Biofabrication of development-inspired scaffolds for regeneration of the annulus fibrosus macro- and microarchitecture

Annulus fibrosus (AF) tissue engineering is a promising strategy for repairing the degenerated intervertebral disc (IVD) and a research area that could benefit from improved tissue models to drive translation. AF tissue is composed of concentric layers of aligned collagen bundles arranged in an angle-ply pattern, an architecture which is challenging to recapitulate with current scaffold design strategies. In response to this need, we developed a strategy to print 3D scaffolds that induce cell and tissue organization into oriented patterns mimicking the AF. Polycaprolactone (PCL) was printed in an angle-ply macroarchitecture possessing microscale aligned topographical cues. The topography was achieved by extrusion through custom-designed printer nozzles which were either round or possessing circumferential sinusoidal peaks. Whereas the round nozzle produced extruded filaments with a slight uniaxial texture, patterned nozzles with peak heights of 60 or 120 m produced grooves, 10.87 {+/-} 3.09 m or 17.77 {+/-} 4.91 m wide, respectively. Bone marrow derived mesenchymal stem cells (BM-MSCs) cultured on the scaffolds for four weeks exhibited similar degrees of alignment within {+/-} 10 {degrees} of the printing direction and upregulation of outer AF markers (COL1, COL12, SFRP, MKX, MCAM, SCX and TAGLN), with no statistically significant differences as a function of topography. Interestingly, the grooves generated by the patterned nozzles induced longitudinal end-to-end alignment of cells, capturing the arrangement of cells during fibrillogenesis. In contrast, topography produced from the round nozzle induced a continuous web of elongated cells without end-to-end alignment. Extracellular collagen I, decorin and fibromodulin were detected in patterns closely following cellular organization. Taken together, we present a single-step biofabrication strategy to induce anisotropic cellular alignments in x-, y-, and z-space, with potential application as an in vitro model for studying AF tissue morphogenesis and growth.