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This study prepared a novel three‐dimensional nanocomposite scaffold by the surface modification of PCL/chitosan nanofiber/net with alginate hydrogel microlayer, hoping to have the privilege of both nanofibers and hydrogels simultaneously. Bead free randomly oriented nanofiber/net (NFN) structure composed of chitosan and polycaprolactone (PCL) was fabricated by electrospinning method. The low surface roughness, good hydrophilicity, and high porosity were obtained from the NFN structure. Then, the PCL/chitosan nanofiber/net was coated with a microlayer of alginate containing neurotrophin‐3 (NT‐3) and conjunctiva mesenchymal stem cells (CJMSCs) as a new stem cell source. According to the cross‐sectional FESEM, the scaffold shows a two‐layer structure with interconnected pores in the range of 20 μm diameter. The finding revealed that the surface modification of nanofiber/net by alginate hydrogel microlayer caused lower inflammatory response and higher proliferation of CJMSCs than the unmodified scaffold. The initial burst release of NT‐3 was 69% in 3 days which followed by a sustained release up to 21 days. The RT‐PCR analysis showed that the expression of Nestin, MAP‐2, and β‐tubulin III genes were increased 6, 5.4, and 8.8‐fold, respectively. The results revealed that the surface‐modified biomimetic scaffold possesses enhanced biocompatibility and could successfully differentiate CJMSCs to the neuron‐like cells.

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