Aqueous Two-Phase Emulsion Bioink-Enabled 3D Bioprinting of Porous Hydrogels

Abstract

3D bioprinting technology provides programmable and customizable platforms to engineer cell-laden constructs mimicking human tissues for a wide range of biomedical applications. However, the encapsulated cells are often restricted in spreading and proliferation by dense biomaterial networks from gelation of bioinks. Herein, a cell-benign approach is reported to directly bioprint porous-structured hydrogel constructs by using an aqueous two-phase emulsion bioink. The bioink, which contains two immiscible aqueous phases of cell/gelatin methacryloyl (GelMA) mixture and poly(ethylene oxide) (PEO), is photocrosslinked to fabricate predesigned cell-laden hydrogel constructs by extrusion bioprinting or digital micromirror device-based stereolithographic bioprinting. The porous structure of the 3D-bioprinted hydrogel construct is formed by subsequently removing the PEO phase from the photocrosslinked GelMA hydrogel. Three different cell types (human hepatocellular carcinoma cells, human umbilical vein endothelial cells, and NIH/3T3 mouse embryonic fibroblasts) within the 3D-bioprinted porous hydrogel patterns show enhanced cell viability, spreading, and proliferation compared to the standard (i.e., nonporous) hydrogel constructs. The 3D bioprinting strategy is believed to provide a robust and versatile platform to engineer porous-structured tissue constructs and their models for a variety of applications in tissue engineering, regenerative medicine, drug development, and personalized therapeutics.

Keywords: 3D bioprinting; aqueous two-phase emulsion; bioink; gelatin methacryloyl (GelMA); porous hydrogel; tissue engineering.

Figure 1.

Schematics showing 3D bioprinting of (a) a porous hydrogel structure using the two-phase aqueous emulsion bioink and (b) a conventional hydrogel structure.

Figure 2.

Characterization of the GelMA-PEO emulsions. (a) Effect of PEO concentration on emulsion droplet size, at GelMA:PEO phase volume ratio of 1:1, where the optical images show the morphologies of the emulsions as the PEO concentrations were varied from 0% to 1.6%. (b) Effect of volume ratio of GelMA (10%) and PEO (1.6%) on emulsion droplet size. Fluorescence micrographs of rhodamine B stained porous GelMA with varied GelMA:PEO volume ratios from 1:1 to 4:1. (c) Pore size distribution of porous GelMA at various GelMA:PEO phase volume ratios. Inset shows average pore size as a function of GelMA:PEO volume ratio. In each case 100 random pores were analyzed. (d) 3D reconstruction confocal fluorescence image of the hydrogel with interconnected pores; inset shows the top view. (e) SEM micrographs showing the porous GelMA hydrogels with the GelMA:PEO volume ratios of 1:1 (left) and 4:1 (right). (f) Young’s modulus of the porous GelMA hydrogels as functions of (i) PEO concentration and (ii) volume ratio. (g) Viscosity of GelMA-PEO emulsions as a function of temperature. Pure GelMA hydrogel at the concentration of 5% was used as the control group. (*p<0.05, **p<0.01, n=3).

Figure 3.

Characterizations of cells encapsulated the porous GelMA constructs (cylinder, 10 mm in diameter and 2 mm in thickness). Standard GelMA hydrogels (5%) were used as the control. (a) Fluorescence micrographs showing viability of encapsulated HepG2 cells on Day 1, Day 3, and Day 7, where live cells were stained in green and dead cells in red (i), and quantifications of cell viability (ii). (b) Quantifications of proliferation of HUVECs on Day 1, Day 3, and Day 7 using the Prestoblue^® assay. (c) Quantifications of NIH 3T3 fibroblast volumes within the GelMA constructs. Graph shows the comparison between cell volumes in standard GelMA and porous GelMA constructs on Day 7. (d) Cell spreading within the GelMA constructs. Confocal fluorescence micrographs show morphologies of NIH/3T3 fibroblasts within (i and iii) standard GelMA constructs and (ii and iv) porous GelMA constructs. The cells were stained for nuclei (blue) and F-actin (green). (*p<0.05, **p<0.01, ***p<0.001, n=3).

Figure 4.

Extrusion bioprinting of the aqueous two-phase emulsion bioink. (a) Designed patterns. (b) Optical micrographs of corresponding printed standard GelMA hydrogel patterns (i and v) and porous GelMA hydrogel pattern (ii-iv, vi-viii) at low magnification (i-iv) and high magnification (v-viii). (c) Photographs of 3D printed multi-layered standard (i-ii) and porous GelMA hydrogel patterns (iii-iv). The bioinks in (i, ii) were stained with rhodamine B prior to bioprinting to aid visualization. (d) Fluorescence micrographs showing bioprinted HUVECs and NIH/3T3 fibroblasts in standard GelMA hydrogel patterns (i-iv) and porous (v-viii) GelMA hydrogel patterns on Day 7 of culture.

Figure 5.

DMD bioprinting of the two-phase emulsion bioink. (a) Principle of the DMD bioprinting. (b) Bioprinted standard GelMA hydrogel pattern (i) and porous (ii) GelMA hydrogel pattern. (c) Fluorescence micrographs showing cell spreading within the standard (i-iii) and porous (iv-vi) GelMA hydrogel patterns on Day 7 of culture. The cells were stained for F-actin (green) and nuclei (blue).