Fabrication of Macroscopic Nanofibrous Multidomain Peptide Hydrogels to Engineer Biological Tissues

Introduction: 3D printing has emerged as a powerful technique for producing hydrogel scaffolds that replicate the geometric complexity of tissues. Biologically derived polymers are commonly used as hydrogel inks in 3D printing due to their inherent bioactivity. However, these polymers lack the mechanical flexibility offered by synthetic counterparts. In addition, many biologically derived polymers lack the fibrous composition that naturally exists at the nano scale within the body. This necessitates the development of hydrogel inks that are both bioactive and fibrous in structure to better replicate tissues. Here, we present the use of Multidomain Peptides (MDPs), a class of self-assembling peptides that form a nanofibrous hydrogel, as a novel ink for 3D printing [1]. After optimizing MDPs for 3D printing, we show that they can be used to create complex, overhanging structures, and multi material prints. In addition, we demonstrate how cationic and anionic MDPs can be strategically patterned to influence cell behavior.

Building upon this work, we have developed a novel fabrication method for producing MDP hydrogels with aligned nanofibers, resembling the alignment present in tissues such as skeletal muscle and peripheral nerve [2]. We show that these anisotropic MDP scaffolds guide cell growth and spreading. Further, by fabricating hydrogel scaffolds with controlled degrees of alignment, we observe that cells exhibit distinct growth patterns in response to nanoscale differences. In total, we have established multiple methods to fabricate multidomain peptide hydrogels and have used biomaterial charge and fibrous alignment cues to direct cellular spreading and engineer tissues.

Materials and

Methods: A variety of chemical characterization (circular dichroism, ATR-FTIR spectroscopy, scanning electron microscopy, polarized light microscopy, Cryo-TEM, 2D Small Angle X Ray Scattering), mechanical characterization (oscillatory rheology, optical coherence elastography, brillouin microscopy), and biological characterization (in vitro work with cell lines and primary cells, immunostaining, confocal microscopy) techniques were used throughout this work.

Results, Conclusions, and Discussions: The first manuscript presents the optimization of MDPs for 3D printing and demonstrates how charge within a 3D printed structure can be used to control cellular growth. In addition, it shows how the self-assembling nature of MDPs makes them an ideal candidate for extrusion 3D printing. This work surpasses the printing complexity achieved during previous attempts to 3D print self-assembling peptides (SAPs) by creating overhanging hydrogel structures and multimaterial prints. More generally, this works presents the full design-to-application pathway and serves as a model for future efforts to expand the library of 3D printable peptide hydrogels.

The second manuscript establishes an extrusion-based fabrication method to control the nanofibrillar alignment of multidomain peptides and demonstrates how their nanoscale cues can be used to direct cell growth. In addition, differences in hydrogel nanostructure are found to lead to differences in bulk mechanical properties, with higher fibrillar alignments leading to stiffer matrices. This is the first demonstration of tunable alignments within any SAP system. In total, this interdisciplinary work establishes how supramolecular design can be harnessed to tune macroscopically aligned nanofibrous hydrogels and more broadly informs the design and fabrication of future supramolecular assemblers and other extracellular-matrix mimetic scaffolds.

Acknowledgements (Optional): [1] A.C. Farsheed, A.J. Thomas, B.H. Pogostin, J.D. Hartgerink, “3D Printing of Self-assembling Nanofibrous Multidomain Peptide Hydrogels” Adv. Mater. (2023), 35, 11, 2210378, https://doi.org/10.1002/adma.202210378
[2] A.C. Farsheed, C. Zevallos-Delgado, L.T. Yu, S. Saeidifard, J.W.R. Swain, J.T. Makhoul, A.J. Thomas, C.C. Cole, E.G. Huitron, K.J. Grande-Allen, M. Singh, K.V. Larin, J.D. Hartgerink, “Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers” ACS Nano. (2024), 18, 19, 12477-12488, https://doi.org/10.1021/acsnano.4c02030 *Featured on Front Cover*