Fully automated, quantitative, noninvasive assessment of collagen fiber content and organization in thick collagen gels

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 de novo 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.