WO2024233707A1

WO2024233707A1 - 3d printing of self-assembling multidomain peptide hydrogels and fabrication of aligned nanofibrous self-assembling peptide hydrogels

Info

Publication number: WO2024233707A1
Application number: PCT/US2024/028436
Authority: WO
Inventors: Jeffrey D. Hartgerink; Adam C. FARSHEED
Original assignee: William Marsh Rice University
Current assignee: William Marsh Rice University
Priority date: 2023-05-09
Filing date: 2024-05-08
Publication date: 2024-11-14
Anticipated expiration: 2025-11-09

Abstract

The present disclosure describes methods for printing hydrogel structures comprising multidomain peptides (MDPs). Also disclosed herein are hydrogel structures formed according to such methods, as well as methods of use thereof and kits comprising the presently disclosed MDPs The present disclosure also describes novel methods for forming hydrogel structures with aligned nanofibers and hydrogel structures formed according to such methods, as well as uses thereof.

Description

DESCRIPTION 3D PRINTING OF SELF-ASSEMBLING MULTIDOMAIN PEPTIDE HYDROGELS AND FABRICATION OF ALIGNED NANOFIBROUS SELF-ASSEMBLING PEPTIDE HYDROGELS STATEMENT OF FEDERALLY SPONSORED RESEARCH [0001] This invention was made with government support under Grant No. R01DE021798 awarded by the National Institutes of Health. The government has certain rights in the invention. PRIORITY CLAIM [0002] This application claims benefit of priority to U.S. Provisional Application Serial No. 63/501,071, filed May 9, 2023, and U.S. Provisional Application Serial No. 63/510,818, filed June 28, 2023, the entire contents of which are hereby incorporated by reference. REFERENCE TO A SEQUENCE LISTING [0003] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on May 8, 2024, is named RICEP0133WO.xml and is 25,877 bytes in size. BACKGROUND 1. Field [0004] This disclosure relates to the fields of materials science, molecular biology, and biotechnology. In particular, methods of forming hydrogel structures via 3D printing are provided. 2. Related Art [0005] 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 1 4863-6540-6909, v.1 structures that better replicate the mechanical properties of biological tissues. Soft materials such as biological tissues 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. Inks used in extrusion 3D printing mainly consist of chemically modified versions of gelatin (Patil et al., 2022; Ataie et al., 2022; Yi et al., 2022; Ahrens et al., 2022; Boularaoui et al., 2021; Soliman et al., 2022; Lee et al., 2020; Kolesky et al., 2014), alginate, (Ding et al., 2022; Kiseleva et al., 2022; Williams et al., 2021; Kajtez et al., 2022) hyaluronic acid, (Dhand et al., 2022; Daly et al., 2021; Davidson et al., 2021; Highley et al., 2015) collagen, (Lee et al., 2019; Szklanny et al., 2021; Brassard et al., 2021) decellularized extracellular matrix, (Huo et al., 2022; Behre et al., 2022; Pati et al., 2014) or combinations of these (Lee et al., 2019; Schmid et al., 2022; Sun et al., 2020; Skylar-Scott et al., 2019; Ouyang et al., 2017; Liu et al., 2017; Colosi et al., 2016; Kang et al., 2016; Kolesky et al., 2016; Rutz et al., 2015; Hinton et al., 2015). The traditional approach for developing 3D printable inks has been to chemically modify naturally-derived hydrogel materials to make them more printable, while attempting to maintain their favorable biological properties. Recently, two generalizable strategies have been proposed to allow for the printing of a wide range of hydrogel materials, which both include a stabilization method to allow for short term print fidelity, followed by a crosslinking method to impart long-term stability (Ouyang et al., 2020; Hull et al., 2022). Despite these advances, naturally derived materials suffer from batch to batch variability, frequently require chemical modification to have sufficient mechanical properties, and have predefined bioactivity. Thus, there exists a need for hydrogels with desirable biological properties that can be used as 3D printable inks. [0006] Multidomain Peptides (MDPs) are a class of self-assembling peptides that form a nanofibrous hydrogel at low concentrations. They have a salt and pH-mediated DVVHPEO\^PHFKDQLVP^^ WKDW^RQFH^ WULJJHUHG^ DOORZV^ IRU^ WKH^SHSWLGHV^ WR^SDFN^ LQWR^ȕ-sheets and elongate into nanofibers. Although the assembly mechanism is well understood, control of the directionality of the nanofibers that form remains elusive. [0007] The extracellular matrix (ECM) consists of fibrillar proteins that support cell attachment and growth. The ECM within distinct tissues differs in its composition and structure. Skeletal muscle and peripheral nerve tissues both have a hierarchical and aligned structure, which is vital for their function. Towards the goal of creating scaffolds that mirror the structure of anisotropic tissues, tissue engineers have worked to create aligned fibrillar 2 4863-6540-6909, v.1 scaffolds that recapitulate the topographical cues and