Challenges remain in the generation of heterogeneous tissues and the repairing of interfacial tissue between soft and hard tissues. The development of tissue engineering scaffolds with gradients in composition, structure, mechanical and chemical properties is essential to modulate cellular behaviors in a graded way and potentially support the growth of functionally graded tissues. Integrated with the three-dimensional (3-D) nanofibrous skeletal structure of native extracellular matrix, electrospun fibers with gradients in amino groups were generated in the current study through an aminolysis process by using a microinfusion pump. Gelatin grafts were constructed to create fibrous scaffolds with gradients in hydroxyapatite (HA) contents, crystal size and mechanical properties through in situ mineralization. Plasmid DNA (pDNA) was included during the mineralization process, and gradations in pDNA loading contents were created on fibrous scaffolds on the basis of HA gradients. Obvious gradients in cell density, osteoblastic differentiation and collagen deposition were demonstrated along the long axis of fibrous mats after cell seeding. Gradients in the amount of pDNA released and the expression of target proteins were indicated on the fibrous mats, which offered a temporally and spatially controlled delivery of growth factors in scaffolds. The creation of gradient futures on 3-D fibrous scaffolds may provide physical, chemical and biological cues and result in efficient regeneration of tissues with spatial distributions of the cell proliferation, differentiation, and matrix secretion. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.