The optimization of cell-substrate and cell-cell interactions is a central objective in tissue engineering applications. There is great potential to modulate and improve such interactions using extra cellular matrices that exhibit a gradient of mechanical properties that mimic accurately the in vivo tissue microenvironment and modulate cell behavior especially at the microscale. Here we show that by applying temperature gradients across a microfluidic channel and exploiting thermophoretic transport effects, it is possible to fabricate biocompatible hydrogels with controllable stiffness and porosity gradients. The elasticity of the hydrogels was evaluated locally by Atomic Force Microscopy revealing values between 20-100 kPa. The hydrogel microstructure was investigated by Scanning Electron Microscopy after supercritical drying and confirms the concentration gradient induced by thermophoresis. Moreover, we show that the stiffness gradient of the biomaterials can be effectively modulated by regulating the temperature difference across the microfluidic device and altering the concentration of gellan gum. Furthermore, the proliferation and level of mineralization of MC3T3 osteoblasts, seeded on the surface of the biomaterial, was monitored over time at different stiffness areas and time points, using live/dead assays and X-Ray Fluorescence technique. Cells show a preferential migration and proliferation towards the stiffer side where they also produce a higher mineralization (i.e., phosphorus and calcium deposits). Taken together, these results establish a new route to controlling the microstructure of cell culture matrices