physical properties of biological tissues (Berns et al., 2014). Fiber spinning techniques (Li et al., 2014; Sleep et al., 2017; Sather et al., 2021) and extrusion 3D printing (Kolberg-Edelbrock et al., 2023; Marshall et al., 2023; McDowall et al., 2021) have emerged as the primary fabrication technologies used and have mainly been used in conjunction with biologically derived polymers that are difficult to chemically tune and possess inherent variability (Wall et al., 2011). Thus, a tissue engineering focus has been to create scaffolds that emulate this organization. Aligned scaffolds have been shown to promote aligned cell growth and maturation, but the fabrication techniques used to make these scaffolds are not scalable and often contain hazardous solvents, and there has yet to be a demonstration using synthetic biomaterials that recapitulates both the nano and macro complexity of biological tissues. Furthermore, a key challenge has been achieving control across multiple length scales and creating macroscopic structures with nanoscale organization. [0008] One promising class of synthetic biomaterials are self-assembling peptides (SAPs), which are rationally designed to form low weight percent nanofibrous hydrogels (Diegelmann et al., 2012). The application of shear force has been demonstrated to align SAP nanofibers and has been used to create hierarchical fibrous hydrogel “noodles” (Marty et al., 2013; Bai et al., 2014; López-Andarias et al., 2015). While the 3D printing of SAPs are known the art (Chen et al., 2018; Leung et al., 2019; Christoff-Tempesta et al., 2021; Kolberg-Edelbrock et al., 2023; Gladman et al., 2016), the alignment of peptide nanofibers can only be achieved in its liquid state and therefore presents difficulties in the construction of geometrically complex hydrogels and/or hydrogels with macroscopic nanofibrous alignment. [0009] Therefore, improved methods for forming scaffolds for biological applications such as tissue engineering are needed. 3 4863-6540-6909, v.1 SUMMARY [0010] As provided herein, the present disclosure relates to methods of printing hydrogel structures with self-assembling multidomain peptides (MDPs). The present disclosure also relates to methods of forming hydrogels with aligned nanofibers. [0011] Also as provided herein, the present disclosure relates to methods for producing hydrogels with aligned nanofibers. In some embodiments, the degree of alignment may be controlled or modulated by the conditions of hydrogel formation. Aligned hydrogels formed according to the presently disclosed methods may be useful for growing cells in a directed manner. Further details on these and other aspects are provided below and in the sections that follow. [0012] In one aspect, the present disclosure provides methods for printing a hydrogel structure, the methods comprising a) forming a multidomain peptide (MDP)-ink composition comprising a multidomain peptide (MDP) comprising a first domain of the formula (X)_m(YZ)_n(X)_m, wherein: X is a positively charged amino acid at pH 7 or a negatively charged amino acid at pH 7; Y is a hydrophilic amino acid and Z is a hydrophobic amino acid or Y is a hydrophobic amino acid and Z is a hydrophilic amino acid m is 1, 2, 3, 4, or 5; and n is 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the MDP-ink composition comprises a solvent that mediates the self- assembly of the MDP; and b) printing a hydrogel structure with the MDP-ink composition. [0013] In some embodiments, X is a positively charged amino acid at pH 7. In other embodiments, X is a negatively charged amino acid at pH 7. In some embodiments, X is selected from among lysine, arginine, glutamic acid, or aspartic acid. In further embodiments, X is selected from among lysine and arginine. In some embodiments, X is lysine. In other embodiments, X is arginine. In some embodiments, X is selected from among glutamic acid and aspartic acid. In further embodiments, X is glutamic acid. In other embodiments, X is aspartic acid. 4 4863-6540-6909, v.1 [0014] In some embodiments, the hydrophobic amino acid is selected from among alanine, leucine, glycine, isoleucine, tryptophan, phenylalanine, proline, methionine, and cysteine and the hydrophilic amino acid is selected from among serine, tyrosine, threonine, asparagine, and glutamine. [0015] In some embodiments, the MDP further comprises an enzymatic cleavage signaling sequence domain, such as leucine-arginine-glycine. In some embodiments, the MDP further comprises a spacer domain. In further embodiments, the spacer domain is a sequence of five or fewer independently selected glycine or serine residues. In some embodiments, the MDP further comprises a bioactive peptide sequence domain. In further embodiments, the bioactive peptide sequence domain has at least 95% sequence identity with SEQ ID NO: 8. In some embodiments, the bioactive peptide sequence domain is SEQ ID NO: 8. In other embodiments, the bioactive peptide sequence domain is selected