Microfluidic 3D biofabrication for the patterning of hierarchical gradient constructs

INTRODUCTION: Functionally graded materials (FGMs) are ubiquitous in human tissues. Nevertheless, there has been limited attempt in replicating FGMs in 3D to generate constructs for tissue engineering and regenerative medicine purposes [1,2]. Here, we describe the fabrication of FGMs harnessing oil-in-water (o-w) and water-in-water (w-w) emulsions using a microfluidic flow- focusing printhead and a fluid-gel support bath, to engineer hierarchically functional implants. METHODS: An organic (cyclohexane) or biocompatible dispersed phase was fractioned in a continuous (Dextran, gelatin, hyaluronic acid methacryloyl (DexMA, GelMA, HAMA)) phase in a flow- focusing microfluidic printhead. Physico-chemical analysis was carried out using scanning electron microscopy (SEM), optical coherence tomography (OCT) and dynamic mechanical analysis (DMA). 3D deposition of FGMs was carried out in an agarose fluid-gel previously described [3]. Human bone marrow stromal (HBMSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) were encapsulated either in the dispersed or the continuous phase of the w-w emulsion, printed and implanted in a chick chorioallantoic membrane (CAM) model. RESULTS: Deposited o-w and w-w fibres were tailored in porous content depending on the ratio of dispersed and continuous phase flows. On-chip characterisation revealed a significant proportionality (p<0.0001) between the diameter of the droplets and the volume fraction of the dispersed phase. Physico-chemical characterisation (SEM, OCT, DMA) of the printed scaffolds revealed hierarchically distributed porosity and preserved architecture following 3D deposition. DexMA, GelMA, HAMA were independently 3D printed comprising a variable set of porous density gradient. HBMSCs were printed when included either in the dispersed or the continuous phase, revealing no significant difference in viability post-printing. VEGF and BMP-2 included in the dispersed phase and patterned in 3D, elicited a tuneable release according to the size and density of the encapsulating droplets. HBMSCs were found functional upon stimulation with VEGF or BMP-2 following implantation in CAM, demonstrating the ability to produce readily implantable skeletal constructs. DISCUSSION & CONCLUSIONS: FGM precursors (o-w, w-w) did not influenced the dispersion formation characteristics and reproducibility, offering a rapid and functional platform for the fabrication of hierarchical constructs. Altogether, we highlighted the synergistic combination of microfluidic technology with a bioprinting platform for the patterning of FGMs, demonstrating the engineering of a biphasic system particularly relevant for the control of densities during the biofabrication of tissue substitutes and implants.