Expanding and optimizing 3D bioprinting capabilities using complementary network bioinks

Abstract

A major challenge in three-dimensional (3D) bioprinting is the limited number of bioinks that fulfill the physicochemical requirements of printing while also providing a desirable environment for encapsulated cells. Here, we address this limitation by temporarily stabilizing bioinks with a complementary thermo-reversible gelatin network. This strategy enables the effective printing of biomaterials that would typically not meet printing requirements, with instrument parameters and structural output largely independent of the base biomaterial. This approach is demonstrated across a library of photocrosslinkable bioinks derived from natural and synthetic polymers, including gelatin, hyaluronic acid, chondroitin sulfate, dextran, alginate, chitosan, heparin, and poly(ethylene glycol). A range of complex and heterogeneous structures are printed, including soft hydrogel constructs supporting the 3D culture of astrocytes. This highly generalizable methodology expands the palette of available bioinks, allowing the biofabrication of constructs optimized to meet the biological requirements of cell culture and tissue engineering.

Fig. 1. Complementary network bioink 3D printing process.

(A) (i) Schematic of the direct 3D printing of nonviscous, photocrosslinkable bioinks, which form droplets during extrusion and undergo spreading once deposited, even with light irradiation during the printing process. (ii) Schematic of the 3D printing of complementary network bioinks in three core steps: cooling-induced deposition mediated by the gelation of gelatin, photocrosslinking of the bioink, and thermal liberation of gelatin during incubation. (B) Oscillatory temperature sweep (between 37°C and 7°C at a rate of 5°C min⁻¹; closed markers indicate cooling and open markers indicate heating) showing reversible cooling-induced gelation of HAMA⁺, while undoped HAMA exhibits a low viscosity across the monitored temperature range. (C) The injection of HAMA⁺ through a needle generates stable filaments in both air and PBS, while undoped HAMA does not (both formulations supplemented with dye for visualization). (D) Storage (G′) and loss (G″) moduli during an oscillatory time sweep of the 2.5 wt % HAMA⁺ bioink during stages of cooling (sharp temperature change from 37°C to 15°C), UV light exposure (10 mW cm⁻²), and heating back to 37°C. (E) Representative images of 3D printed tubular and lattice constructs using 2.5 wt % HAMA and HAMA⁺ (containing 5 wt % Fluorescein-gelatin). A frequency of 1.5 Hz and a strain of 1% were used for oscillatory tests. Scale bars, 5 mm (C and E, left) and 1 mm (E, right). Credit for all photographs in this figure: Liliang Ouyang, Imperial College London.

Fig. 2. 3D printing a library of complementary network bioinks.

(A) Representative images and (B) measured outer diameters and heights of 3D printed tubular constructs (upper row, oblique view; bottom row, top view) using various complementary network bioinks, all containing 5 wt % Fluorescein-gelatin. (C) Representative images (top row, top view; bottom row, side view) of 3D complex 3D printed structures (from left to right: lattice, ear-shape, brain-shape, pyramid, and trifurcated tubular constructs) using 2.5 wt % PEGA⁺ bioink (containing 5 wt % Rhod-gelatin). (D) Side (left) and top (right) views of a printed lattice structure (2.5 wt % PEGA⁺) with the intensity profile showing structural uniformity. (E) Printed heterogeneous tubular and (F) tracheal-esophageal models before (0 day) and after incubation (1 and 4 days) at 37°C. HAMA⁺ (2.5 wt %; clear phase) and GelMA⁺ (5 wt %; green phase) were printed alternately along the longitudinal or transverse directions. Scale bars, 5 mm. Credit for all photographs in this figure: Liliang Ouyang, Imperial College London.

Fig. 3. 3D printing soft hydrogel constructs.

(A) Representative images of 3D printed tubular structures using HAMA⁺ bioinks across various concentrations of HAMA. (B) Comparison of 3D printed tubes before (0 day) and after incubation (1 day in air or PBS) using either 0.5 or 1 wt % HAMA⁺. (C) The compressive moduli of cylinders cast from HAMA formulations (with and without 5 wt % gelatin) across various concentrations before (0 day) and after (1 day) incubation. Two-tailed Mann-Whitney test, *P < 0.05; n.s., not significant (n ≥ 3). (D) Comparison of 3D printed tubes before (0 day) and after incubation (1 day in air or PBS) using 1.5 wt % CSMA⁺ and 1.5 wt % PEGA⁺. Fluorescein-gelatin was used for visualization. Scale bars, 5 mm. Credit for all photographs in this figure: Liliang Ouyang, Imperial College London.

Fig. 4. Bioprinting and culture of constructs containing astrocytes.

(A) Fluorescence microscopy images (green, phalloidin; blue, DAPI) and (B) metabolic activity of astrocytes (2 × 10⁶ cells ml⁻¹) encapsulated in GelMA⁺ hydrogels with varied GelMA concentrations (2.5 to 10 wt %). a.u., arbitrary unit. (C) Confocal laser scanning microscopy images (green, phalloidin; blue, DAPI) of printed multilayer lattices with 90° or 60° angles between adjacent layers (2 days). (D) Quantified cell viability and (E) LIVE/DEAD-stained astrocytes (green, calcein AM; red, ethidium homodimer-1) after bioprinting and culture for up to 7 days. Two-tailed Mann-Whitney test (n = 3). (F) High-magnification fluorescence microscopy images of astrocytes (green, phalloidin; blue, DAPI) in printed constructs (2 days). GelMA⁺ (2.5 wt %) and astrocytes (2 × 10⁶ ml⁻¹) were used for bioprinting in (C) to (F). Scale bars, 100 μm (A, E, and F) and 500 μm (C).

Fig. 5. Exploring complementary network bioinks for tissue engineering.

(A) Bioprinting a porous cylinder construct (diameter, 2 cm; height, 1 cm) with Saos-2 cells in the bioink. Numbers on the images indicate the printing process. (B) Images of a printed cylinder before (0 day) and after culture (14 days). The microscopy image (hematoxylin and eosin staining) of the cylinder cross section indicates the maintenance of a uniform pattern during tissue formation. (C) Alizarin Red S staining of printed samples (14 days). The higher-magnification images indicate the top and bottom layers along the height of the cylinder. (D) Trifurcated tubular bioprinted constructs that replicate their digital design. (E) Normalized ALP activity and (F) Alizarin Red S staining of samples at different positions along the length of the trifurcated tube after 14 days of culture. GelMA⁺ (5 wt %), Saos-2 (7.5 × 10⁶ ml⁻¹), and osteogenic medium were used throughout. Scale bars, 5 mm (A, B, and D), 1 mm (C, left, and F), and 100 μm (C, right). Credit for all photographs in this figure: Liliang Ouyang, Imperial College London.