from among Arg- Gly-Asp (SEQ ID NO: 11), SEQ ID NO: 9, SEQ ID NO: 10, or is a sequence derived from fibronectin, vitronectin, laminin, collagen, VEGF, FGFs, PDGF, HGF, TGF-ȕ^ RU^%03^^ In further embodiments, the bioactive peptide sequence domain has at least 95% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31. In some embodiments, the bioactive peptide sequence domain is selected from among SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31. [0016] In some embodiments of the present disclosure, m is 2, 3, or 4. In some embodiments, n is 4, 5, 6, 7, or 8. [0017] In some embodiments, the MDP is N-terminally acetylated or C-terminally amidated. In further embodiments, the MDP is N-terminally acetylated. In still further embodiments, the MDP is C-terminally amidated. 5 4863-6540-6909, v.1 [0018] In some embodiments, the first domain has at least 95% sequence identity with SEQ ID NO: 1. In further embodiments, the first domain is SEQ ID NO: 1. In other embodiments, the first domain has at least 95% sequence identity with SEQ ID NO: 2. In further embodiments, the first domain is SEQ ID NO: 2. [0019] In some embodiments, the solvent of the present methods is suitable for cell culture. In some embodiments, the solvent is selected from among Hank’s Balanced Salt Solution (HBSS), Phosphate Buffered Saline (PBS), cell culture medium, a biological fluid, or any solution that buffers. In further embodiments, the solvent is Hank’s Balanced Salt Solution (HBSS). In some embodiments, the solvent is water. In some embodiments, the solvent further comprises an additive, a crosslinker, a stiffener, a synthetic material, an inorganic salt, a gelling agent, or any combination thereof. In some embodiments, the solvent further comprises cells, such as myoblasts. [0020] In some embodiments, the precursor solution is between 0.1%-10% MDP by weight. In further embodiments, the precursor solution is between 1%-5% MDP by weight. In some embodiments, the precursor solution has less than about a 5% change in storage modulus G` between a temperature of about 4C and about 37C at a strain of about 1% and at a frequency of about 1 rad/s. In some embodiments, the precursor solution is characterized by a storage modulus G` greater than about 1000 Pa at a strain of about 1% and at a frequency between about 0.01 rad/s and about 100 rad/s. In some embodiments, the precursor solution is characterized by a storage modulus G` to loss modulus G`` ratio that is greater than 10 at a strain of 1% and at a frequency of 1 rad/s. In some embodiments, the precursor solution is characterized by a storage modulus G` to loss modulus G`` crossover at a strain between 10% and 100% and at a frequency of 1 rad/s. In some embodiments, the precursor solution is characterized by being able to recover 80% of its initial storage modulus G` at a strain of 1% and at a frequency of 1 rad/s after being sheared at a strain of 500% and at a frequency of 1 rad/s for one minute. In some embodiments, the precursor solution is stable at a temperature of about 4C for at least about 1 month. In some embodiments, the precursor solution is characterized by a negative slope of its viscosity as a function of increasing shear rates between 0.001 s-1 and 100 s-1. [0021] In some embodiments, the hydrogel structures further comprise layers of a second MDP comprising at least a second domain. In some embodiments, the second domain has at least 95% sequence identity with SEQ ID NO: 1. In further embodiments, the second 6 4863-6540-6909, v.1 domain is SEQ ID NO: 1. In other embodiments, the second domain has at least 95% sequence identity with SEQ ID NO: 2. In further embodiments, the second domain is SEQ ID NO: 2. [0022] In some embodiments, the printing comprises 3D printing. [0023] In some embodiments, the present methods further comprise contacting the printed hydrogel structure with one or more cells in vitro. [0024] In another aspect, the present application discloses hydrogel structures made according to the methods disclosed herein. [0025] In yet another aspect, the present disclosure provides methods of using hydrogels disclosed herein and hydrogels made according to presently disclosed methods, wherein the methods comprise the hydrogel contacting one or more cells in vitro. In further embodiments, the presently disclosed methods comprise seeding and growing cells on the hydrogel structure in vitro. In some embodiments, the present methods further comprise using the seeded and grown cells in vivo. In further embodiments, the cells are administered to animals. In still further embodiments, the cells are administered to humans. [0026] In still another aspect, the present disclosure provides a kit for printing a hydrogel structure, the kit comprising: (a) a first container comprising a multidomain peptide as disclosed herein; and (b) a second container comprising a solvent as disclosed herein. [0027] In yet another aspect, the present disclosure provides methods of forming