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

从再生医学和医疗器械到疫苗佐剂和药物输送平台，一系列广泛的生物材料正在被开发用于医疗应用。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，也是监管部门批准新材料和医疗器械所必需的。这些研究严重依赖活体动物模型，并涉及组织学和免疫组织化学等终末期方法。这项建议的主要目标是开发一种基于漫反射拉曼光谱(DRS)的新型便携式设备，用于监测活体动物模型的FBR。这将允许纵向测量同一动物的FBR(而不是在特定时间点牺牲动物)。尽管有一系列活体动物成像方法可用，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弥漫性光子迁移。利用近红外光(波长为785-1000 nm)，DRS非常适合探测皮肤内部和皮下，以测量FBR过程中的生物分子过程。在我们之前的工作中，我们展示了拉曼光谱可以检测皮肤炎症和监测胶原密度的变化，这是FBR的两个主要特征。然而，该项目的主要挑战是优化DRS仪器的设计，以便达到检测极限和精度，以便能够在体内量化这些变化。这个项目建立在我们使用光在组织中传播的模型的最新结果的基础上，这些结果表明，通过优化DRS探头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由聚合物生物材料皮下植入引发的FBR的小鼠模型。尽管FBR在组织中诱导了一系列生物分子变化，但在这个项目中，我们将重点放在两个关键读出上，我们已经证明了这两个读出的原理，这两个参数是从组织学获得的主要参数：通过量化胶原浓度获得的纤维包膜厚度，以及通过量化DNA信号获得的炎症。如果成功，这项新技术将允许科学家和工程师在同一动物身上非侵入性地跟踪FBR，而不必在每个时间点牺牲动物。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敏感性，通过减少研究中使用的动物数量，具有伦理和经济利益。这项技术有可能将研究中的动物数量减少至少50%(典型的两个终点研究)。然而，为了更好地了解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，我们预计可以实现75%-80%的削减。

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.