立，控制rhBMP 2使用引起的炎症的挑战在 存在免疫调节性系统性疾病（如T2 DM），存在显著的知识差距， 限制临床结果。基于炎症的初步研究告知MSC EV microRNA（miRNA） 组合物和对免疫调节和骨再生的作用，我们假设， 使用具有抗炎特性的工程化MSC EV和rhBMP 2的组合递送系统 自组装亮氨酸拉链（LZ）肽基水凝胶和藻酸盐珠可以增强骨愈合， 2型糖尿病。我们提出了三个具体目标来检验这一假设。在aim 1中，我们将测试我们的初步数据驱动 假设在预处理的MSC EV中改变的miRNA的子集有助于增强细胞内的细胞增殖。 炎症预处理的MSC衍生EV的免疫调节功能。我们还将产生 过表达具有抗炎特性的候选miRNA的工程化MSC EV，以进一步研究 单个EV相关miRNA控制免疫调节功能的机制方面。在aim中 2，我们将创建一种新型的双重递送系统，其中包含LZ-水凝胶和含有工程EV的藻酸盐珠 和rhBMP 2。我们将测试我们的假设，1）具有抗炎特性的电动汽车（产生 可以在愈合的炎性阶段（长达7天）从自组装LZ-1中释放， 水凝胶和2）包封在藻酸盐珠粒中具有延长释放特性（4-6周）的rhBMP 2可 提供长期的骨诱导信号以促进/增强骨再生。在目标3中，使用糖尿病患者 在小鼠颅骨缺损模型中，我们将测试含有抗炎剂的工程化MSC EV miRNA将抑制创伤后的炎症，而持续释放的rhBMP 2促进骨愈合。 总的来说，工程化和MSC EV rhBMP 2的组合使用的这些第一次研究的结果将 为MSC EV介导的免疫调节和再生免疫调节的持续定义提供了基础。 双重输送系统在T2 DM骨愈合中的特性和适用性。此外，这些研究还将 为生长因子和工程化EV的组合用于组织工程提供框架 应用.

Project Summary/Abstract The inflammatory conditions and the complex physiological changes associated with chronic immunological responses to wound healing in type 2 diabetes (T2DM) challenge the outcomes of the treatments for bone regeneration. The related morbidity and costs associated with bone healing failures are considerable and increasing. Recent discoveries have demonstrated that the immune system is tightly linked to bone physiology and that MSC derived extracellular vesicles (EVs) play a prominent role in immunomodulation of bone repair. While recombinant human bone morphogenetic protein 2 (rhBMP2) mediated bone regeneration is well established, the challenges of controlling inflammation arising from rhBMP2 usage is exacerbated in the presence of immunomodulating systemic diseases such as T2DM present a significant knowledge gap that limit clinical outcomes. Based on preliminary studies of inflammation informed MSC EV microRNA (miRNA) composition and effects on immunomodulation and bone regeneration, we hypothesize that a dual delivery of engineered MSC EVs with anti-inflammatory properties and rhBMP2 using a combinatorial delivery system with self-assembling leucine zipper (LZ) peptide-based hydrogels and alginate beads can enhance bone healing in T2DM. We propose three specific aims to test this hypothesis. In aim 1, we will test our preliminary data driven hypothesis that a subset of altered miRNAs in preconditioned MSC EVs contributes to the enhanced immunomodulatory function of the inflammatory preconditioned MSC derived EVs. We will also generate engineered MSC EVs that overexpress candidate miRNA with anti-inflammatory properties to further study the mechanistic aspects by which individual EV related miRNAs control the immunomodulation functionality. In aim 2, we will create a novel dual delivery system with LZ-hydrogels and alginate beads that contain engineered EVs and rhBMP2 respectively. We will test our hypothesis that 1) EVs with anti-inflammatory properties (generated from aim 1) can be released during the inflammatory phase of healing (up to 7 days) from a self-assembling LZ- hydrogel and 2) rhBMP2 that is encapsulated in alginate beads with an extended-release profile (4-6 weeks) can provide a long term osteoinductive signal to promote/enhance bone regeneration. In aim 3, using a diabetic mouse calvarial defect model, we will test the hypothesis that engineered MSC EVs containing anti-inflammatory miRNA will suppress inflammation upon wounding while sustained released rhBMP2 promote bone healing. Overall, the results of these first studies of the combinatorial usage of engineered and MSC EVs rhBMP2 will provide a foundation for continued definition of MSC EV-mediated immunomodulation, and the regenerative properties and applicability of the dual delivery system to bone healing in T2DM. Further, these studies will also provide a framework for the use of growth factors and engineered EVs in combination for tissue engineering applications.