hydrogels with aligned nanofibers, the methods comprising: a) obtaining a multidomain peptide (MDP) with a sequence comprising: a first domain of the formula X_m; a second domain of the formula (YZ)_n; and a third domain of the formula Xm; wherein: X is a negatively or positively charged amino acid, 7 4863-6540-6909, v.1 Y is a hydrophilic amino acid and Z is a hydrophobic amino acid or Y is a hydrophobic amino acid and Z is a hydrophilic amino acid; m is 1, 2, 3, or 4; and n is 2, 3, 4, 5, 6, 7, 8, 9 or 10; b) dissolving the multidomain peptide in a solvent to form a precursor solution; and c) extruding the precursor solution into a gelation bath to form a hydrogel with aligned nanofibers. [0028] In some embodiments, the sequence of the multidomain peptide comprises a sequence of the formula Xm(YZ)nXm. In some embodiments, X is selected from the group consisting of glutamic acid, aspartic acid, arginine, histidine, and lysine. In some embodiments, X is selected from the group consisting of lysine, arginine, and histidine. In other embodiments, X is selected from the group consisting of glutamic acid and aspartic acid. In some embodiments, X is lysine. In other embodiments, X is arginine. In still other embodiments, X is histidine. In yet other embodiments, X is glutamic acid. In other embodiments, X is aspartic acid. [0029] In some embodiments, the hydrophobic amino acid is selected from the group consisting of alanine, leucine, glycine, isoleucine, tryptophan, phenylalanine, proline, methionine, and cysteine, and the hydrophilic amino acid is selected from the group consisting of serine, tyrosine, threonine, asparagine, and glutamine. In some embodiments, Y or Z is serine. In some embodiments, Y or Z is leucine. In some embodiments, m is 2, 3, or 4. In further embodiments, m is 2. In some embodiments, n is 6, 7, 8, 9, or 10. In further embodiments, n is 6. In some embodiments, Y is serine or leucine, Z is serine or leucine, and n is 6, 7, 8, 9, or 10. In further embodiments, Y is serine or leucine, Z is serine or leucine, and n is 6. [0030] In some embodiments, the sequence of the multidomain peptide comprises SEQ ID NO: 1. In other embodiments, wherein the sequence of the multidomain peptide comprises SEQ ID NO: 2. In some embodiments, wherein the sequence of the multidomain peptide comprises SEQ ID NO: 3. In other embodiments, wherein the sequence of the multidomain peptide comprises SEQ ID NO: 4. In some embodiments, wherein the sequence of the multidomain peptide comprises SEQ ID NO: 5. 8 4863-6540-6909, v.1 [0031] In some embodiments, the multidomain peptide comprises a spacer domain. In some embodiments, the spacer is selected from the group consisting of aminohexanoic acid, polyethyleneglycol, 5 or fewer glycine residues, and 3 or fewer of SEQ ID No. 7. [0032] In some embodiments, the multidomain peptide comprises a bioactive peptide sequence domain. In further embodiments, the bioactive peptide sequence domain has at least 95% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31. In some embodiments, the bioactive peptide sequence domain is selected from among SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.In some embodiments, the bioactive peptide sequence is derived from fibronectin, vitronectin, laminin, collagen, VEGF, FGFs, PDGF, HGF, TGF-ȕ^ and BMP. In some embodiments, a bioactive peptide sequence is positioned at the N-terminal end of the multidomain peptide. In other embodiments, a bioactive peptide sequence is positioned at the C-terminal end of the multidomain peptide. In further embodiments, a bioactive peptide sequence is positioned at the N-terminal and the C-terminal end of the multidomain peptide. [0033] In some embodiments, the multidomain peptide comprises an enzymatic cleavage signaling sequence domain. In some embodiments, the enzymatic cleavage signaling sequence is LRG (SEQ ID NO: 6). [0034] In some embodiments, the multidomain peptide has a net positive or a net negative charge in an aqueous solution of a pH of between about 5 and about 9. In some embodiments, wherein the multidomain peptide further comprises a lipid, a polysaccharide, or a polymer. [0035] In some embodiments, the solvent of the precursor solution is water. In some embodiments, the concentration of the multidomain peptide in the precursor solution is 9 4863-6540-6909, v.1 between about 0.1 mg/ml to about 100 mg/ml. In some embodiments, the concentration of the multidomain peptide in the precursor solution is between about 1% and about 5% by weight. [0036] In some embodiments, the gelation bath comprises a salt or buffer. In some embodiments, the gelation bath comprises a gelling agent. In some embodiments, the gelling agent is temperature sensitive. In some embodiments, the gelling agent is a non-ionic copolymer surfactant. In further embodiments, the gelling agent is Pluronic F-127. In some embodiments, the gelling agent is gelatin microparticles. In some embodiments, the gelling agent is Poly(ethylene