Live monitoring of foreign-body response in animals by diffuse Raman spectroscopy

从再生医学和医疗设备到疫苗佐剂和药物输送平台，各种各样的生物材料正在开发用于医疗应用。植入生物材料的异物反应（FBR）是导致医疗器械失效的主要原因。评估FBR不仅是生物材料开发的一个组成部分，也是新材料和医疗器械监管部门批准的必要条件。这些研究严重依赖于体内动物模型，并涉及到组织学和免疫组织化学等后期方法。本提案的主要目标是开发一种基于漫射拉曼光谱（DRS）的新型便携式设备来监测活体动物模型中的FBR。这将允许在同一动物身上进行FBR的纵向测量（而不是在特定时间点牺牲动物）。尽管有一系列的活体动物成像技术，但它们缺乏监测FBR的分子特异性、灵敏度或分辨率。DRS依赖于结缔组织中的漫射光子迁移。DRS使用近红外光（785-1000 nm波长），非常适合探测皮肤深处和皮下，以测量FBR过程中的生物分子过程。在我们之前的工作中，我们表明拉曼光谱可以检测皮肤炎症并监测胶原蛋白密度的变化，这是FBR的两个主要标志。然而，这个项目的主要挑战是优化DRS仪器的设计，以达到检测极限和准确性，以便在体内量化这些变化。该项目建立在我们利用组织中光传播建模的最新结果的基础上，该结果表明，通过优化DRS探针的设计以及与高光通量光谱仪和探测器的集成，可以实现这种高水平的性能。虽然DRS是一种平台技术，但该项目的重点是用于研究皮下植入聚合物生物材料引发的快速反应的小鼠模型。虽然FBR在组织中诱导了一系列的生物分子变化，但在这个项目中，我们将重点关注两个关键的读数，我们已经证明了原理证明，这是从组织学中获得的主要参数：胶原浓度定量所得的纤维化囊的厚度，以及DNA信号定量所得的炎症。如果成功，这项新技术将使科学家和工程师能够在同一动物身上无创地跟踪快流反应器，而不必在每个时间点都牺牲动物。这将提供独特的高质量数据，具有无与伦比的时间分辨率和分子灵敏度，通过减少用于研究的动物数量，具有伦理和经济效益。该技术有可能将研究中的动物数量减少至少50%（典型的双终点研究）。然而，由于需要更好地了解FBR中随时间变化的分子变化，越来越多的研究需要四个或更多的时间终点。在这种情况下，我们设想可以减少75-80%。

A broad range of biomaterials are being developed for medical applications, from regenerative medicine and medical devices to vaccine adjuvants and drug delivery platforms. Foreign body response (FBR) to implanted biomaterials is the leading cause of medical device failure. Assessing FBR is not only an integral part of biomaterials development but also it is required for regulatory approval of new materials and medical devices. These studies heavily rely on in vivo animal models and involve end stage methodologies such as histology and immunohistochemistry. The main goal of this proposal is to develop a new portable device based on diffuse Raman spectroscopy (DRS) to monitor FBR in live animal models. This will allow longitudinal measurements of FBR on the same animal (rather than sacrificing animals at specific time points). Although a range of in-vivo animal imaging are available, they lack the molecular specificity, sensitivity or resolution to monitor FBR. DRS relies on diffuse photon migration in connective tissue. Using near-infrared light (785-1000 nm wavelength), DRS is ideally-suited for probing deeper in and under skin to measure the biomolecular processes during FBR. In our previous work we showed that Raman spectroscopy can detect inflammation in skin and monitor changes in collagen density, two main hallmarks of FBR. However, the main challenge in this project is to optimise the design of the DRS instrument in order to achieve a detection limit and accuracy to allow quantification of these changes in-vivo. This project builds on our latest results using modelling of light propagation in tissue that show that this high level of performance can be achieved by optimising the design of the DRS probe and integration with high optical throughput spectrometers and detectors.While DRS is a platform technology, the focus of this project is on a mouse model used for investigation FBR triggered by subcutaneous implantation of polymer biomaterials. Although FBR induces a range of biomolecular changes in tissue, in this project we will focus on two key read-outs, for which we already have demonstrated proof-of-principle, and which are the main parameters obtained from histology: the thickness of the fibrotic capsule from quantification of collagen concentration, and inflammation from quantification of DNA signals. If successful, this new technology would allow scientists and engineers to follow FBR non-invasively, on the same animal, without having to sacrifice animals at each time point. This will provide unique high quality data, with unmatched time resolution and molecular sensitivity, with ethical and economic benefits by reducing the number of animals used in research.The technology has the potential to reduce the number of animals in research by a minimum 50% (typical two end-points studies). However, the need for better understanding of the time-dependent molecular changes in FBR, more and more studies require four and more time end points. In such cases we envisage that reduction by 75-80% may be achieved.