Understanding the mechanisms by which the topographical cues of extracellular matrix (ECM) affect cellular responses (proliferation, adhesion, growth, orientation, and differentiation) is fundamentally important for tissue engineering / regenerative medicine applications. Although a growing body of literature supports that the substrate’s topography influences the cell proliferation and differentiation, the underlying cellular and molecular mechanisms are poorly understood. In our attempt to approach this issue we study the cell response dependence on tunable topography, mechanical properties and defined chemistry. Our experimental findings also indicate that depending on the relation of the direction of flow with respect to the orientation of topographical features, wall shear stress gradients act in a synergistic or antagonistic manner to topography in promoting a guided morphological cell response. Towards this scope ultra-fast lasers as tools for 3D structuring were used. The laser fabricated substrates presented here could be used as model scaffolds for the systematic exploration of the role of 3D microenvironment on cell adhesion, proliferation and differentiation, with the ultimate goal of providing mechanistic insight to guide the development of clinically relevant strategies for tissue/organ repair.