A multimaterial bioink method for 3D printing tunable, cell-compatible hydrogels

Abstract

A multimaterial bio-ink method using polyethylene glycol crosslinking is presented for expanding the biomaterial palette required for 3D bioprinting of more mimetic and customizable tissue and organ constructs. Lightly crosslinked, soft hydrogels are produced from precursor solutions of various materials and 3D printed. Rheological and biological characterizations are presented, and the promise of this new bio-ink synthesis strategy is discussed.

Keywords: 3D printing; biofabrication; bioprinting; hydrogels; tissue engineering.

Figure 1

3D bioprinting PEGX-gelatin a) extrusion through 200 μm tip b) 15×15 mm square printed, 4 layers c) 20 layer porous hexagon shape ~ 5 mm thick d) inner structure of 4 layer printed structure, scale bar 500 μm, struts ~350–450 μm diameter e) warm gelatin (no PEGX) printed on 5 °C substrate with 200 μm tip, 2 layers, scale bar 500 μm, struts ~1.1–1.3 mm diameter.

Figure 2

a) Phase plot of varying gelatin concentrations with varying PEGX:gelatin (m:m) ratios, circled formulas were rheologically tested. b) Gelation profile of 5 w/v% gelatin and 0.1 PEGX:gelatin (m:m) after addition of PEG cross-linker and c) response to increasing strains, failure (G′_c) at 1590% strain.

Figure 3

a) Phase plot of fibrinogen b) phase plot of 4 arm PEG amine (10,000 g/mol) c) phase plot of gelatin cross-linked with physical PEGX variant, PEG-SVA (1000 g/mol). Scanning electron micrographs of d) PEGX-gelatin (5 w/v%) at 0.1 PEGX:polymer mass ratio e) PEGX-gelatin (3 w/v%)-atelocollagen (0.06 w/v%) at 0.2 PEGX:polymer mass ratio f) PEGX-gelatin (2.25 w/v%)-peptide amphiphiles (0.75 w/v%) at 0.4 PEGX:polymer mass ratio, Ca²⁺-treated, scale bars 50 nm.

Figure 4

a) PEGX-gelatin (red) and PEGX-fibrinogen (blue) co-printed cylinder, 15 mm diameter b) PEGX-gelatin (red) and PEGX-fibrinogen (blue) co-printed construct with inner structure patterned, struts ~650 μm diameter c) Cell viability using Live/Dead assay at one day post-printing using 3 w/v% fibrinogen at a 0.2 PEG ratio, scale bars 200 μm d) PEGX-PEG and PEGX-gelatin co-printed structure seeded with HDFs (green, calcein AM). Cells preferentially adhere to PEGX-gelatin e) XY projection of 3D reconstruction. HUVECs (CellTracker^™ Red) printed in 5 w/v% gelatin and 0.1 PEGX:gelatin (m:m) seeded with hMSCs (CellTracker^™ Green) into internal porous structure. hMSCs fill empty spaces of inner structure, inset: whole construct, scale bar 400 μm. hMSCs spread into open spaces of construct and onto printed bioink strands at Day 4.

Scheme 1

a) Polymer or polymer mixtures can be linear (e.g. gelatin), branched (e.g. 4 arm PEG amine) or multi-functional (e.g. gelatin methacrylate). In example, the red circles may represent amines, blue triangles methacrylate groups, and the yellows stars SVA groups of PEGX, as demonstrated in this paper. b) PEGX can be linear or multi-arm and can be various chain lengths. c) Cells can be optionally incorporated by d) mixing with polymers and PEGX to form the bioink. e) Optional, secondary cross-linking to increase mechanical robustness may be performed post-printing. f) By changing the reactive groups of PEGX, polymers of other functional groups may be cross-linked. For example, purple polygons could represent thiols, cross-linkable with maleimide PEGX (pink squares) and green ellipses could represent alkynes, cross-linkable with azide PEGX (orange hexagon). g) Printing process of PEGX bioink method and corresponding phase: PEGX with or without cells are mixed within the polymer solution and loaded into the printing cartridge. After gel formation and stable mechanical properties are achieved, gels can be 3D printed into multi-layer structures.