oxide). In some embodiments, the gelling agent is agarose. [0037] In some embodiments, the gelation bath comprises a buffering agent, an additive, a crosslinker, an inorganic salt, or any combination thereof. In some embodiments, the gelation bath comprises PBS, HBSS, cell culture medium, or a biological fluid, or any combination thereof. In some embodiments, the gelation bath comprises PBS. In some embodiments, the gelation bath comprises between about 10 mM Phosphate buffer and about 100 mM Phosphate buffer and about 140 mM NaCl. In some embodiments, the gelation bath comprises about 10mM Phosphate buffer and about 140mM NaCl. In other embodiments, the gelation bath comprises about 30mM Phosphate buffer and about 140mM NaCl. In still other embodiments, the gelation bath comprises about 50mM Phosphate buffer and about 140mM NaCl. [0038] In some embodiments, the precursor solution is extruded into the gelation bath with a 3D printer. In some embodiments, the hydrogel is a multilayer structure. In some embodiments, the method does not comprise heating the precursor solution. [0039] In some embodiments, the precursor solution further comprises cells. In some embodiments, the method further comprises seeding or growing one or more cells on the hydrogel with aligned nanofibers. In another aspect, the present disclosure provides methods of seeding and/or growing cells on a hydrogel with aligned nanofibers, such as a hydrogel with aligned nanofibers formed according to the presently disclosed methods. In some embodiments, the cells grow longitudinally along the aligned nanofibers. In some embodiments, the cells grow circumferentially about the aligned nanofibers. In some embodiments, wherein the cells are muscle cells, nerve cells, fibroblast cells, endothelial cells, cardiac cells, bone cells, fat cells, epithelial cells, pancreatic cells, stem cells, immune cells, or cancerous cells. In some embodiments, the cells are muscle cells. In some 10 4863-6540-6909, v.1 embodiments, the cells are nerve cells. In some embodiments, the cells are fibroblast cells. In some embodiments, the cells are endothelial cells. In some embodiments, the cells are cardiac cells. In some embodiments, the cells are bone cells. In some embodiments, the cells are fat cells. In some embodiments, the cells are epithelial cells. In some embodiments, the cells are pancreatic cells. In some embodiments, the cells are stem cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are cancerous cells. [0040] In some embodiments, the cells remodel the aligned hydrogel and deposit their own matrix. In some embodiments, the method further comprises growing the cells into tissue. In some embodiments, the tissue is suitable for use as a replacement tissue for an injury to a human. In some embodiments, the cells or tissue are suitable for use as a platform for drug candidate testing. [0041] In another aspect, the present disclosure provides hydrogels with aligned nanofibers formed according to the methods described above. In some embodiments, the hydrogel is biocompatible. In some embodiments, the hydrogel remains intact at pH 3-11. In some embodiments, the hydrogel remains intact at physiological pH. [0042] In another aspect, the present disclosure provides pharmaceutical compositions comprising: (a) a hydrogel as provided above; and (b) a therapeutic or prophylactic molecule. [0043] In some embodiments, the molecule is a protein. In some embodiments, the protein is an antibody. [0044] In still another aspect, the present disclosure provides methods of treating or preventing a disease or disorder in a patient in need thereof comprising administering a hydrogel with aligned nanofibers as provided above or the pharmaceutical composition as provided above. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human. In some embodiments, the disease or disorder is a cancer. [0045] In another aspect, the present disclosure provides methods of treating an injury in a patient in need thereof comprising administering a hydrogel with aligned nanofibers as disclosed above or the pharmaceutical composition as disclosed above. In some 11 4863-6540-6909, v.1 embodiments, the patient is a mammal. In further embodiments, the patient is a human. In some embodiments, the disease or disorder is a muscle injury, such as volumetric muscle loss. In other embodiments, the disease or disorder is a nerve injury, such as peripheral nerve transection. [0046] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. 