3D printing of annulus fibrosus anatomical equivalents recapitulating angle-ply architecture for intervertebral disc replacement

Abstract

Structural recapitulation is one of the prime prerequisites for the biomechanics of a complex tissue like annulus fibrosus (AF). Design and fabrication of a biomimetic AF anatomical structure has garnered immense interest among tissue engineers owing to its potential in restoration of intervertebral disc function. Three-dimensional (3D) printing technology, in recent times has demonstrated its applicability to fabricate such a complex tissue architecture. Here, we fabricated angle-ply AF anatomical equivalents by depositing silk-carrageenan filaments in a layer-by-layer fashion using micro-extrusion based 3D printing strategy. The 3D-printed constructs exhibited fiber alignment that mimicks the native tissue when flipped by 90° across the periphery. The 3D printed constructs were physiochemically characterized including swelling and degradation. The cross-aligned structure was revealed by field-emission electron microscopy and fluorescence studies. Infrared spectroscopy analysis confirmed the induction of stable secondary β-sheet structure in the composite blend constructs. The 3D printed constructs exhibited higher mechanical strength (~78 kPa) than the native porcine AF tissue (~37 kPa). The constructs supported survival, growth and proliferation of both porcine primary AF cells (~1.43-fold) and adipose derived stem cells (ADSCs) (~1.32-fold) over the period of 14 days. Histological, biochemical and gene expression studies subsequently revealed the deposition of AF specific extracellular matrix on the constructs when maintained in chondrogenic medium. The developed biomaterial ink showed minimal-immunogenicity as depicted by the subcutaneous in vivo implantation assessment. Hence, the strategy adopted here to fabricate AF anatomical 3D printed equivalents may provide a new direction towards disc replacement therapy.