Development of nonlinear endomicroscopy: toward assessing articular cartilage repair In vivo

摘要 关节软骨的修复和再生仍然是一个临床和科学挑战。可靠的评估 评估软骨修复结果的工具对于改进现有方法和 新技术的发展。活检的组织学分析是修复评估的金标准； 然而，活组织检查是侵入性检查，因此仅限于临床评估和研究。 软骨修复。常用的医学成像方法，如x射线照相、计算机断层扫描 (CT)、磁共振成像(MRI)和超声波可以非破坏性地执行成像；然而， 它们的空间分辨率不足以揭示关节软骨复杂的细胞和基质结构。 虽然有些成像技术是非破坏性的，可以对组织成像，如关节镜、激光 扫描共焦关节镜(LSCA)和光学相干断层扫描(OCT)，它们都是作为 由于使用了粗大的内窥镜探头，因此需要进行外科手术。无法进行临床活体成像 对高空间分辨率的软骨组织的研究仍然是一个问题。为了解决这个问题，我们建议 研制一种基于非线性光学显微镜(NLOM)的软骨内窥镜系统 体内修复。在软骨组织的NLOM成像中，二次谐波产生(SHG)信号提供了高分辨率 双光子激发荧光(TPEF)使胶原原纤维结构的分辨率信息 软骨细胞和弹性蛋白纤维的可视化。然而，目前的软骨NLOM成像设备都使用 桌面系统体积太大，无法直接用于临床应用。因此，我们的长期目标是 将这项技术转化为临床成像工具，用于评估关节软骨修复和治疗 在细胞水平上。在本申请中，我们将重点关注以下三个具体目标。(1)我们将确定 NLOM在评价关节软骨形态变化中的作用使用自发的办公自动化 作为豚鼠关节软骨病理模型，我们将检测NLOM成像是否能定量检测到 早期骨性关节炎软骨组织的差异。(二)设计建设紧凑、高性能的大桥。 以细棒物镜为成像探头的SPEED NLOM成像系统。一个数值模拟模型将 将被开发以帮助优化系统设计。(3)随着内窥镜的发展，我们将首先 通过执行类似于Aim1中所述的定量成像研究来评估其性能 骨性关节炎豚鼠的软骨组织。然后我们将使用内窥镜和桌面系统来 对软骨修复模型进行定量成像研究，以测试内窥镜是否可以检测到 未处理和微骨折处理的缺损区组织之间的形态差异。随着成功的到来 通过这项研究，我们将能够确定使用NLOM评估软骨的有用性和局限性。 修复并进行进一步的活体动物研究，为未来软骨修复的临床研究做准备 使用先进的内窥镜系统。

SUMMARY Repair and regeneration of articular cartilage remains a clinical and scientific challenge. Reliable assessment tools for evaluating outcomes of cartilage repair are critical for both refinement of existing methods and development of new techniques. Histological analysis of biopsies is the gold standard for repair assessment; however, biopsies are invasive procedures and therefore limited in clinical evaluation and studies of the cartilage repair. The common medical imaging methods, such as x-ray radiography, computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound, can perform imaging non-destructively; however, their spatial resolutions are not sufficient to reveal the complex cell and matrix architecture of articular cartilage. Though some imaging techniques are non-destructive and can image tissue, such as arthroscopy, laser scanning confocal arthroscopy (LSCA) and optical coherence tomography (OCT), they are all performed as surgical procedures because of using thick endoscopy probes. The inability to perform clinical in vivo imaging on cartilage tissue with high spatial resolution remains a problem. To solve the problem, we propose to develop a nonlinear optical microscopy (NLOM) based endomicroscopy system for assessment of cartilage repair in vivo. In NLOM imaging of cartilage tissue, second harmonic generation (SHG) signal provides high- resolution information of fibril organization of collagen while two-photon excited fluorescence (TPEF) enables visualization of chondrocytes and elastin fibers. However, the current cartilage NLOM imaging devices all use tabletop systems that are too bulky to be used directly in clinical applications. Thus, our long-term goal is to translate this technology into a clinical imaging tool for assessment of articular cartilage repair and treatment at the cellular level. In this application, we will focus on three specific aims as follows. (1) We will determine the efficacy of using NLOM to evaluate morphological changes of articular cartilage. Using spontaneous OA guinea pigs as an articular cartilage pathology model, we will test if NLOM imaging can detect the quantitative differences among the early stages of OA cartilage tissues. (2) We will design and build a compact and high- speed NLOM imaging system with a thin rod objective as the imaging probe. A numerical simulation model will be developed to help optimize the system design. (3) With the developed endomicroscope, we will first evaluate its performance by performing a similar quantitative imaging study as described in Aim1 on excised cartilage tissues from guinea pigs with OA. We will then use the endomicroscope and tabletop system to perform a quantitative imaging study on a cartilage repair model to test if the endomicroscope can detect morphological differences between tissues in non-treated and microfracture treated defects. With the success of this study, we will be able to determine the usefulness and limitation for using NLOM to assess cartilage repair and to perform further in vivo animal study in preparation for future clinical studies on cartilage repair with the developed endomicroscope system.