Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.

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 ͑SHG͒ 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 ͑OI͒, 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 collage...