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

不愈合的伤口影响数百万美国人，每年需要数十亿美元的护理费用。这些慢性伤口通常归因于病理学，例如心血管疾病和糖尿病，其可以改变伤口内的细胞代谢并产生许多其他病理学后遗症。本研究计划的长期目标是建立无创、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。本提案的目的是发展 使用受损伤口愈合的体外和体内模型，观察改变的代谢和组织微结构的生物标志物。具体来说，我们计划使用非线性光学显微镜能够双光子激发荧光，二次谐波产生，共焦反射定义真皮和表皮细胞的氧化还原状态，胶原组织，和血管生成只使用内源性的对比度。中心假设是改变的细胞代谢可以提供受损伤口愈合的早期标志物，并且胶原蛋白重塑、血管生成和上皮再形成的定量生物标志物可以为开发适当的治疗方案提供背景。为了初步测试该假设，并开发受损愈合的生物标志物，将在目标1中使用三维人类皮肤等效模型来评估光学氧化还原比对氧和葡萄糖水平变化的敏感性。在目的2和3中，将使用伤口愈合的糖尿病小鼠模型在体内评价受损愈合。除了目标1中体外建立的代谢生物标志物外，还将开发技术来量化糖尿病伤口中改变的胶原基质组织和减少的血管生成。总的来说，这些目标将提供一套光学生物标志物，以便能够早期检测伤口中改变的细胞代谢，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成像技术将能够对伤口代谢和组织进行时空评估，这是目前实验室或临床上无法实现的。由于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应用立即应用于慢性伤口评估。 通过完成研究和培训计划，申请人将从塔夫茨，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。这种强化培训和职业发展计划将补充申请人在非线性光学方面的背景，并使其成功过渡到独立调查员。

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.