Bayan Christopher, Levitt Jonathan M, Miller Eric, Kaplan David, Georgakoudi Irene
J Appl Phys. 2009 May 15;105(10):102042. doi: 10.1063/1.3116626. Epub 2009 May 19.
Collagen is the most prominent protein of human tissues. Its content and organization define to a large extent the mechanical properties of tissue as well as its function. Methods that have been used traditionally to visualize and analyze collagen are invasive, provide only qualitative or indirect information, and have limited use in studies that aim to understand the dynamic nature of collagen remodeling and its interactions with the surrounding cells and other matrix components. Second harmonic generation (SHG) imaging emerged as a promising noninvasive modality for providing high-resolution images of collagen fibers within thick specimens, such as tissues. In this article, we present a fully automated procedure to acquire quantitative information on the content, orientation, and organization of collagen fibers. We use this procedure to monitor the dynamic remodeling of collagen gels in the absence or presence of fibroblasts over periods of 12 or 14 days. We find that an adaptive thresholding and stretching approach provides great insight to the content of collagen fibers within SHG images without the need for user input. An additional feature-erosion and feature-dilation step is useful for preserving structure and noise removal in images with low signal. To quantitatively assess the orientation of collagen fibers, we extract the orientation index (OI), a parameter based on the power distribution of the spatial-frequency-averaged, two-dimensional Fourier transform of the SHG images. To measure the local organization of the collagen fibers, we access the Hough transform of small tiles of the image and compute the entropy distribution, which represents the probability of finding the direction of fibers along a dominant direction. Using these methods we observed that the presence and number of fibroblasts within the collagen gel significantly affects the remodeling of the collagen matrix. In the absence of fibroblasts, gels contract, especially during the first few days, in a manner that allows the fibers to remain mostly disoriented, as indicated by small OI values. Subtle changes in the local organization of fibers may be taking place as the corresponding entropy values of these gels show a small decrease. The presence of fibroblasts affects the collagen matrix in a manner that is highly dependent on their number. A low density of fibroblasts enhances the rate of initial gel contraction, but ultimately leads to degradation of collagen fibers, which start to organize in localized clumps. This degradation and reorganization is seen within the first days of incubation with fibroblasts at a high density and is followed by collagen fiber deposition by the fibroblasts. These collagen fibers are more highly oriented and organized than the fibers of the original collagen gel. These initial studies demonstrate that SHG imaging in combination with automated image analysis approaches offer a noninvasive and easily implementable method for characterizing important features of the content and organization of collagen in tissuelike specimens. Therefore, these studies could offer important insights for improving tissue engineering and disease diagnostic efforts.
胶原蛋白是人体组织中最主要的蛋白质。其含量和组织结构在很大程度上决定了组织的力学性能及其功能。传统上用于可视化和分析胶原蛋白的方法具有侵入性，只能提供定性或间接信息，并且在旨在了解胶原蛋白重塑的动态性质及其与周围细胞和其他基质成分相互作用的研究中用途有限。二次谐波产生（SHG）成像作为一种有前景的非侵入性方法出现，可用于提供厚标本（如组织）中胶原纤维的高分辨率图像。在本文中，我们提出了一种全自动程序，以获取有关胶原纤维含量、取向和组织结构的定量信息。我们使用该程序在有无成纤维细胞的情况下监测胶原凝胶在12天或14天内的动态重塑。我们发现，一种自适应阈值化和拉伸方法无需用户输入即可深入了解SHG图像中胶原纤维的含量。一个额外的特征腐蚀和特征膨胀步骤对于保留低信号图像中的结构和去除噪声很有用。为了定量评估胶原纤维的取向，我们提取取向指数（OI），这是一个基于SHG图像空间频率平均二维傅里叶变换功率分布的参数。为了测量胶原纤维的局部组织，我们获取图像小区域的霍夫变换并计算熵分布，该分布表示沿主导方向找到纤维方向的概率。使用这些方法，我们观察到胶原凝胶中成纤维细胞的存在和数量显著影响胶原基质的重塑。在没有成纤维细胞的情况下，凝胶会收缩，尤其是在最初几天，收缩方式使得纤维大多保持无序取向，如小的OI值所示。随着这些凝胶的相应熵值略有下降，纤维的局部组织可能正在发生细微变化。成纤维细胞的存在对胶原基质的影响高度依赖于它们的数量。低密度的成纤维细胞会提高初始凝胶收缩率，但最终会导致胶原纤维降解，这些纤维开始在局部聚集中组织起来。这种降解和重组在与高密度成纤维细胞孵育的最初几天内可见，随后成纤维细胞会沉积胶原纤维。这些胶原纤维比原始胶原凝胶的纤维具有更高的取向性和组织性。这些初步研究表明，SHG成像与自动图像分析方法相结合，为表征组织样标本中胶原蛋白含量和组织的重要特征提供了一种非侵入性且易于实施的方法。因此，这些研究可为改进组织工程和疾病诊断工作提供重要见解。
