Dual Delivery of Engineered EVs and Growth Factor for Bone Regeneration

项目摘要/摘要 与慢性免疫相关的炎症条件和复杂的生理变化 2型糖尿病(T2 DM)患者对伤口愈合的反应挑战骨治疗的结果 再生。与骨愈合失败相关的发病率和成本是相当可观的， 越来越多。最近的发现表明，免疫系统与骨骼生理密切相关。 MSC来源的细胞外小泡(EVS)在骨修复的免疫调节中起重要作用。 而重组人骨形态发生蛋白2(RhBMP2)介导的骨再生效果良好 已经确立，控制因使用重组人骨形态发生蛋白2而引起的炎症的挑战在 存在免疫调节全身性疾病，如T2 DM，存在显著的知识差距 限制临床结果。基于炎症诱导的MSC EV microRNA(MiRNA)的初步研究 成分和对免疫调节和骨再生的影响，我们假设双重递送 具有抗炎特性的工程化MSC EVS和使用组合递送系统的rhBMP2 自组装亮氨酸拉链(LZ)多肽水凝胶和藻酸盐微珠可促进兔骨愈合 T2 DM。我们提出了三个具体目标来检验这一假设。在目标1中，我们将测试我们的初步数据驱动 假设MSC EV中改变的miRNAs的子集有助于增强 炎性预适应MSC来源的EVS的免疫调节功能。我们还将生成 过表达具有抗炎特性的候选miRNA的工程MSC Evs以进一步研究 单个EV相关miRNAs控制免疫调节功能的机制方面。在AIM 2，我们将创建一种新型的双重递送系统，使用LZ-水凝胶和含有工程电动汽车的藻酸盐微珠 和rhBMP2。我们将检验我们的假设：1)具有抗炎特性的EV(产生 可在愈合的炎症阶段(长达7天)从自组装的LZ- 水凝胶和2)包裹在海藻酸盐珠中的具有缓释特征(4-6周)的rhBMP2可以 提供长期的骨诱导信号，以促进/增强骨再生。在目标3中，使用了一位糖尿病患者 小鼠颅骨缺损模型，我们将验证工程MSC EVS含有抗炎作用的假说 MiRNA可抑制创伤后的炎症反应，而持续释放的rhBMP2可促进骨愈合。 总体而言，这些关于工程化和MSC EVS组合使用的首批研究结果将是 为继续定义MSC EV介导的免疫调节和再生 双给药系统在2型糖尿病骨愈合中的应用此外，这些研究还将 为将生长因子和工程化EVS结合用于组织工程提供框架 申请。

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.