The generation of cell gradients is critical for understanding many biological systems and realizing the unique functionality of many implanted biomaterials.However,most previous work can only control the gradient of cell density and this has no effect on the gradient of cell orientation,which has an important role in regulating the functions of many connecting tissues.Here,we report on a simple stretched inverse opal substrate for establishing desired cell orientation gradients.It was demonstrated that tendon fibroblasts on the stretched inverse opal gradient showed a corresponding alignment along with the elongation gradient of the substrate.This “random-to-aligned” cell gradient reproduces the insertion part of many connecting tissues,and thus,will have important applications in tissue engineering.