Abstract: AbstractAdipose-derived stem cells (ADSCs) have considerable potential for bone regeneration. However, their performance is limited by a lack of scaffolds that adequately mimic the hierarchical structure of bone to promote proliferation and osteogenic differentiation of ADSCs. In this study, nanofiber membranes composed of polycaprolactone, poly(lactide-co-glycolide), and hydroxyapatite (HAp) were prepared via electrospinning, and the membranes curled after responding to temperature stimuli in an aqueous solution. Transmission electron microscopy and scanning electron microscopy observations indicated that needle-like HAp nanoparticles with an average diameter of 57 ± 39 nm and a length-diameter ratio of 7.4 ± 1.56 were entrapped in the nanofiber matrix and did not affect the surface morphology of fibers. After cutting and deformation, the nanofibers changed from straight to bent, and the diameters increased; they were 1105 ± 200 nm for BPLG85-H and 1120 ± 199 nm for BPLG80-H. Additionally, tubular units with a single layer (BPLG-H(1.5)) or multiple layers (BPLG-H(3.5)) were obtained by controlling the initial shape and size of the membranes. rADSCs on the concave surface of BPLG-H(3.5) proliferated faster and exhibited better osteogenic activity than those on the convex side of BPLG-H(3.5) and both surfaces of BPLG-H(1.5), which was correlated with the higher expressions of vascular endothelial growth factor and bone morphogenetic protein 2. Additionally, rADSCs on both units maintained osteogenic activity after storage at -80 °C for 20 d. In rat skull defect (diameter of 8 mm) models, rADSC-loaded BPLG-H(3.5) units fixed using gelatin hydrogel (ADSC@BHM) exhibited 84.1 ± 6.6% BV/TV after implantation for 12 weeks, which was 155.6% higher than that of the Blank group. H&E and Masson’s staining results demonstrated that there was more bone regeneration at the defect center of ADSC@BHM than in the BHM and Blank groups. In conclusion, rADSC-loaded BPLG-H(3.5) with an osteon-mimic structure provides a potential strategy to repair bone defects.