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了600万至800万人类失明病例。在所有这些情况下，伤口愈合是维持或恢复体内平衡和确保最佳视力结果的基本要素。角膜创面愈合是一个复杂的过程，细胞必须同时整合可溶性细胞外信号环境中细胞活性因子提供的多种信号，以及细胞外基质提供的生物物理信号。这一过程的失调或延迟可导致慢性不愈合的伤口，烟雾形成和视觉损害。传统的医学和外科治疗有时不足以产生最佳结果。目前迫切需要改进角膜创伤的治疗方法。因此，提出了一种新颖、通用和可推广的工程方法来促进良好的角膜伤口愈合结果。通过利用蛋白质偶联化学、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。与使用治疗药物对角膜伤口进行常规局部治疗相比，将细胞活性因子直接整合到角膜伤口床中可以使用更少的化合物，这种方法提供了更低的细胞毒性可能性，具有更大的安全边际，并且在治疗计划的执行中节省了显着的成本。可生物降解的材料将被用于暂时控制角膜伤口中的细胞活性因子。将细胞活性因子直接整合到角膜创面中，可以最大限度地减少由长期持续的细胞活性因子信号传导引起的有害影响的可能性。抗菌因子如银和人类b-防御素-3（一种天然存在的宿主肽）将被用来测试它们直接整合到角膜伤口中可以在不损害愈合的情况下提供抗菌活性的新命题。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创面的表面化学和生物物理特性，以促进良好的愈合结果。在假设1中，将利用负载纳米亚微米微珠的聚电解质多层膜（PEMs）将抗菌化合物和细胞活性因子整合到角膜创面中。令人兴奋的初步数据已经证明了通过冲压将功能化PEMs转移到伤口床上的可行性，并表明PEMs内的亚微米珠子需要有效地转移到柔软材料（如角膜伤口床）上。通过优化PEMs和珠状材料的生物降解，可以实现抗菌因子和细胞活性因子在角膜创面中的短暂停留。银和b-防御素-3的动力学、抗菌活性和细胞毒性将在加入微球和/或PEMs后进行研究。在假设2中，蛋白质连锁化学将直接将抗菌化合物和细胞活性因子整合到角膜创面中。最近的研究表明，共价固定细胞活性因子在各种模型表面的能力，同时保持其生物活性。首先，将确定用于角膜细胞的各种蛋白连锁化学物质的安全性。然后，将确定细胞活性因子EGF调节或短暂停留的最佳连锁化学，以及将EGF固定在角膜创面床的最佳工艺。最后，将共价固定EGF的效果与传统的表皮生长因子局部治疗进行比较。如果成功，这笔拨款的结果将对人类和动物角膜伤口的管理产生巨大影响。

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.