In nature anisotropic geometries are present in several tissues such as muscle fibers, tendon or cerebral cortex. Since different tissues present different specificities as organization, mechanical properties, and extracellular matrix (ECM), engineering techniques are needed to tune specific in vitro tissues models, especially for 3-D1 anisotropic arranges. While progress was made to obtain cells aligned in two-dimensions (2-D), the reported techniques for 3-D alignment are limited and present limitations controlling mechanical and porosity properties2,3. Here, we describe the use of a hybrid polymeric system mimicking ECM while controlling mechanical and geometrical properties in 3-D. Controlling polymeric ratios, and using crosslinking and freezing as a mechanism to guide porosity, 3-D structures are programmed regarding stiffness, shape and directionality with tunable micro-porosity to direct cellular responses. We demonstrate that crosslinking plus freezing temperature uniaxial gradients grant control over 3-D anisotropy, pore diameter, and structure stiffness, showing broad applications in modelling human tissues, such as 3-D neural constructs. Using methacrylated gelatin and gellan gum as a polymeric blend and running a precise fabrication technique, we achieved in vitro anisotropic neurites outgrowth as it happens in brain cortex. Furthermore, the applied strategy also allow the alignment of other cell types arranged in cylindrical clusters, as tested with a co-culture of mesenchymal stem cells and endothelial cells.
This study received funding from Portuguese Foundation for Science and Technology (FCT) through the project OsteoCart (PTDC/CTMBPC/115977/2009). The authors also thank FCT for the Ph.D. scholarship provided to R. F. Canadas (SFRH/BD/92565/2013). This work also had the support of the LusoAmerican Development Foundation (FLAD)