Non-invasive label-free quantitative imaging for chronic wound characterization

描述(由申请人提供)：无法愈合的伤口影响着数百万美国人，需要护理，每年的费用高达数十亿美元。这些慢性伤口通常被归因于病理，如心血管疾病和糖尿病，这些疾病会改变伤口内的细胞代谢，并产生一系列其他病理后遗症。这项研究计划的长期目标是建立非侵入性、实时、定量的光学生物标志物 以识别和表征体内受损的愈合。这项提议的目标是发展 利用创伤愈合受损的体外和体内模型研究代谢和组织微结构改变的生物标记物。具体地说，我们计划使用能够双光子激发荧光、二次谐波产生和共聚焦反射的非线性光学显微镜来确定真皮和表皮细胞的氧化还原状态、胶原组织和血管生成，仅使用内源对比源。中心假说是细胞代谢改变可以提供创面愈合受损的早期标志物，而胶原重塑、血管生成和再上皮化的定量生物标志物可以为开发适当的治疗方案提供背景。为了初步验证这一假设，并开发受损愈合的生物标记物，目标1将利用三维人体皮肤等效模型来评估光学氧化还原比率对氧气和葡萄糖水平变化的敏感性。在AIMS 2和3中，将使用糖尿病小鼠伤口愈合模型在体内评估受损的愈合。除了目标1中体外建立的代谢生物标记物外，还将开发技术来量化糖尿病伤口胶原基质组织的改变和血管生成的减少。总的来说，这些AIMS将提供一套光学生物标记物，以早期检测伤口内细胞代谢的变化，以及可用于指导伤口护理的各种其他微观结构和生化结果。这些非侵入性3D成像技术将使目前实验室或临床无法获得的伤口新陈代谢和组织的时空评估成为可能。随着非线性显微镜技术已经可用于临床，本提案中开发的成像方法将使临床立即应用于慢性伤口评估。通过完成研究和培训计划，申请者将从塔夫茨、麻省理工学院和贝丝以色列女执事医疗中心的合作者那里获得不同伤口愈合研究方法的丰富经验。这项密集的培训和职业发展计划将补充申请者在非线性光学方面的背景，并使其能够成功过渡到独立调查人员。

DESCRIPTION (provided by applicant): Non-healing wounds affect millions of Americans and require care with annual costs in the billions of dollars. These chronic wounds are often attributed to pathologies, such as cardiovascular disease and diabetes, which can alter cell metabolism within the wound and produce a host of other pathological sequelae. The long-term goal of this research plan is to establish non-invasive, real-time, quantitative optical biomarkers to identify and characterize impaired healing in vivo. The objective of this proposal is to develop biomarkers of altered metabolism and tissue microstructure using in vitro and in vivo models of impaired wound healing. Specifically, we plan to use non-linear optical microscopes capable of two photon excited fluorescence, second harmonic generation, and confocal reflectance to define dermal and epidermal cell redox states, collagen organization, and angiogenesis using only endogenous sources of contrast. The central hypothesis is that altered cell metabolism can provide an early marker of impaired wound healing, and quantitative biomarkers for collagen remodeling, angiogenesis, and re-epithelialization can provide context for developing appropriate treatment regimens. To initially test this hypothesis, and develop biomarkers of impaired healing, three- dimensional human skin equivalent models will be utilized in Aim 1 to evaluate the sensitivity of an optical redox ratio to changes in oxygen and glucose levels. In Aims 2 and 3, impaired healing will be evaluated in vivo using a diabetic mouse model of wound healing. In addition to the metabolic biomarkers established in vitro in Aim 1, techniques will be developed to quantify altered collagen matrix organization and reduced angiogenesis in diabetic wounds. Collectively, these aims will provide a suite of optical biomarkers to enable the early detection of altered cell metabolism in the wound, and a variety of other microstructural and biochemical outcomes that can be used to guide wound care. These non-invasive 3D imaging techniques will enable spatiotemporal assessments of wound metabolism and organization not currently available in the laboratory or clinic. With non-linear microscopy technology already available for the clinic, the imaging methods developed in this proposal will enable immediate clinical application to chronic wound assessments. Through the completion of the research and training plans, the applicant will gain extensive experience in different wound healing research methods from collaborators at Tufts, MIT, and Beth Israel Deaconess Medical Center. This intensive training and career development plan will supplement the applicant's background in non-linear optics and enable the successful transition to independent investigator.