Unraveling the Mechanism of Mechanotransduction in Hierarchical Collagen Fiber Formation

揭示分层胶原纤维形成中的力传导机制

• 批准号: 10637410
• 负责人: Jennifer Puetzer
• 金额: $ 32.61万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10637410
• 关键词: Actomyosin; Cells; Chemicals; Cicatrix; Closure by clamp; Collagen; Collagen Fiber; Collagen Fibril; Cues; Cytoskeleton; Development; Devices; Diameter; Embryo; Engineering; Environment; Fascicle; Fiber; Fibroblasts; Focal Adhesion Kinase 1; Gel; Goals; Human; In Vitro; Individual; Injury; Integrins; Ion Channel; Knowledge; Length; Ligaments; Mechanical Stimulation; Mechanics; Meniscus structure of joint; Methods; Musculoskeletal; Natural regeneration; Neonatal; Pain; Periodicity; Piezo 1 ion channel; Play; Proteoglycan; Protocols documentation; Rehabilitation therapy; Role; Signal Pathway; Signal Transduction; Source; Stretching; Surface; System; Tendon structure; Tissue Engineering; Tissues; Translating; Work; cell growth regulation; cellular development; clinically relevant; crosslink; density; in vivo; insight; loss of function; mechanical signal; mechanotransduction; meter; mouse model; novel; postnatal; repaired; replacement tissue; response; restraint; sensor; therapeutic target; tissue regeneration; transmission process

PROJECT SUMMARY Collagen fibers are the primary source of strength and function in tissues throughout the body, particular tendons, ligaments, and menisci. Cells organize these fibers hierarchically, assemble them from nm-wide fibrils, into larger fibers and fascicles, growing in size and strength with increasing mechanical demand. Injuries disrupt this organization, resulting in loss of function, pain, and decreased mobility. Unfortunately, collagen fibers do not regenerate after injury, nor in engineered replacements, creating a lack of repair options for torn tendons, ligaments, and menisci. Our long-term goal is to understand how cells regulate collagen fiber formation so to engineer functional replacements and drive repair in vivo for musculoskeletal tissues throughout the body. As a step towards this goal, the objective of this proposal is to explore how mechanical cues transmitted via cellular contraction and stretch-activated ion channels regulate ligament fibroblast’s development of hierarchical fibers. While cellular contraction forces, highly regulated by integrins, focal adhesion kinase (FAK), and the actomyosin network, are well established to play a major role in collagen fibril alignment, recent work has suggested stretch- activated ion channels, TRPV4 and Piezo1, also play independent and transient roles in regulating collagen organization. However, this work is largely confined to 2D surfaces, unorganized collagen gels, or mouse models, which all lack the larger fibers and fascicles that dominate human musculoskeletal tissues. Recently, we developed a novel culture device that guides cells to produce native-size hierarchically organized fibrils, fibers, and fascicles over 6 weeks of culture. This novel system provides the unique ability to finally dissect the mechanical cues of development and investigate how these forces guide cells to produce strong hierarchical fibers. We hypothesize mechanical signals via integrin-based contraction and stretch-activated ion channels are critical to cell-driven hierarchical fiber formation, with each regulating different aspects of fiber maturation, both with and without dynamic load. Specifically, we hypothesize that while cellular contraction via FAK is critical to progressive hierarchical development, TRPV4 will regulate alignment and remodeling at the fibril level, and Piezo1 will regulate matrix maturation via collagen crosslinking at the fiber and fascicle level. In Aim 1 we will evaluate the contribution of FAK, TRPV4, and Piezo1 in passive static culture when cell-generated contraction forces drive fiber formation and in Aim 2 we will evaluate how their contribution changes with dynamic mechanical stimulation. In both aims, we will investigate how each signaling mechanism, when inhibited or activated, alters collagen organization at the fibril, fiber, and fascicle length-scale, proteoglycan accumulation, and collagen crosslinking, all important to overall tissue function and mechanics. A better understanding of how mechanical cues drive cells to produce hierarchical fibers is integral not only to creating functional replacements, but also identifying therapeutic targets for regenerating collagen fibers after injury, reducing scar formation, and developing optimal rehabilitation protocols for regenerating musculoskeletal tissues throughout the body.

项目摘要 胶原蛋白纤维是整个身体组织中强度和功能的主要来源，特定肌腱， 韧带和半月板。细胞通过分层组织这些纤维，从NM宽的原纤维组装成较大的纤维 纤维和筋膜，随着机械需求的增加，尺寸和强度增长。伤害破坏了这一点 组织，导致功能丧失，疼痛和增加的流动性。不幸的是，胶原蛋白纤维没有 受伤后再生或工程置换术，从而使肌腱缺乏维修选择， 韧带和半月板。我们的长期目标是了解细胞如何调节胶原蛋白纤维形成，从而 工程师的功能替换并驱动体内修复整个身体的肌肉骨骼组织。作为 朝着这个目标迈出的一步，该提案的目的是探索如何通过蜂窝传输机械提示 收缩和拉伸激活的离子通道调节韧带成纤维细胞的层次纤维发展。 而细胞收缩力，由整合素，局灶性粘合剂激酶（FAK）和肌动蛋白高度调节 网络已经确立了在胶原原纤维对齐中发挥重要作用的良好确定，最近的工作表明伸展运动 - 激活的离子通道TRPV4和Piezo1在调节中也起着独立和短暂的作用 组织。但是，这项工作在很大程度上仅限于2D表面，无组织的胶原蛋白凝胶或鼠标 模型都缺乏主导人肌肉骨骼组织的较大纤维和束。最近，我们 开发了一种新型的培养装置，可指导细胞产生天然大小的层次结构组织的原纤维，纤维和 在6周的培养物中束。这个新颖的系统提供了最终剖析机械的独特能力 发育线索和研究这些迫使细胞如何引导细胞产生强大的层次纤维。我们 通过基于整合素的收缩和拉伸激活的离子通道假设的机械信号对 细胞驱动的层次纤维形成，每个控制纤维成熟的不同方面，既有和 没有动态载荷。特别是，我们假设虽然通过FAK的细胞收缩对于渐进式至关重要 层次结构的开发，TRPV4将在原纤维水平调节对齐和重塑，而Piezo1将 通过胶原蛋白在纤维和筋膜水平上交联调节基质成熟。在AIM 1中，我们将评估 当细胞生成的收缩力驱动时，FAK，TRPV4和PIEZO1在被动静态培养中的贡献 纤维形成，在目标2中，我们将评估它们的贡献如何随动态机械刺激而变化。 在这两个目标中，我们都将研究每种信号传导机制如何在抑制或激活时如何改变胶原蛋白 在原纤维，纤维和筋膜长度尺度上的组织，蛋白聚糖的积累和胶原蛋白交联， 所有对整体组织功能和机械的都重要。更好地了解机械提示如何驱动细胞 生产层次纤维不仅是创建功能替代品的组成部分，而且还可以识别 受伤后再生胶原蛋白纤维的治疗靶标，减少疤痕形成并发展最佳 康复方案，用于再生整个身体的肌肉骨骼组织。