(A-4) 3D Printing of Self-Assembling Nanofibrous Multidomain Peptide Hydrogels

Introduction:: 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, we establish the printability of Multidomain Peptides (MDPs), a class of self-assembling peptides that form a nanofibrous hydrogel at low concentrations. MDPs with different charge functionalities are optimized as distinct inks and are used to create complex three-dimensional 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.


Materials and Methods:: The cationic MDP used in this work, named K2, has the sequence K₂(SL)₆K₂ whereas the anionic MDP, named E2, has the sequence E₂(SL)₆E₂ (Figure 1). They were synthesized via Solid Phase Peptide Synthesis and confirmed with MALDI mass spectrometry. The secondary structure of the peptides were analyzed using CD and ATR-FTIR Spectroscopy. SEM was used to observe the fibrous network that forms in both K2 and E2 hydrogels.
Rheological testing was performed on both K2 and E2 to assess their potential use as extrudable inks. A frequency sweep was performed on 1 – 4 wt% K2 and E2 to understand how increasing peptide concentration affects the resulting gel storage modulus (G′). 4 wt% K2 and E2, which had the highest G′ values, were subjected to additional rheological testing to ensure they were shear thinning (shear sweep), shear yielding (strain sweep), and rapidly self-healing (oscillatory high and low strains), which are essential properties for a 3D printable ink.
3D printing of MDPs was performed on an Allevi 3 bioprinter. A series of calibration lines at varying printhead speeds and an “overhang test” were performed to optimize and demonstrate the printability of multiple MDP inks, respectively. Then, a series of 3D structures with increasing complexity were printed using MDPs.
C2C12 Cells were seeded onto printed constructs, and cell viability and morphology were analyzed over time. Live/dead staining and immunostaining of actin filaments were performed to analyze cell viability and cell morphology, respectively.


Results, Conclusions, and Discussions:: Both K2 and E2 adopt a β-sheet secondary structure and form a nanofibrous network (Figure 2). They also demonstrate around a 2X increase in storage modulus for each additional wt% (Figure 3a, e). 4 wt% K2 and E2 are shear thinning (Figure 3b, f), shear yielding (Figure 3c, g), and rapidly self-healing (Figure 3d, h). Frequency sweeps on 3 replicates of each ink showed reproducible rheological properties that matched closely to initial rheological testing (Figure 3i). In addition, the MDP inks showed stable G′ values between 4 - 37°C, with the two 4% inks averaging just a ~5% change over the temperature sweep (Figure 3j). The MDP inks also showed long term stability when stored at 4°C (Figure 3k).
For each MDP ink, a series of calibration lines at varying printhead speeds were printed and the width of calibration lines was measured as a function of printhead speed to determine optimal printing conditions (Figure 4a). Using optimized pressures and print speeds, all three inks were successfully printed over a 16mm overhang (Figure 4b). To extend the proof-of-concept test prints, a series of 3D structures with increasing complexity were printed using MDPs (Figure 4c – f). The successfully printed 2x2 and 1x1 log piles were stored in HBSS and had no change in pore dimensions for over 10 days (Figure 4h).
C2C12 cells seeded onto K2 and E2 constructs had high cell viability (Figure 5a – c), but charge-dependent differences in cell behavior and morphology (Figure 5d – f).
In this study, we establish MDPs as a new class of 3D printable hydrogels. Following characterization of their supramolecular assembly, MDPs are shown to possess the necessary rheological properties to be used for extrusion 3D printing. Printing optimization is then undertaken and utilized to create complex, overhanging structures and multi-material prints. This work represents one of very few 3D printing papers involving self-assembling peptides and surpasses the level of printing complexity that has been previously accomplished. In addition, in vitro characterization of cationic and anionic MDP constructs revealed charge-dependent differences in cell morphology and charge-independent high cell viability.


Acknowledgements (Optional): : This work was supported by funding from the National Institute of Health (NIH) (R01 DE021798). We gratefully acknowledge additional funding from the National Science Foundation Graduate Research Fellowship Program that supports A.C.F and B.H.P. We also acknowledge the support of Rice University’s Biomaterials Lab.


References (Optional): : A.C. Farsheed, A.J. Thomas, B.H. Pogostin, J.D. Hartgerink, “3D Printing of Self-assembling Nanofibrous Multidomain Peptide Hydrogels” Adv. Mater. (2023), https://doi.org/10.1002/adma.202210378