Repairing annulus fibrosus (AF) defects is one of the most challenging topics in intervertebral disc disease treatment research. The highly oriented native structure offers mechanical functionality to the spine, however manufacturing scaffolds with such a structure still presents a challenge for tissue engineering. Here, a three-dimensional (3D) multi-lamellar scaffold with hierarchically aligned nano- and micro-fibers for AF tissue engineering was successfully developed. Aligned polycaprolactone (PCL) nano-fiber sheets, which were fabricated by electrospinning, were inserted into fused-deposit-modeling (FDM) micro-fibers to build a layer-by-layer structure, with the thickness of each layer being 0.7 mm and the angle of fiber alignment in each adjacent layer being 60°. Human mesenchymal stem cells (hMSCs) were used for in vitro compatibility studies. The architecture of the scaffold was characterized by scanning electron microscopy (SEM). Uniaxial tensile testing showed closed mechanical properties of the scaffold to native AF tissue. The XTT cell viability and DNA quantification of the cells on the multi-lamellar scaffold were found to be significantly higher than the FDM scaffolds without nano-fibers. Confocal microscopy demonstrated that the cells spread evenly on the surface of the electrospun sheet and oriented along the nano-fiber direction. This 3D multi-lamellar scaffold has the advantages of stability from the FDM micro-fibers, and unique characteristics from the aligned electrospun nano-fibers, such as mimicking the extracellular matrix (ECM), and an ultrahigh surface area for improved hMSC attachment, proliferation and contact guidance of cell morphology. The newly designed scaffold mimics the native structure of AF and has a great potential as a substrate for the regeneration of AF.
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