12 4863-6540-6909, v.1 BRIEF DESCRIPTION OF THE DRAWINGS [0047] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0048] FIGS. 1A & 1B show a schematic of MDP assembly and 3D printing. FIG. 1A: Structure of MDPs with one hydrophilic face, one hydrophobic face, and charged domains at either side. Under physiological conditions MDPs self-DVVHPEOH^DQG^XQGHUJR^ȕ- sheet fibrilization. This results in the formation of MDP nanofibers and gelation. FIG. 2B: Development of the presently disclosed methods included the assessment of MDPs as a 3D printable ink candidate, optimization of 3D printing 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. [0049] FIGS. 2A-2B show structures of the peptides used in certain embodiments of the present disclosure. FIG. 2A: “K2”, K2(SL)6K2 and FIG. 2B: “E2”, E2(SL)6E2. K = Lysine, S = Serine, L = Leucine, E = Glutamic Acid. [0050] FIGS. 3A-3B show matrix-assisted laser desorption/ionization (MALDI) spectra of K2 (FIG 3A) and E2 (FIG. 3B). K2 has an expected mass = 1773 g/mol and E2 has an expected mass = 1776 g/mol. These spectra confirmed the successful synthesis of each MDP. These peptides have been prepared (and published) previously and were prepared several times over the course of this study. The above data is representative of batch characterization. [0051] FIGS. 4A-4F show the characterization of MDP secondary structure and nanofiber network. FIG. 4A: Circular dichroism of K2 and E2 from 180 to 250 nm. FIG. 4B: Attenuated total reflectance Fourier transform infrared spectroscopy of K2 and E2 from 1500 to 1750 cm^í1. Scanning electron microscopy of K2 (FIG. 4C and FIG. 4D) and E2 (FIG. 4E and FIG. 4F) nanofilaments that formed a hydrogel. (Scale bars = 5 µm in FIG. 4C, FIG. 4E and 1 µm in FIG. 4D and FIG. 4F. 13 4863-6540-6909, v.1 [0052] FIGS. 5A-5K show the rheology of MDP gels and MDP inks. Frequency sweeps from 0.1 to 100 rad s^í1 on 1, 2, 3, 4 wt% K2 (FIG. 5A) and E2(FIG. 5E). Shear sweeps from 0.01 to 100 s^í1 on 4 wt% K2 (FIG. 5B) and E2 (FIG. 5F). Strain sweeps from 0.1 to 100% on 4 wt% K2 (FIG. 5C) and E2 (FIG. 5G). Oscillatory high and low strains on 4 wt% K2 (FIG. 5D) and E2 (FIG. 5H). White regions represent 1% strain and grey regions represent 500% strain. FIG. 5I: Frequency sweeps from 0.1 to 100 rad s^í1 on MDP inks (n = 3). FIG. 5J: Temperature sweeps from 4 to 37 °C on MDP inks. FIG. 5K: Frequency sweeps from 0.1 to 100 rad s^í1 on MDP inks after storage at 4 °C for >1 month. [0053] FIGS. 6A-6B show the fiber formation tests on 4% K2 (FIG. 6A) and 4% E2 inks (FIG. 6B) through 25G, 27G, and 30G needles. [0054] FIGS. 7A-7H show MDP 3D printing optimization and printed structures. FIG. 7A: Printhead speed calibration curves for 4% K2, 4% E2, and 3% K2 MDP inks (n = 3–9). FIG. 7B: 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). FIG. 7C: Cylinder, FIG. 7D: 2 × 2 log pile, FIG. 7E: 1 × 1 log pile, and FIG. 7F: multimaterial 1 × 1 log pile. Top and side views of each 3D printed structure. 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). FIG. 7G: Modified 2 × 2 log pile with visible overhanging layers (Scale bars = 2 mm). FIG. 7H: Top view images of 2 × 2 log pile and 1 × 1 log pile after being incubated in HBSS for 1 day. [0055] FIGS. 8A-8B show a modified 2x2 log pile with visible overhanging layers (Scale bars = 1 mm). [0056] FIGS. 9A-9B show the 4% E2 printing attempts of (FIG. 9A) 2x2 log pile and (FIG. 9B) 1X1 log pile (Scale bars = 2 mm). [0057] FIG. 10 shows the 4% K2 overhang test (left) before and (right) after drying. [0058] FIG. 11 shows two graphs comparing the measured dimensions of the pores for each design (left, 2 x 2 log pile; right, 1 x 1 log pile) at dry conditions (directly after printing), after 1 day of storage in HBSS, and after 10 days of storage in HBSS (n=9). The statistical analyses used were multiple unpaired t tests. 14 4863-6540-6909, v.1 [0059] FIGS. 12A-12B show 2x2 (FIG. 12A) and 1x1 (FIG. 12B) log piles that have been stored in HBSS for 10 days after printing (Scale bars = 2 mm). [0060] FIGS. 13A-13F show the 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 1 day (FIG. 13A) and 10 days (FIG. 13B) of culture ^6FDOH^EDUV^ ^^^^^^P^^^$OO^ LPDJHV^DUH^PD[LPXP^ LQWHQVLW\^SURMHFWLRQV^RI^^^^^^P^]-stacks. FIG. 13C: 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. FIG. 13D: Number of cells that adhered to and grew on 3D printed MDPs over5 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. FIG. 13E: 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. FIG. 13F: Immunostaining of cells on 3D printed MDPs after 10 days of culture ^6FDOHV^EDU^ ^^^^^^P^^^7KH^UHG^GDVKHG^OLQHV^UHSUHVHQW^UHJLRQV^ZKHUH^.^^ZDV^SULQWHG^^ZKLOH^ 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. [0061] FIGS. 14A-14B show MDP bioprinting. FIG. 14A: Live/dead staining 1 day after printing within K2 and E2. FIG. 14B: Quantification of cell viability of bioprinting after 1 day (n=3). [0062] FIG. 15 shows manual pipette extrusion of an aligned multidomain peptide (MDP) hydrogel. An MDP (K2) was dissolved in Milli-Q water to form a MDP precursor solution (4wt% K2), which was then extruded into a gelation bath to form a MDP hydrogel with nanofibrillar alignment. The MDP precursor solution in this instance also comprised a blue dye. Inset contains the corresponding polarized light microscopy image. (Scale bars = ^PDLQ^^^PP^^^LQVHW^^^^^^P^^ [0063] FIG. 16 illustrates the effect of gelation bath composition on