3D Printing of Self-Assembling Nanofibrous Multidomain Peptide Hydrogels

Abstract

3D printing has become one of the primary fabrication strategies used in biomedical research. Recent efforts have focused on the 3D printing of hydrogels to create structures that better replicate the mechanical properties of biological tissues. These pose a unique challenge, as soft materials are difficult to pattern in three dimensions with high fidelity. Currently, a small number of biologically derived polymers that form hydrogels are frequently reused for 3D printing applications. Thus, there exists a need for novel hydrogels with desirable biological properties that can be used as 3D printable inks. In this work, the printability of multidomain peptides (MDPs), a class of self-assembling peptides that form a nanofibrous hydrogel at low concentrations, is established. MDPs with different charge functionalities are optimized as distinct inks and are used to create complex 3D structures, including multi-MDP prints. Additionally, printed MDP constructs are used to demonstrate charge-dependent differences in cellular behavior in vitro. This work presents the first time that self-assembling peptides have been used to print layered structures with overhangs and internal porosity. Overall, MDPs are a promising new class of 3D printable inks that are uniquely peptide-based and rely solely on supramolecular mechanisms for assembly.

Keywords: 3D printing; biomaterials; hydrogels; self-assembling peptides; supramolecular chemistry.

Figure 1.

MDP assembly and 3D printing schematic. a) Structure of MDPs with one hydrophilic face (blue), one hydrophobic face (pink), and charged domains at either side (purple). Under physiological conditions MDPs self-assemble and undergo β-sheet fibrilization. This results in the formation of MDP nanofibers and gelation. b) The process undertaken in this study, which includes the assessment of MDPs as a 3D printable ink candidate, 3D printing optimization with multiple MDP inks, and the printing of constructs with increasing difficulty (including multimaterial printing). Finally, MDP inks with opposite charge were used to create 3D structures and observe differing in vitro characteristics.

Figure 2.

Characterization of MDP secondary Structure and nanofiber network. a) Circular Dichroism of K2 and E2 from 180 – 250 nm. b) Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy of K2 and E2 from 1500 – 1750 cm⁻¹. c-f) Scanning Electron Microscopy of K2 (c,d) and E2 (e,f) nanofilaments that formed a hydrogel. (Scale bars = 5 μm in c, e and 1 μm in d, f).

Figure 3.

Rheology of MDP gels and MDP inks. Frequency sweeps from 0.1 – 100 rad/s on 1 – 4 wt% a) K2 and e) E2. Shear sweeps from 0.01 – 100 s⁻¹ on 4 wt% b) K2 and f) E2. Strain sweeps from 0.1 – 100% on 4 wt% c) K2 and g) E2. Oscillatory high and low strains on 4 wt% d) K2 and h) E2. White regions represent 1% strains and grey regions represent 500% strain. i) Frequency sweeps from 0.1 – 100 rad/s on MDP inks (n=3). j) Temperature sweeps from 4 – 37°C on MDP inks. k) Frequency sweeps from 0.1 – 100 rad/s on MDP inks after storage at 4°C for >1 month.

Figure 4.

MDP 3D printing optimization and printed structures. a) Printhead speed calibration curves for 4% K2, 4% E2, and 3% K2 MDP inks (n=3 – 9). b) Overhang tests for MDP inks, where orange, blue, and yellow correspond to 4% K2, 4% E2, and 3% K2 MDP inks (Scale bars = 8 mm). c) Cylinder, d) 2×2 log pile, e) 1×1 log pile, and f) multimaterial 1×1 log pile. (Top to bottom) 3D printed structures imaged next to a dime for scale, top views, and side views for each of the constructs. The first three structures were printed with 4% K2 alone and the fourth had alternating 4% K2 and 4% E2 at each layer (Scale bars = 2 mm). g) Modified 2×2 log pile with visible overhanging layers (Scale bars = 2 mm). h) Top view images of 2×2 log pile and 1×1 log pile after being incubated in HBSS for 1 day.

Figure 5.

In vitro characterization of 3D printed MDP log pile hydrogels with differing charge. Live/dead staining of C2C12 cells seeded onto 3D printed K2, E2, and K2/E2 hydrogels after a) 1 day and b) 10 days of culture (Scale bars = 300 μm). All images are maximum intensity projections of 300 μm z-stacks. c) Cell viability of cells seeded onto 3D printed MDPs over 10 days (n = 3). The statistical analyses used were multiple one-way ANOVAs with Tukey’s multiple comparisons test between time points of the same group. d) Number of cells that adhered to and grew on 3D printed MDPs over 5 days (n=3). The statistical analyses used were multiple two-way ANOVAs with Tukey’s and Sidak’s multiple comparisons tests within gels at each timepoint and across gels for each timepoint, respectively. e) Length of longest axis of all live cells on each 3D printed MDP after 10 days in culture (n=3). The statistical analysis used was an unpaired t test and the whiskers represent min to max values. f) Immunostaining of cells on 3D printed MDPs after 10 days of culture (Scales bar = 300 μm). The red dashed lines represent regions where K2 was printed, while the white dashed lines represent where E2 gel was printed over the K2. Significance is represented as: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.