Multiscale Engineered Heterogeneous Hydrogel Composites for Digital Light Processing 3D Printing

Abstract

Hydrogel-based bioinks are widely adopted in digital light processing (DLP) 3D printing. Modulating their mechanical properties is especially beneficial in biomedical applications, such as directing cell activity toward tissue regeneration and healing. However, in both monolithic and granular hydrogels, the tunability of mechanical properties is limited to parameters such as cross-linking or packing density. Herein, we present a bioink platform with multiscale heterogeneity for DLP printing, fabricated by incorporating microgels within a cross-linked polymer matrix to form a mechanically tunable heterogeneous hydrogel composite. The properties of the separate components as well as their interactions can be efficiently tailored from both chemical and physical perspectives, enabling control across both nano and micro scales. Monodisperse, spherical gelatin methacryloyl (GelMA) microgels with a stiffness that can be tuned through polymer concentration or cross-link density are fabricated by a high-throughput microfluidic device. Microgels that have been precross-linked through chemical or physical methods are then embedded in a continuous GelMA matrix, where they influence the biomechanical and biochemical characteristics of composites through particle density and encapsulation of cells. Modulation of microgel volume and selecting different printing parameters enables tailoring of the composite compressive modulus across a range of 29 to 244 kPa. Using this composite hydrogel platform as a DLP ink allows for the fabrication of complex 3D structures with macroscale heterogeneity, providing the potential to mimic tissue- and organ-level complexity. This study presents a unique approach to designing heterogeneous hydrogel composites with tunable properties at the nano-, micro-, and macro-scales, and introduces a highly modular hydrogel platform for DLP 3D printing.

Keywords: biomaterials; digital light processing (DLP) 3D printing; hydrogel composite; microgels; regenerative medicine.

1

Fabricating heterogeneous hydrogel composites. (A) Preparation of microgels on a microfluidic chip under optical microscopy (top). Representative fluorescence image of microgels in suspension and average particle size of microgels with different GelMA concentrations (bottom). (B) Representative 3D reconstructions of confocal z-stacks of heterogeneous hydrogel composites with 1:5 (left) and 1:1 (right) μgel/bulk volume ratios. (C) AlamarBlue viability assay result for dermal fibroblast cells encapsulated in microgels after 3, 5, and 7 days of culture: normalized percentage alamarBlue reduction of cell-laden microgels. (D) Representative microscopic images of cell-laden composites after 1, 3, and 5 days of culture: brightfield (top) and green fluorescence (bottom). Statistical analysis performed using a one-way ANOVA, *p < 0.05.

2

Influence of polymer concentration on heterogeneous hydrogel composite properties. (A) Schematic overview of influencing hydrogel composite properties by controlling microgel GelMA concentration. (B) Rheological characterization of heterogeneous hydrogel composites with varying microgel GelMA concentrations, including representative temperature sweeps for preprint composite bioinks (left, 4–45 °C) and strain sweeps for printed composites (right, 0.1–100%). (C) Compression test of heterogeneous hydrogel composites with varying microgel GelMA concentrations (n = 3): compressive moduli. (D) Schematic overview of influencing hydrogel composite properties by controlling bulk GelMA concentration. (E) Rheological characterization of heterogeneous hydrogel composites with varying bulk GelMA concentrations, including representative temperature sweeps for preprint composite bioinks (left, 4–45 °C), and strain sweeps for printed composites (right, 0.1–100%). (F) Compression test of heterogeneous hydrogel composites with varying bulk GelMA concentrations (n = 3): compressive moduli. (G) Representative printability heat maps for heterogeneous hydrogel composites with varying microgel GelMA concentrations (left) and bulk GelMA concentrations (right), each accompanied by images corresponding to the highest and lowest printability values. Images in (G) are reused in Figures S1B and S3B for fidelity and printability comparison. Statistical analysis performed using a one-way ANOVA, ns = no significance, *p < 0.05, **p < 0.01.

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Influence of photopolymerization on heterogeneous hydrogel composite properties. (A) Schematic overview of influencing photopolymerization by controlling projector power level and tartrazine concentration. (B) Rheological characterization of heterogeneous hydrogel composites with varying tartrazine concentrations: representative results of UV-induced cross-linking using an 80% projector power level. (C) Assessment of the maximum photopolymerization depth under 0.5, 1, and 2 mM Tartrazine. (D) Compression test of heterogeneous hydrogel composites with varying tartrazine concentrations (n = 3): compressive moduli. (E) Rheological characterization of preprint hydrogel composite bioink: representative results of UV-induced cross-linking using 40, 60, and 80% projector power levels. (F) Assessment of the maximum photopolymerization depth under 40, 60 and 80% projector power levels. (G) Compression test of heterogeneous hydrogel composites printed using 40, 60 and 80% projector power levels (n = 3): compressive moduli. (H) Representative printability heat maps for heterogeneous hydrogel composites with varying tartrazine concentrations (left) and under varying projector power levels (right), each accompanied by images corresponding to the highest and lowest printability values, as well as failed printing attempts. All experiments were performed with 12.5% GelMA for both microgel and bulk concentrations. Images in (H) are reused in Figures S4B and S5B for fidelity and printability comparison. Statistical analysis performed using a one-way ANOVA, ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

4

Influence of microgel-bulk interactions on heterogeneous hydrogel composite properties. (A) Schematic overview of microfluidic droplet generation and cross-linking mechanism of microgels via photo-cross-linking and physical cross-linking. (B) ¹HNMR spectra of un-cross-linked GelMA and microgels both photo-cross-linked and physically cross-linked (left), and quantification of unconsumed methacrylate groups in photo-cross-linked and physically cross-linked microgels (right). (C) Rheological characterization of preprint hydrogel composite bioink containing photo-cross-linked microgels (left) and physically cross-linked microgels (right): representative results of UV-induced cross-linking using 80% projector power level. (D) Compression test of heterogeneous hydrogel composites containing photo-cross-linked and physically cross-linked microgels (n = 3): compressive moduli. (E) Representative printability heat map for heterogeneous hydrogel composites with varying microgel cross-link types and microgel to bulk volume ratios (top), and images corresponding to printability values closest to 1 are considered to exhibit the best printing performance. (bottom). All experiments were performed with 12.5% GelMA for both microgel and bulk concentrations. Images in (E) are reused in Figures S7C and S9B for fidelity and printability comparison. Statistical analysis performed using a one-way ANOVA, ns = no significance, ***p < 0.001.

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Constructs printed using the heterogeneous hydrogel composite bioinks. (A) 3D models and printed constructs of cross-sectional structure of human tissues: small intestine (top) and hepatic lobules (bottom). (B) 3D models and printed constructs of organ-analogues: nose (left), left ear (top right), and little finger (bottom right). (C) 3D model obtained from the scanning app and printed structure of a knee joint anatomical model. (D) Schematic overview of dual-material printing using 2 bioinks (left), and the printed structure (right). (E) Structure printed using 3 different bioinks (left), interfaces between different bioinks (center), and compressive modulus of regions corresponding to each bioink (right). Statistical analysis performed using a one-way ANOVA, ns = no significance, **p < 0.01.