Poster N4 - 3D Printing Anisotropic Tissue Scaffolds with Nanofibrous Peptide Hydrogels

Introduction: Biomaterials researchers are looking for ways to recreate the nanofibrillar structure and mechanical properties of natural tissues to create tissue engineering scaffolds. Although progress has been made using biologically derived polymers, there has been limited success in making synthetic biocompatible polymers that can direct cellular growth. Towards this goal, we have designed a class of self-assembling peptides, called Multidomain Peptides (MDPs), in which the degree of nanofibrous scaffold alignment can be tuned using ions using a simple and elegant extrusion technique [1]. Here, we show that varying the degree of nanofibrillar alignment results in unique cell behaviors.

In this work, support bath-based 3D printing is used to scale up the fabrication of aligned matrices that direct cell alignment while demonstrating sophisticated architectures [2]. We have optimized a support bath for 3D printing that possesses both the rheological and ionic properties to physically support the scaffold during printing as well as promote and control the self-assembly of MDP nanofibers. Specifically, we designed a self-healing support bath that was viscous enough to support the printing process, while also of the proper ionic strength to initiate aligned hydrogel fabrication. With the ability to control nanofibrillar alignment in a complex 3D print, we now can fabricate cell scaffolds that not only promote cell growth but also influence the direction of cell proliferation. Cellular behavior on these prints is directionally dependent on the alignment of the nanofibers, paving the way for macroscopic and functional muscle and nerve scaffolds.

Materials and

Methods: Manual solid-phase peptide synthesis and techniques such as circular dichroism and infrared spectroscopy were used to characterize the multidomain peptide itself. An Allevi 3 bioprinter was used to execute manually written G-code instructions for extrusion 3D printing into an agarose-based support bath. Scanning electron microscopy, polarized light microscopy, and cryo-TEM were all used to image and assess the nanofibrous matrix formed by the peptide ink. Mechanical properties of peptide hydrogels were measured with oscillatory rheology, optical coherence elastography, and Brillouin microscopy. Confocal microscopy and immunohistochemistry were used to characterize cellular behavior on the extruded noodles as well as 3D printed scaffolds.

Results, Conclusions, and Discussions: The published manuscript establishes the reliable method of inducing and controlling nanofibrillar alignment in extruded MDPs. Additionally, the degree of nanofibrillar alignment is shown to affect both the mechanical properties of the extruded MDP as well as the way cells behave on the surface. More aligned nanofibers lead to a stiffer overall hydrogel. This work demonstrates how MDP self-assembly can be tuned using ions to achieve varying degrees of nanofibrous alignment, laying the foundation for more complex control of self-assembly mechanisms that could more closely replicate the extracellular matrix.

Applying this principle to extrusion 3D printing, the successful development and optimization of an agarose-based support bath allows for mechanical stabilization of 3D printed MDPs as well as induces nanofibrillar alignment. Reliable liberation of 3D printed scaffolds from the support bath and subsequent cell culture revealed that cells still can recognize matrix alignment on the multi-layered prints. Curved and freeform printed scaffolds are also shown to be mechanically stable after liberation. This work opens the door to complex, multilayered anisotropic tissue scaffolds that direct cell alignment. Future possibilities include the fabrication of functional muscle and nerve tissues on a supramolecular MDP scaffold.

Acknowledgements (Optional): References
1. 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*
2. J.T. Makhoul*, A.C. Farsheed*, D. Chew-Martinez*, et al. “3D Printing Anisotropic Tissue Scaffolds with Nanofibrous Peptide Hydrogels” Manuscript in preparation.

* indicates authors with equal contribution