alignment. An MDP (K2) was dissolved in Milli-Q water to form an MDP precursor solution (3wt% K2), then extruded into a series of gelation baths (listed above images) and imaged with polarized light microscopy. The ionic strengths (I) and pH values for each of the gelation baths are also listed. The MDP precursor solution in this instance also comprised an orange dye. Checks 15 4863-6540-6909, v.1 indicate that hydrogels could be grasped with tweezers and check pluses indicate that hydrogels could be lifted out of solution. PB: Phosphate Buffer; HBSS: Hank’s Balanced Salt 6ROXWLRQ^^^6FDOH^EDU^ ^^^^^P^^ [0064] FIG. 17 shows the effect of incubation time on alignment. Top: An MDP (K2) was dissolved in Milli-Q water to form an MDP precursor solution (3wt% K2), then extruded into a series of gelation baths (listed above images) and imaged with polarized light microscopy. The ionic strengths (I) and pH values for each of the gelation baths are also listed. The MDP precursor solution in this instance also comprised an orange dye. Images represent hydrogels at time of formation (time 0). Bottom: Hydrogels from top were incubated in HBSS buffer for 2.5 hours at 4C and then reimaged. Checks indicate that hydrogels could be grasped with tweezers and check pluses indicate that hydrogels could be lifted out of solution. PB: Phosphate Buffer; HBSS: Hank’s Balanced Salt Solution; (Scale EDU^ ^^^^^P^^^ [0065] FIG. 18 illustrates the effect of MDP precursor solution additives on alignment. An MDP (K2) was dissolved in phosphate buffer of varying concentrations to form MDP precursor solutions (3wt% K2), extruded into a gelation bath (50mM Phosphate Buffer, 140mM NaCl), and imaged with polarized light microscopy. PB: Phosphate Buffer; 0.1PB = 1mM PB; 0.5PB= 5 mM PB; 1PB = 10mM PB. (Scale bar = 500^m). [0066] FIG. 19 shows the effect of MDP precursor solution pH on alignment. An MDP (K2) dissolved in 10mM Phosphate buffer at varying pH values (listed above images) to form multiple MDP precursor solutions (3wt% K2), extruded into 100mM phosphate buffer at pH 7.4 (listed above images), and imaged with polarized light microscopy. Checks indicate that hydrogels could be grasped with tweezers and check pluses indicate that hydrogels could be lifted out of solution. PS: Precursor Solution; GB: Gelation Bath; PB: 3KRVSKDWH^%XIIHU^^^6FDOH^EDU^ ^^^^^P^^^ [0067] FIG. 20 illustrates the effect of MDP precursor solution concentration on alignment. An MDP (K2) dissolved at varying concentrations (listed above images) in Milli- Q water to form MDP precursor solutions, then extruded into a gelation bath (50mM Phosphate Buffer, 140mM NaCl), and imaged with polarized light microscopy. The MDP SUHFXUVRU^VROXWLRQ^LQ^WKLV^LQVWDQFH^DOVR^FRPSULVHG^D^SXUSOH^G\H^^^6FDOH^EDU^ ^^^^^P^^ [0068] FIG. 21 shows the effect of MDP precursor solution concentration and sequence on alignment. MDPs (K2 and E2) dissolved at varying concentrations (listed above 16 4863-6540-6909, v.1 images) in Milli-Q water to form MDP precursor solutions, then extruded into a gelation bath (50mM Phosphate Buffer, 140mM NaCl), and imaged with polarized light microscopy. The K2 and E2 precursor solutions further comprised an orange and purple dye, respectively. ^6FDOH^EDU^ ^^^^^P^^^ [0069] FIG. 22 illustrates the effect of MDP precursor solution concentration, sequence, and solvent on alignment. MDPs (K2 and E2) dissolved at various concentrations in various solvents (listed above images) to form MDP precursor solutions, then extruded into a gelation bath (50mM Phosphate Buffer, 140mM NaCl), and imaged with brightfield and polarized light microscopy. The K2 and E2 precursor solutions further comprised an orange DQG^SXUSOH^G\H^^UHVSHFWLYHO\^^^6FDOH^EDU^ ^^^^^P^^ [0070] FIG. 23 shows aligned multidomain peptide (MDP) hydrogels fabricated using various sized extrusion nozzles. An MDP (K2) was dissolved in Milli-Q water to form an MDP precursor solution (3wt% K2), then extruded into a gelation bath (50mM Phosphate Buffer, 140mM NaCl) using different sized nozzles (listed above images), and imaged with brightfield and polarized light microscopy. Small, medium, and large correspond to a nozzle LQQHU^GLDPHWHU^RI^^^^^P^^^^^^P^^DQG^^^^^P^^UHVSHFWLYHO\^^7KH^0'3^SUHFXUVRU^VROXWLRQ^LQ^ WKLV^LQVWDQFH^DOVR^FRPSULVHG^DQ^RUDQJH^G\H^^^6FDOH^EDU^ ^^^^^P^^ [0071] FIG. 24 illustrates aligned multidomain peptide (MDP) hydrogels fabricated using a 3D printer in conjunction with various gelation-support baths. An MDP (K2) was dissolved in Milli-Q water to form an MDP precursor solution (3wt% K2), then automatically extruded by a 3D printer into various support baths (listed above images), and imaged with polarized light microscopy. Each of the support baths were created with a gelation bath (50mM Phosphate Buffer, 140mM NaCl) as the solvent to create joint gelation-support baths. The MDP precursor solution in this instance also comprised an orange dye. (Scale bar = ^^^^P^^ [0072] FIG. 25 shows scanning