Macrophage Phenotype Transition as the Biological Mechanism of Chronic Wound Healing Treated with Non-Thermal, Non-Cavitational Therapeutic Ultrasound

项目摘要/摘要 在美国，慢性伤口影响着大约650万名患者。现行标准 伤口处理方案并不保证愈合，而是专注于维持伤口 有利于被动自愈的环境。因此，有必要开发替代方案 促进主动愈合和缩短愈合时间的治疗方法，从而降低成本。我们有 此前有报道称，采用低频(20-100 kHz)、低强度(50-150 mW/cm2)治疗 与伤口相比，超声波(Lfli US)显著(p&lt；0.03)减少体内静脉性溃疡的大小。 用假装置治疗。这项建议旨在确定通过什么生物机制 Lfli US在体外促进慢性伤口愈合。有证据表明，愈合受损的原因 巨噬细胞表型的失调，尤其是从亲核细胞到巨噬细胞的缺陷转化。 炎性(M1)到促愈合(M2)巨噬细胞。我们对清创组织的表征 慢性创面的愈合表明，愈合的慢性创面含有比M2样更高比例的M1-样细胞。 就像巨噬细胞。此外，整合素下游的信号蛋白rac2和焦点黏附 激酶激活是巨噬细胞机械转导的关键调节因子，并促进 巨噬细胞从M1表型向M2表型的转变。拟议的研究将系统地 用体外培养的巨噬细胞检测激光照射对巨噬细胞表型的影响 三维(3D)脚手架。我们假设Ifli US直接和间接地刺激 促炎症的M1巨噬细胞通过rac2向促进愈合的M2巨噬细胞转变。这个项目 将加强我们对慢性伤口愈合的理解，以及治疗性超声波的潜力 加速愈合。目的1阐明激光超声对巨噬细胞功能和功能的直接影响。 LFli-US直接处理炎性巨噬细胞的表型及功能鉴定 变化(增殖、迁移和吞噬)、蛋白质/细胞因子分泌和基因表达。 同时，我们将验证rac2是促进M1的潜在的机械转导途径 通过共聚焦分析整合素、粘着斑激酶和rac2向M2巨噬细胞的转化 显微镜和RNA表征。[AIM 2将使用我们的 先前对接受治疗的慢性创面患者清创组织的诊断M1/M2评分 与Ifli US合作。]目的3阐明Lfli-US对巨噬细胞功能的间接影响和 3D巨噬细胞成纤维细胞共培养表型。这项研究的结果将为最佳 设计LFli超声治疗方案，为慢性疾病提供个性化、积极的治疗方案 可减少伤口，加速慢性伤口愈合，并有助于降低年度伤口护理成本。

PROJECT SUMMARY/ ABSTRACT Chronic wounds affect approximately 6.5 million patients in the United States. Current standard protocols for wound management do not guarantee healing and focus on maintaining a wound environment that is conducive to passive self-healing. Hence, there is a need to develop alternative treatments that promote active healing and shorten healing time leading to reduced costs. We have previously reported that treatment with low-frequency (20-100 kHz), low-intensity (50-150 mW/cm2) ultrasound (LFLI US) significantly (p<0.03) reduces venous ulcer size in vivo as compared to wounds treated with a sham device. This proposal aims at determining the biological mechanisms by which LFLI US promotes chronic wound healing in vitro. There is evidence that the cause of impaired healing is the dysregulation of macrophage phenotype, especially the defective transition from pro- inflammatory (M1) to pro-healing (M2) macrophages. Our characterization of tissue debrided from chronic wounds has shown that healing chronic wounds contain higher proportions of M1-like than M2- like macrophages. Additionally, the signaling protein Rac2, downstream of integrin and focal adhesion kinase activation, is a key regulator of mechanotransduction in macrophages and facilitates the transition of macrophages from the M1 to M2 phenotype. The proposed study will systematically examine the effects of LFLI ultrasound on macrophage phenotype, using macrophages cultured in three-dimensional (3D) scaffolds. We hypothesize that LFLI US directly and indirectly stimulates the transition of pro-inflammatory M1 macrophages to pro-healing M2 macrophages via Rac2. This project will enhance our understanding of chronic wound healing and the potential of therapeutic ultrasound to accelerate healing. Aim 1 will elucidate the direct effects of LFLI US on macrophage function and phenotype by treating inflammatory macrophages directly with LFLI US and characterizing functional changes (proliferation, migration, and phagocytosis), protein/cytokine secretion, and gene expression. Concurrently, we will validate Rac2 as the potential mechanotransduction pathway which promotes M1 to M2 macrophages transition by analyzing integrins, focal adhesion kinases, and Rac2 via confocal microscopy and RNA characterization. [Aim 2 will validate the in vitro findings from Aim 1 using our previously developed diagnostic M1/M2 score on debrided tissue from chronic wound patients treated with LFLI US.] Aim 3 will elucidate the indirect effects of LFLI US on macrophage function and phenotype via a 3D macrophage fibroblast co-culture. The results of this study will inform the optimal design of LFLI ultrasound therapy protocols, lead to a personalized, active treatment for chronic wounds, accelerate chronic wound healing, and contribute to reduced annual wound care costs.