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The construction of scaffolds and subsequent incorporation of cells and biologics have been widely investigated to regenerate damaged tissues. Scaffolds act as a template to guide tissue formation, and their characteristics have a considerable impact on the regenerative process. Whereas many technologies exist to induce specific two-dimensional (2D) morphologies into biomaterials, the introduction of three-dimensional (3D) micromorphologies into individual pore walls of scaffolds produced from biological molecules such as collagen poses a challenge. We here report the use of dicarboxylic acids to induce specific micromorphologies in collagen scaffolds and evaluate their effect on cellular migration and differentiation. Insoluble type I collagen fibrils were suspended in monocarboxylic and dicarboxylic acids of different concentrations, and unidirectional and random pore scaffolds were constructed by freezing and lyophilization. The application of various acids and concentrations resulted in variations in 3D micromorphologies, including wall structure, wall thickness, and pore size. The use of dicarboxylic acids resulted in acid-specific micromorphologies, whereas monocarboxylic acids did not. Dicarboxylic acids with an odd or even number of C-atoms resulted in frayed/fibrillar or smooth wall structures, respectively, with varying appearances. The formation of micromorphologies was concentration-dependent. In vitro analysis indicated the cytocompatibility of scaffolds, and micromorphology-related cell behavior was indicated by enhanced myosin staining and myosin heavy chain gene expression for C2C12 myoblasts cultured on scaffolds with frayedlike micromorphologies compared to those with smooth micromorphologies. In conclusion, porous collagen scaffolds with various intrawall 3D micromorphologies can be constructed by application of dicarboxylic acids, superimposing the second level of morphology to the overall scaffold structure. Acid crystal formation is key to the specific micromorphologies observed and can be explained by the odd/even theory for dicarboxylic acids. Scaffolds with a 3D micrometer-defined topography may be used as a screening platform to select optimal substrates for the regeneration of specific tissues.

Introduction of Specific 3D Micromorphologies in Collagen Scaffolds Using Odd and Even Dicarboxylic Acids