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Three‐dimensional (3D) homogenous scaffolds composed of natural biopolymers have been reported as superior candidates for bone tissue engineering. There are still remaining challenges in fabricating the functional scaffolds with gradient structures to similar with natural bone tissues, as well as high mechanical properties and excellent affinity to surround tissues. Herein, inspired by the natural bone structure, a gradient‐structural scaffold composed of functional biopolymers was designed to provide an optimized 3D environment for promoting cell growth. To increase the interactions among the scaffolds, dopamine (DA) was employed to modify alginate (Alg) and needle‐like nano‐hydroxyapatite (HA) was prepared with quaternized chitosan as template. The obtained dopamine‐modified alginate (Alg‐DA) and quaternized chitosan‐templated hydroxyapatite (QCHA) were then used to fabricate the porous gradient scaffold by “iterative layering” freeze‐drying technique with further crosslinking by calcium ions (Ca2+). The as‐prepared Alg‐DA/QCHA gradient scaffolds were possessed seamlessly integrated layer structures and high levels of porosity at around 77.5%. Moreover, the scaffolds showed higher compression modules (1.7 MPa) than many other biopolyermic scaffolds. The gradient scaffolds showed appropriate degradation rate to satisfy with the time of the bone regeneration. Both human chondrocytes and fibroblasts could adhesive and growth well on the scaffolds in vitro. Furthermore, an excellent osteogenetic activity of the gradient scaffold can effectively promote the regeneration of the bone tissue and accelerate the repair of the bone defects in vivo, compared with that of the scaffold with the homogenous structure. The novel multilayered scaffold with gradient structure provided an interesting option for bone tissue engineering. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1615–1627, 2019.

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