Ultrasound enhanced platelet-like particle therapy for accelerated wound repair

项目摘要 仅在美国，慢性伤口就影响了超过650万患者，并导致超过200万美元的健康成本。 每年250亿美元。适当的伤口愈合是大量相互关联的生物事件的结果， 是根据损伤微环境在时间上进行协调的。在受伤后，血凝块 其涉及嵌入纤维蛋白网内的血小板栓的形成。血小板结合倍数 随着时间的推移，血小板通过肌动蛋白驱动的机制收缩这种纤维蛋白网， 通过稳定纤维蛋白网络有助于随后的伤口愈合，进一步防止失血， 恢复血流通过阻塞性血栓。血小板介导的血栓收缩， 减小凝块大小、改变凝块组织并增加凝块硬度。增加的基质刚度已被 参与激活参与伤口愈合的重要机械敏感途径，包括Rho GT3信号转导、肌动蛋白细胞骨架结合和转化生长因子β的机械活化 (TGFβ），进而促进成纤维细胞迁移到伤口床和细胞外基质（ECM）中 生产重要的是，慢性不愈合伤口的特征在于显著降低的 这些细胞活动。这项提案的长期目标是利用最近开发的血小板样- 血小板颗粒（PLPs），其模拟天然血小板的这种凝块收缩特征，以促进慢性非血小板性疾病的愈合。 愈合伤口PLP介导的凝块收缩通过集体布朗扳手机制发生， 局部纤维蛋白基质，诱导整体和细胞规模的变形，最终导致整体凝血 崩溃颗粒可变形性和高纤维蛋白亲和力对于实现PLP介导的凝块收缩至关重要。 然而，PLP介导的凝块收缩的动力学比天然血小板慢得多（天 （小时）。为了在伤口修复中获得血块收缩的益处，关键是增加PLP介导的血栓形成的速率。 凝块收缩，以更接近地概括天然血小板的时间尺度。因此， 该提议的目的是利用PLP的超声（US）刺激来增加PLP的变形 以便以精细控制的方式增加凝块收缩的速率。我们 中心假设是1）US刺激将增加纤维蛋白网络内的PLP变形， 从而增加与纤维蛋白网络的相互作用以及PLP介导的凝块的速率和程度 收缩;和2）增强的凝块收缩将增加凝块硬度，促进成纤维细胞迁移到血管中， 通过激活Rho GT3信号传导，增加ECM产生，并增加伤口闭合 率在体外和体内。我们将在以下目标中探索这一假设：1）确定最佳 US序列可使PLP变形最大化并增加PLP介导凝块的动力学 收回2)表征PLP-US治疗对体外伤口愈合结局的影响， in vivo.我们所提出的工作的意义是发展一种简单的和可翻译的技术 从而能够有效治疗不愈合的伤口。

PROJECT SUMMARY Chronic wounds affect over 6.5 million patients in the United States alone and result in health costs of more than $25 billion annually. Proper wound healing is the result of a large number of interrelated biological events, which are orchestrated temporally in response to the injury microenvironment. Immediately following injury, a clot is produced which involves the formation of a platelet plug embedded within a fibrin mesh. Platelets bind multiple fibrin fibers and overtime, platelets contract this fibrin mesh through actin driven mechanisms, which contributes to subsequent wound healing by stabilizing the fibrin network, further preventing blood loss, and restoring blood flow past the otherwise obstructive thrombi. Platelet-mediated clot retraction, significantly decreases clot size, alters clot organization, and increases clot stiffness. Increased matrix stiffness has been implicated in activation of important mechanically sensitive pathways involved in wound healing, including Rho GTPase signaling, actin cytoskeleton engagement and mechanical activation of transforming growth factor beta (TGFβ), which in turn promote fibroblast migration into the wound bed and extracellular matrix (ECM) production. Importantly, chronic non-healing wounds are characterized by significantly decreased activation of these cellular events. The long-term objective of this proposal is to utilize recently develop platelet-like- particles (PLPs) that mimic this clot retraction feature of native platelets to promote healing in chronic non- healing wounds. PLP-mediated clot retraction occurs via a collective Brownian wrench mechanism to collapse the local fibrin matrix, inducing both global and cell-scale deformations, which ultimately leads to global clot collapse. Particle deformability and high fibrin affinity are critical to achieving PLP-mediated clot retraction. However, the dynamics of PLP-mediated clot retraction are much slower (days) than that of natural platelets (hours). To obtain the benefits of clot retraction in wound repair, it is critical to increase the rate of PLP-mediated clot retraction, to more closely recapitulate the time scale of natural platelets. Therefore, the overarching objective of this proposal is to utilize ultrasound (US) stimulation of PLPs to increase the deformation of PLPs within the fibrin network in order to increase the rate of clot retraction in a finely controlled manner. Our central hypothesis is that 1) US stimulation will increase PLP deformations within the fibrin network, thereby increasing interactions with the fibrin network and the rate and degree of PLP-mediated clot retraction; and 2) enhanced clot retraction will increase clot stiffness, promote fibroblast migration into the wound bed through activation of Rho GTPase signaling, increase ECM production, and increase wound closure rates in vitro and in vivo. We will explore this hypothesis in the following aims: 1) Determine the optimal US sequence to maximize PLP deformation and increase kinetics of PLP-mediated clot retraction. 2) Characterize the effect of PLP-US therapy on wound healing outcomes in vitro and in vivo. The significance of our proposed work is the development of a simple and translatable technology enabling the effective treatment of non-healing wounds.