Engineering the open porous nanofibrous microsphere integrated fibrillar hydrogel for the co-delivery of antibacterial and angiogenic agents aimed at the rapid diabetic wound repair

项目总结 这项提议旨在设计一种由开放的多孔纳米纤维组成的多因素创面敷料 微球(NMS)与刺激响应性纤维水凝胶(FHG)相结合，将治疗药物释放到 加速糖尿病伤口愈合。所提出的创面敷料中开放的多孔结构可以增强细胞 迁移和肉芽组织形成，以快速闭合伤口。首先，将制造出多孔的NMS 使用我们最新发明的核壳电喷雾技术，从电纺短纳米纤维片段 泡泡技术。之后，多肽拴系基质金属蛋白酶(MMPs)反应的FHG将 与多孔性纳米管结合，形成可注射的杂化凝胶，然后进行光交联。此前，我们 证明了多孔纳米纤维支架在细胞迁移和细胞/组织方面显示出巨大的改善 整合。受此结果启发，我们将开发一种新的伤口敷料平台，以改善伤口 修复过程的几个方面；(I)NMS的细胞外基质(ECM)模拟多孔性结构可以 在愈合过程中加速细胞迁移。(Ii)用甲基丙烯酸酯明胶设计ECM模拟物FHG (GelMA)和基质金属蛋白酶反应性多肽连接物可以提供维持潮湿的条件和持续的输送 局部的治疗剂。(3)基因工程多肽偶联NMS结合FHG可预防糖尿病 通过持续释放抗菌肽和血管生成多肽来调节血管生成。至 为了实现这些目标，我们将加入抗菌LL-37模拟W379多肽，并与血管内皮细胞 用生长因子(VEGF)模拟QK多肽将NMS整合成FHG。具体地说，我们已经确定了这个漏洞 NMS的特性可以通过多孔微结构调节细胞迁移，促进伤口愈合 与无孔纳米管相比，纳米管复合凝胶。纤维状水凝胶网络将提供潮湿的条件 与细胞外基质相似的伤口和伤口环境中多肽的受控释放。我们将进行评估和优化 材料特性采用以下表征工作流程：NMS的孔隙率、混合体的可注入性 凝胶，体外细胞毒性定量，体外凝胶降解与不同的基质金属蛋白酶细胞反应(存活， 增殖、迁移)、W379多肽的体外抗菌作用、QK的体外血管生成特性 多肽、体内免疫反应(流式细胞术分析)、体内物质降解(通过 组织学)和体内组织愈合/再生(免疫组织学分析)。之后， 我们将专注于一个 感染糖尿病小鼠(db/db)夹板创面愈合模型的建立 不含多肽 。我们希望看到通过开放的多孔NMS加速伤口愈合 可注射性水凝胶与市售Geistlich derma gideTM可注射剂组成的比较 水凝胶。该项目有可能开发一类新的生物材料，并创造出一种廉价和 糖尿病创面的有效治疗通过增加我们对如何抑制生物被膜形成和 糖尿病伤口闭合中孔洞诱导的细胞迁移。

PROJECT SUMMARY This proposal aims to engineer a multifactorial wound dressing composed of open porous nanofibrous microspheres (NMs) integrated with a stimuli-responsive fibrillar hydrogel (FHG) to release therapeutics to accelerate diabetic wound healing. The open porous structure in the proposed wound dressing can enhance cell migration and granulation tissue formation for rapid wound closure. Initially, the porous NMs will be fabricated from electrospun short nanofiber segments using our newly invented core-shell electrospray technique with bubble technology. Afterward, the peptide-tethered matrix metalloproteinases (MMP) responsive FHG will be integrated with porous NMs to develop an injectable hybrid gel followed by photocrosslinking. Previously, we demonstrated that porous nanofiber scaffolds show tremendous improvements in cell migration and cell/tissue integration. Inspired by the results, we will develop a new wound dressing platform that improves the wound healing process in several aspects; (i) The extracellular matrix (ECM) mimetic porous structure of the NMs can accelerate the cell migration during the healing. (ii) Engineering an ECM mimetic FHG with methacrylate gelatin (GelMA) and MMP responsive peptide linker can offer to maintain the moist conditions and sustained delivery of therapeutic agents locally. (iii) The engineered peptide conjugated NMs integrated FHG can prevent diabetic wound infection and tune the angiogenesis by sustained release of antibacterial and angiogenic peptides. To achieve these goals, we will incorporate antibacterial LL-37 mimic W379 peptide, and vascular endothelial growth factor (VEGF) mimetic QK peptide to the NMs integrated FHG. Specifically, we have identified that porous NMs properties can modulate cell migration through the porous microarchitecture and improve wound healing compared with nonporous NMs composed gel. The fibrillar hydrogel network will provide moist conditions in the wound similar to ECM and the controlled release of peptides in the wound milieu. We will evaluate and optimize material properties using the following characterization workflow: the porosity of NMs, the injectability of hybrid gel, in vitro cytotoxicity quantification, in vitro gel degradation with different MMP cell responses (survival, proliferation, and migration), in vitro antibacterial efficacy of W379 peptide, in vitro angiogenic properties of QK peptide, in vivo immune response (analysis by Flow cytometry), in vivo material degradation (analysis by histology), and in vivo tissue healing/regeneration (analysis by immunohistology). Afterward, we will focus on an infected diabetic mouse (db/db) splinted wound healing model to evaluate the efficacy of the dressing with and without peptides . We expect to see accelerated wound healing through the open porous NMs composed of the injectable hydrogel compared with commercially available Geistlich Derma gideTM composed of the injectable hydrogel. This project has the potential to develop a new class of biomaterial and create an inexpensive and effective treatment for diabetic wounds by increasing our understanding of how to inhibit biofilm formation and porosity-induced cell migration in diabetic wound closure.