Collagen/Κ-Carrageenan-Based Scaffolds as Biomimetic Constructs for In Vitro Bone Mineralization Studies

Tissue engineering offers attractive strategies to develop three-dimensional scaffolds mimicking the complex hierarchical structure of the native bone. The bone is formed by cells incorporated in a molecularly organized extracellular mat r i x made of an inorganic phase, called biological apatite, and an organic phase mainly made of collagen and noncollagenous macro-molecules. Although many strategies have been developed to replicate the complexity of bone at the nanoscale in vitro, a critical challenge has been to control the orchestrated process of mineralization promoted by bone cells in vivo and replicate t h e anatomical and biological properties of na t i v e bone. In this study, we used type I collagen to fabricate mineralized scaffolds mimicking the microenvironment of the native bone. The sulfated poly-saccharide kappa-carrageenan was added to the scaffolds to fulfi l l the role of noncollagenous macromolecules in the organization and mineralization of the bone matrix and cell adhesion. Scanning electron microscopy images of the surface of the collagen/kappa-carrageenan scaffolds showed the presence of a dense and uniform network of intertwined fibrils, while images of the scaffolds' lateral sides showed the presence of collagen fibri l s with a parallel alignment, which is characteristic of dense connective tissues. MC3T3-E1 osteoblasts were cultured in the collagen scaffolds and were viable after up to 7 days of culture, both in the absence and in the presence of kappa-carrageenan. The presence of kappa-carrageenan in the collagen scaffolds stimulated the maturation of the cells to a mineralizing phenotype, as suggested by the increased expression of key genes related to bone mineralization, including alkaline phosphatase (Alp), bone sialoprotein (Bsp), osteocalcin (Oc), and osteopontin (Opn), as well as the abi l i t y to mineralize the extracellular matri x after 14 and 21 days of culture. Taken together, the results described in this study shed l i g h t on the potential use of collagen/kappa-carrageenan scaffolds to study the role of the structural organization of bone-mimetic synthetic matrices in cell function.