Development of a nanocomposite bioink library for the 3D bioprinting of skeletal tissue to aid bone repair

INTRODUCTION: Nanoclays are a versatile family of biocompatible and bioactive nanomaterials. Indeed, nanoclays can: (i) assemble and gelate rapidly in the presence of ions, (ii) demonstrate shear- thinning properties in suspension in an aqueous solvent and, (iii) the capacity to retain and localise encapsulated compounds [1]. Herein, we explore a number of nanocomposites (comprising alginate, methylcellulose, gellan gum, GelMA) for bone regeneration. The current studies demonstrate the ability of Laponite® (LAP) nanoclay to support the 3D bioprinting of skeletal implants and stimulate the formation of bone tissue in vitro and ex vivo. Specifically, we demonstrate the use of human bone decellularised and demineralised extracellular matrix (hbECM) in combination with nanoclay filler for skeletal tissue repair. METHODS: hbECM was prepared from donated bone tissue. The mineral and cellular fractions were removed following a series of washes with 2%P/S followed by digestion in 0.5 N HCl with trypsin/EDTA. Silicate nanoclay, namely Laponite® (LAP, BYK, UK) was blended with alginate to facilitate ink printability. Material inks were tested for physical properties (SEM, mass loss, swelling ratio), print fidelity and the ability to support HBMSCs viability (live-dead) and phenotype (alkaline phosphatase (ALP) staining). Ex vivo studies examined HBMSC-laden and cell-free controls in 3D scaffolds in the chicken chorioallantoic membrane (CAM) model for 7 days following incorporation of vascular endothelial growth factor (VEGF) or bone morphogenetic protein 2 (BMP-2) to stimulate angiogenesis and osteogenesis. RESULTS: Nanocomposite inks exhibited negligible swelling behaviour compared to the nanoclay-free material, while demonstrating higher viscoelastic properties ideal for 3D printing. Acellular material inks were tested for printability, identifying the nanocomposite as suitable for cell printing. HBMSCs were printed at 106 cell ml-1 and cultured up to 21 days, demonstrating a higher proliferation rate (p<0.001) compared to the nanoclay-free controls. The presence of nanoclay did not affect cell viability allowing the sustained retention of cells within the printed hydrogel structure up to 21 days with evidence of proliferation compared to LAP-free controls. CAM implantation revealed that the inclusion of nanoclay with alginate and bone ECM facilitated the sustained release of VEGF, promoting a vasculature network significantly larger (p<0.001) compared to VEGF-free and LAP-free controls. DISCUSSION & CONCLUSIONS: The current studies demonstrate the synergistic combination of nanoclay and biomimetic materials, (alginate and hbECM) support the formation of osteogenic tissue both in vitro and ex vivo and offer promising novel 3D bioprint approach to repair skeletal tissue.