Fabrication of endothelial cell-laden carrageenan microfibers for microvascularized bone tissue engineering applications

Recent achievements in the area of tissue engineering (TE) have enabled the development of three-dimensional (3D) cell-laden hydrogels as in vitro platforms that closely mimic the 3D scenario found in native tissue. These platforms are extensively used to evaluate cellular behavior, cell-cell interactions and tissue-like formation in highly defined settings. In this study, we propose a scalable and flexible 3D system based on micro-sized hydrogel fibers that might be used as building blocks for the establishment of 3D hydrogel constructs for TE applications. For this purpose, chitosan (CHT) coated kappa-carrageenan (κ-CA) microfibers were developed using a two-step procedure involving ionotropic gelation (for the fiber formation) and polyelectrolyte complexation with CHT (for the enhancement of fiber stability). The performance of the obtained fibers was assessed regarding their swelling and stability profiles, as well as their ability to carry and, subsequently, promote the outward release of microvascular-like endothelial cells (ECs), without compromising their viability, phenotype and in vitro functionality. Finally, the possibility of assembling and integrating these cell-laden fibers within a 3D hydrogel matrix containing osteoblast-like cells was evaluated. Overall, the obtained results demonstrate the suitability of the micro-sized κ-CA fibers to carry and deliver functional microvascular-like ECs. Furthermore, it is shown that it is possible to assemble these cell-laden micro-sized fibers into 3D heterotypic hydrogels constructs. This in vitro 3D co-culture platform provides a versatile approach to investigate the interactions between multiple cell types in controlled settings, which may open up novel 3D in vitro culture techniques to better mimic the complexity of tissues. Overall, the obtained results demonstrated the suitability of the proposed fibers for delivering microvascular-like endothelial cells and/or support the possibility of assembling these cell-laden micro-sized fibers into 3D hydrogels heterotypic constructs that may be used in bone tissue engineering approaches with improved vascularization