Engineering the Corneal Wound Bed to Promote Healing

描述（由申请人提供）：在世界范围内，主要由感染、创伤和手术并发症引起的角膜疾病导致600万至800万例人类患者失明。在所有这些情况下，伤口愈合是维持或恢复体内平衡并确保最佳视觉结果的基本要素。角膜伤口愈合是一个复杂的过程，其中细胞必须同时整合由可溶性细胞外信号环境中的细胞活性因子提供的多种线索以及由细胞外基质提供的生物物理线索。该过程的失调或延迟可导致慢性不愈合伤口、混浊形成和视觉损害。传统的医疗和手术治疗有时不足以产生最佳结果。在角膜伤口的治疗中迫切需要改进的疗法。因此，提出了一种新的、通用的和可推广的工程方法来促进有利的角膜伤口愈合结果。通过利用蛋白质结合化学，界面科学和纳米-亚微米制造技术的最新进展，我建议从根本上改变角膜伤口，以促进愈合。与用治疗剂对角膜伤口的常规局部治疗相比，将细胞活性因子直接整合到角膜伤口床中使得能够使用显著更少的化合物，这种方法提供了低得多的细胞毒性可能性，具有大得多的安全裕度，并且在治疗计划的执行中呈现显著的成本节约。生物可降解材料将用于获得对角膜伤口中细胞活性因子持续性的时间控制。将细胞活性因子直接整合到角膜伤口中将使由长期、持续的细胞活性因子信号传导引起的有害作用的可能性最小化。抗微生物因子如银和人b-防御素-3（一种天然存在的宿主肽）将用于测试新的命题，即它们直接整合到角膜伤口中可以提供抗微生物活性而不损害愈合。本提案的总体目的是确定如何利用界面材料工程的新方法从根本上改变角膜伤口床的表面化学和生物物理特性，以促进有利的愈合结果。在假设1中，将利用装载有纳米-亚微米珠粒的纳米多层膜（PEM）将抗微生物化合物和细胞活性因子整合到角膜伤口床中。令人兴奋的初步数据已经证明了通过冲压将功能化PEM转移到伤口床中的可行性，并表明PEM内的亚微米珠粒是有效转移到软材料如角膜伤口床所必需的。通过优化PEM和珠粒材料的生物降解，可以实现整合到角膜伤口床中的抗微生物和细胞活性因子的短暂驻留。然后，将银和b-防御素-3掺入微珠和/或PEM后，研究其动力学、抗微生物活性和细胞毒性。在假设2中，蛋白质连接化学将直接将抗菌化合物和细胞活性因子整合到角膜伤口床中。最近的研究已经证明了在各种模型表面上共价结合细胞活性因子的能力，同时保留其生物活性。首先，将确定用于角膜细胞的各种蛋白质连接化学的安全性。然后，将确定使细胞活性因子EGF能够调节或瞬时驻留的最佳连接化学以及将EGF固定到角膜伤口床的最佳过程。最后，将共价固定的EGF的功效与传统的EGF局部治疗进行比较。如果成功，这笔赠款的结果将对人类和动物角膜伤口的管理产生巨大影响。 公共卫生相关性：角膜创伤和感染是常见的，导致全球600万至800万例失明。不良的角膜伤口愈合可导致角膜透明度的永久性丧失，并且可用于解决该问题的药物和手术治疗有限。这项提案的目的是开发新的治疗方法，以帮助角膜伤口愈合使用工程启发的方法;这些新的疗法可能会深刻改变我们如何治疗人类和动物的角膜伤口。

DESCRIPTION (provided by applicant): Worldwide, corneal diseases primarily resulting from infection, trauma, and surgical complications are responsible for 6 to 8 million cases of blindness in human patients. In all of these cases, wound healing is an essential element to maintaining or restoring homeostasis and ensuring optimal visual outcomes. Corneal wound healing is a complex process wherein cells must simultaneously integrate multiple cues provided by the cytoactive factors in the soluble extracellular signaling environment as well as biophysical cues supplied by the extracellular matrix. Dysregulation or delay of this process can result in chronic non-healing wounds, haze formation, and visual compromise. Conventional medical and surgical treatments are sometimes insufficient in producing optimal outcomes. There is an urgent need for improved therapies in the treatment of corneal wounds. Therefore, a novel, versatile and generalizable engineering approach is proposed to promote favorable corneal wound healing outcomes. By utilizing recent advances in protein-conjugation chemistry, interfacial science, and nano-submicron fabrication technologies, I propose to fundamentally change the corneal wound to promote healing. Compared to conventional topical treatment of a corneal wound with therapeutic agents, the direct integration of cytoactive factors into the corneal wound bed enables the use of significantly less compound, an approach that provides a much lower likelihood of cytotoxicity, has a much greater safety margin, and presents significant cost savings in the execution of the therapeutic plan. Biodegradable materials will be used to gain temporal control over cytoactive factor persistence in the corneal wound. Direct integration of cytoactive factor(s) into the corneal wound will minimize the probability of deleterious effects resulting from long-standing, persistent cytoactive factor signaling. Antimicrobial factors such as silver and human b-defensin-3, a naturally occurring host peptide, will be used to test the novel proposition that their direct integration into the corneal wound can provide antimicrobial activity without impairing healing. The overall purpose of this proposal is to determine how novel approaches to interfacial materials engineering can be utilized to fundamentally alter the surface chemistry and biophysical characteristics of the corneal wound bed to promote favorable healing outcomes. In hypothesis 1, polyelectrolyte multilayers (PEMs) loaded with nano-submicron beads will be utilized to integrate antimicrobial compounds and cytoactive factors into the corneal wound bed. Exciting preliminary data already have documented the feasibility of transferring functionalized PEMs into wound beds by stamping and shown that submicron beads within the PEMs are required for efficient transfer to soft materials such as corneal wound beds. By optimizing the biodegradation of PEMs and bead materials, transient residence of antimicrobial and cytoactive factors integrated into the corneal wound bed can be achieved. The kinetics, antimicrobial activity, and cytotoxicity of silver and b-defensin-3 will then be investigated following incorporation into beads and/or PEMs. In hypothesis 2, protein linkage chemistries will directly integrate antimicrobial compounds and cytoactive factors into the corneal wound bed. Recent studies have demonstrated the ability to covalently immobilize cytoactive factors on various model surfaces while preserving their bioactivity. First, the safety of various protein linkage chemistries for use with corneal cells will be determined. Then, the optimal linkage chemistry to enable tuning or transient residence of a cytoactive factor, EGF, will be determined as well as the best process for immobilizing EGF to the corneal wound bed. Lastly, the efficacy of covalently immobilized EGF will be compared to traditional topical treatment of EGF. If successful, the outcomes of this grant will have a dramatic impact on the management of corneal wounds in humans and animals. PUBLIC HEALTH RELEVANCE: Corneal trauma and infection are common and responsible for 6 to 8 million cases of blindness worldwide. Poor corneal wound healing can result in permanent loss of corneal transparency, and there are limited medical and surgical treatments available to address this problem. The purpose of this proposal is to develop novel treatments to aid in corneal wound healing using an engineering-inspired approach; these new therapies may profoundly alter how we treat corneal wounds in people and animals.