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

项目总结/摘要 在美国，慢性伤口影响大约650万患者。现行标准 伤口处理的方案不能保证愈合， 有利于被动自我修复的环境。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，从而降低成本。我们有 先前报道，低频率（20-100 kHz）、低强度（50-150 mW/cm 2） 与伤口相比，超声波（LFLI US）显著（p<0.03）减小了体内静脉溃疡的大小 用假器械治疗该建议旨在确定生物学机制， LFLI US在体外促进慢性伤口愈合。有证据表明导致愈合不良的原因 是巨噬细胞表型的失调，特别是从亲- 炎性（M1）至促愈合（M2）巨噬细胞。我们的组织特征是 慢性伤口已经表明，愈合的慢性伤口含有比M2更高比例的M1样蛋白。 比如巨噬细胞此外，信号蛋白Rac 2，整合素和粘着斑下游， 激酶激活，是巨噬细胞中机械转导的关键调节因子，并促进巨噬细胞的机械转导。 巨噬细胞从M1表型转变为M2表型。拟议的研究将系统地 使用培养在以下环境中的巨噬细胞，检查LFLI超声对巨噬细胞表型的影响： 三维（3D）支架。我们假设LFLI US直接和间接刺激了 促炎性M1巨噬细胞通过Rac 2向促愈合性M2巨噬细胞的转变。这个项目 将增强我们对慢性伤口愈合的理解，以及治疗性超声的潜力， 加速愈合。目的1：阐明低频超声波对巨噬细胞功能的直接影响， 通过直接用LFLI US治疗炎性巨噬细胞并表征功能表型， 变化（增殖、迁移和吞噬作用）、蛋白质/细胞因子分泌和基因表达。 同时，我们将验证Rac 2作为促进M1的潜在机械转导途径 通过共聚焦显微镜分析整合素、粘着斑激酶和Rac 2， 显微镜和RNA表征。[Aim 2将验证目标1的体外结果，使用我们的 先前对接受治疗的慢性伤口患者的清创组织制定的诊断M1/M2评分 关于LFLI US）目的3：阐明低频超声波对巨噬细胞功能的间接影响， 表型通过3D巨噬细胞成纤维细胞共培养。这项研究的结果将告知最佳的 LFLI超声治疗方案的设计，导致个性化，积极治疗慢性 伤口，加速慢性伤口愈合，并有助于降低每年的伤口护理成本。

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.