Microphysiological Models to Evaluate the Role of Age-Dependent Fibrinogen Sialylation in Wound Healing

伤口愈合是一个复杂的过程。伤口愈合的第一步是凝血。形成的凝块停止出血，并为细胞重建受损组织提供支持。伤口愈合在婴儿和成人之间也是不同的，但这些差异还没有被很好地理解。该项目将开发新的工具，以更好地了解(1)凝血在婴儿和成人之间是如何不同的，以及(2)凝血的差异如何导致伤口愈合的差异。许多类型的细胞参与修复，包括血小板和成纤维细胞。血小板在凝血中起着重要作用，成纤维细胞在止血后帮助重建受损的组织。该项目还将测量成纤维细胞和血小板在伤口愈合过程中的相互作用。更好地了解伤口愈合有望为美国近600万凝血功能受损和无法愈合的伤口患者确定新的治疗靶点。该项目的教育部分包括对本科生和研究生进行工程生物医学系统方面的培训，包括与临床医生合作。外联活动包括为K-12科学博览会竞赛开发实践教育单元和活动，并为一年一度的兽医学院开放日和该地区吸引K-12学生的北卡罗来纳州科学咖啡馆制作示范工具包。创伤修复涉及生物化学和机械信号的复杂协调，这些信号指导着多种细胞类型的行为。由于创面愈合的复杂性，目前尚不清楚创面愈合受损的机制。受伤后，会形成纤维蛋白凝块，作为支持组织修复的细胞渗透的临时支架，从而止血并促进愈合。纤维蛋白基质的缺乏可能导致无法愈合的伤口。虽然已知衰老影响愈合，但尚不清楚与年龄相关的纤维蛋白特性差异如何影响愈合结果。最近的研究发现，成人和新生儿之间的纤维蛋白网络特性存在着广泛的差异。例如，与成人纤维蛋白相比，成纤维细胞在新生儿纤维蛋白上的附着和迁移更好；在小鼠全层损伤模型中，与成人纤维蛋白支架相比，新生纤维蛋白支架在体内促进了更好的愈合。虽然翻译后修饰的许多差异可能导致与年龄相关的纤维蛋白原功能的差异，但最近的数据强调，新生儿纤维蛋白原中唾液酸(Sia)含量的增加显著地导致了与年龄相关的凝块结构、力学以及聚合和降解动力学的差异。这项工作的主要假设是，与成人纤维蛋白原相比，新生儿纤维蛋白原中SIA含量的增加会导致纤维蛋白聚合机制、凝块结构和机械性能的改变，从而推动改善愈合结果。该项目将研究新生儿和成人纤维蛋白原网络+/-SIA中的SIA含量如何在体外影响成人和新生儿成纤维细胞反应。下一步，这个项目将调查SIA含量如何影响伤口愈合中的细胞串扰。创新的组织工程技术和微流控技术将被用于研究创伤愈合过程中血小板和成纤维细胞之间的相互作用的微生理系统。MPS提供了一种方法来生成可操作、可翻译的数据，方法是用必要的主要人体细胞类型、显微解剖学、细胞-细胞相互作用和微机械线索来概括选定的人类组织功能。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Wound healing is a complex process. The first step in wound healing is clotting. The clot that forms stops bleeding and provides support for cells to rebuild damaged tissue. Wound healing is also different between babies and adults, but these differences are not well understood. This project will develop new tools to better understand (1) how clotting is different between babies and adults and (2) how differences in clotting lead to differences in wound healing. Many cells types are involved in healing, including platelets and fibroblasts. Platelets play important roles in clotting and fibroblasts help rebuild damaged tissue after bleeding stops. The project will also measure interactions between fibroblasts and platelets in wound healing. Better understanding of wound healing is expected to lead to identification of novel therapeutic targets for the nearly 6 million people in the United States suffering from impaired coagulation and non-healing wounds. The project's education component involves training undergraduate and graduate students in engineering biomedical systems, including working with clinicians. Outreach activities include developing hands-on educational modules and activities that will be innovated for K-12 science fair competitions and constructing a demonstration kit for the Annual College of Veterinary Medicine Open House Day and the North Carolina Science Café, which engages K-12 students in the region. Wound healing involves the complex orchestration of biochemical and mechanical signals directing behavior of multiple cell types. Due to the complex nature of wound healing, much remains unknown about mechanisms underlying impaired healing. After injury, a fibrin clot is formed that stops bleeding and promotes healing by serving as a provisional scaffold for infiltrating cells that support tissue repair. A deficient fibrin matrix can result in non-healing wounds. While it is known that aging influences healing, it is unknown how age related differences in fibrin properties may influence healing outcomes. Recent studies have identified extensive differences in fibrin network properties between adults and neonates. For example, fibroblasts attach and migrate better on neonatal fibrin compared to adult fibrin, and neonatal fibrin scaffolds promote better healing in vivo in a murine full thickness injury model compared to adult fibrin scaffolds. While many variances in post-translational modifications likely contribute to age-related differences in fibrinogen function, recent data highlights that increased sialic acid (Sia) content in neonatal fibrinogen contributes significantly to age-related differences in clot structure, mechanics, and polymerization and degradation dynamics. The overarching hypothesis of this work is that increased Sia content in neonatal fibrinogen results in altered fibrin polymerization mechanisms, clot structure, and mechanical properties compared to adult fibrinogen, which drives improved healing outcomes. This project will investigate how Sia content in neonatal and adult fibrinogen networks +/- Sia influence adult and neonatal fibroblast responses in vitro. Next, this project will investigate how Sia content influences cellular crosstalk in wound healing. Innovative tissue engineering techniques and microfluidics will be employed to develop a microphysiological system (MPS) to study the interactions of platelets and fibroblasts during wound healing. MPS provide a means to generate actionable, translatable data by recapitulating select human tissue functions with the requisite primary human cell types, microanatomy, cell-cell interactions and micromechanical cues.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.