Tailoring the assembly of collagen fibers in alginate microspheres

The application of microspheres instead of bulk hydrogels in cell-laden biomaterials offers multiple advantages such as a high surface-to-volume-ratio and, consequently, a better nutrition and oxygen transfer to and from cells. The preparation of inert alginate microspheres is facile, quick, and well-established and the fabrication of alginate-collagen microspheres has been previously reported. However, no detailed characterization of the collagen fibrillogenesis in the alginate matrix is available. We use second-harmonic imaging microscopy reflection microscopy and turbidity assay to study assembly of collagen in alginate microspheres. We show that the assembly of collagen fibers in a gelled alginate matrix is a complex process that can be aided by addition of small polar molecules, such as glycine and by a careful selection of the gelling buffer used to prepare alginate hydrogels. Highlights In situ characterization of collagen fiber assembly in a gelled alginate matrix using collagen-specific second harmonic generated microscopy Collagen fibrillogenesis is positively influenced by the presence of small molecules in the solution prior microsphere preparation The ratio and amount of calcium, sodium and chloride ions used for the alginate gelling has also a crucial impact on the development of a collagen fiber network