electron microscopy images of an MDP (K2) dissolved in Milli-Q water to form an MDP precursor solution (3wt% K2), then extruded into a 10mM Phosphate Buffer, 140mM NaCl gelation bath. The aligned MDP hydrogels were IRUPHG^XVLQJ^D^VPDOO^^^^^^P^^DQG^PHGLXP^^^^^^P^^GLDPHWHU^QR]]OH^^UHVSHFWLYHO\^^^6FDOH^ EDUV^ ^^OHIW^^^^P^^^ULJKW^^^^P^^ [0073] FIG. 26 shows scanning electron microscopy images of an MDP (K2) dissolved in Milli-Q water to form an MDP precursor solution (3wt% K2), then extruded into 17 4863-6540-6909, v.1 a 30mM Phosphate Buffer, 140mM NaCl gelation bath. The aligned MDP hydrogels were SUHSDUHG^XVLQJ^D^VPDOO^^^^^^P^^GLDPHWHU^QR]]OH^^^6FDOH^EDUV^ ^^OHIW^^^^P^^^ULJKW^^^^P^^ [0074] FIG. 27 shows scanning electron microscopy images of an MDP (K2) dissolved in Milli-Q water to form an MDP precursor solution (3wt% K2), then extruded into a 50mM Phosphate Buffer, 140mM NaCl gelation bath. The aligned MDP hydrogels were SUHSDUHG^XVLQJ^D^VPDOO^^^^^^P^^GLDPHWHU^QR]]OH^^^6FDOH^EDUV^ ^^OHIW^^^^P^^^ULJKW^^^^P^^ [0075] FIG. 28 shows scanning electron microscopy images of an MDP (K2) dissolved in Milli-Q water to form an MDP precursor solution (3wt% K2), then extruded into a 50mM Phosphate Buffer, 140mM NaCl gelation bath. The aligned MDP hydrogels were SUHSDUHG^XVLQJ^D^PHGLXP^^^^^^P^^GLDPHWHU^QR]]OH^^^6FDOH^EDUV^ ^^OHIW^^^^^QP^^^ULJKW^^^^P^^ [0076] FIGS. 29A-29D illustrate a concentration screen of extruded MDP precursor solution. Polarized light microscopy of (FIG. 29A) 1wt%, (FIG. 29B) 2wt%, (FIG. 29C) 3wt%, and (FIG. 29D) 4wt% K2 in Milli-Q water precursor solutions extruded through a 100-1000 ^/^SLSHWWH^WLS^LQWR^D^SKRVSKDWH-EXIIHUHG^VDOLQH^JHODWLRQ^EDWK^^VFDOH^EDU^ ^^^^^^P^^ [0077] FIGS. 30A-30D relate to equilibration of precursor solutions described herein. (FIG. 42A) Rheology shear sweep between 0.1 and 10 s-1, (FIG. 30B) Attenuated total reflectance Fourier transform infrared spectroscopy between 1500 and 1750 cm-1, and (FIG. 30C) dynamic light scattering z-average measurements (n = 3; mean ± standard deviation; *P < 0.05 by one-way ANOVA and Tukey’s multiple comparisons test) of 3wt% K2 in Milli-Q water precursor solution at days 0, 1, 7, and 21. (FIG. 30D) Cryo-transmission electron microscopy of 3wt% K2 in Milli-Q water precursor solution at days (I) 0, (II) 1, and (III) 14 (scale bar = 200 nm). [0078] FIGS. 31A-31X illustrate the effect of additional gelation bath compositions on alignment. Gelation Bath Composition Screen. Polarized light microscopy of 3wt% K2 in Milli-Q water precursor solution extruded through a 0.1-^^^^/^SLSHWWH^WLS^LQWR^D^VHULHV^RI^S+^ ^^JHODWLRQ^EDWKV^^VFDOH^EDU^ ^^^^^^P^^^A green checkmark indicate that hydrogels could be lifted from solution without fracturing, whereas a red X indicate that hydrogels fractured when lifted from solution. (FIG. 31A) 5 mM HEPES/ 0 mM NaCl. (FIG. 31B) 10 mM HEPES/ 0 mM NaCl. (FIG. 31C) 50 mM HEPES/ 0 mM NaCl. (FIG. 31D) 5 mM phosphate buffer (PB)/ 0 mM NaCl. (FIG. 31E) 10 mM PB/ 0 mM NaCl. (FIG. 31F) 50 mM PB/ 0 mM NaCl. (FIG. 31G) 5 mM HEPES/ 70 mM NaCl. (FIG. 31H) 10 mM HEPES/ 70 mM NaCl. (FIG. 31I) 50 mM HEPES/ 70 mM NaCl. (FIG. 31J) 5 mM PB/ 70 mM NaCl. (FIG. 31K) 10 18 4863-6540-6909, v.1 mM PB/ 70 mM NaCl. (FIG. 31L) 50 mM PB/ 70 mM NaCl. (FIG. 31M) 5 mM HEPES/ 140 mM NaCl. (FIG. 31N) 10 mM HEPES/ 140 mM NaCl. (FIG. 31O) 50 mM HEPES/ 140 mM NaCl. (FIG. 31P) 5 mM PB/ 140 mM NaCl. (FIG. 31Q) 10 mM PB/ 140 mM NaCl. (FIG. 31R) 50 mM PB/ 140 mM NaCl. (FIG. 31S) 5 mM HEPES/ 700 mM NaCl. (FIG. 31T) 10 mM HEPES/ 700 mM NaCl. (FIG. 31U) 50 mM HEPES/ 700 mM NaCl. (FIG. 31V) 5 mM PB/ 700 mM NaCl. (FIG. 31W) 10 mM PB/ 700 mM NaCl. (FIG. 31X) 50 mM PB/ 700 mM NaCl. [0079] FIGS. 32A-32F illustrates the results of a precursor solution extrusion rate screen. Polarized light microscopy of 3wt% K2 in Milli-Q water precursor solution extruded at (FIG. 32A, FIG. 32D) 0.3 mL/min, (FIG. 32B, FIG. 32E) 0.6 mL/min, and (FIG. 32C, FIG. 32F) 0.9 mL/min into (FIG. 32A-32C) 10 mM phosphate buffer (PB)/ 140 mM NaCl, pH 7 and (FIG. 32D-32F^^^^^3%^^^^^^P0^1D&O^^S+^^^JHODWLRQ^EDWKV^^VFDOH^EDU^ ^^^^^^P^^ [0080] FIGS. 33A-33J show the results of a gelation bath phosphate buffer screen. Polarized light microscopy of (FIG. 33A, FIG. 33F) pregelled, (FIG. 33B, FIG. 33G) 1X PB, (FIG. 33C, FIG. 33H) 3X PB, (FIG. 33D, FIG. 33I) 5X PB, and (FIG. 33E, FIG. 33J) 10X 3%^ K\GURJHOV^ ^VFDOH^ EDU^ ^ ^^^^ ^P^^^ ^FIG. 33A- FIG. 335E) 100-^^^^^ ^/^ DQG^ ^FIG. 33F- FIG. 33J) 0.1-^^^^/^SLSHWWH^WLSV^ZHUH^XVHG^WR^PDQXDOO\^H[WUXGH^WKH^SUHFXUVRU^VROXWLRQV^ PB: Phosphate Buffer; 1X PB: 10 mM phosphate buffer/ 140 mM NaCl, pH 7; 3X PB: 30 mM phosphate buffer/ 140 mM NaCl, pH 7; 5X PB: 50 mM phosphate buffer/ 140 mM NaCl, pH 7; 10X PB: 100 mM phosphate buffer/ 140 mM NaCl, pH 7. [0081] FIGS. 34A-34E show the tunable alignment of nanofibrous MDP hydrogels. (FIG. 34$^^ 3RODUL]HG^ OLJKW^ PLFURJUDSKV^ ^VFDOH^ EDUV^ ^ ^^^^ ^P^^^ ^),*^^ 34B, FIG. 34C) VFDQQLQJ^HOHFWURQ^PLFURJUDSKV^^VFDOH^EDUV^ ^^^^P^DQG^^^^^QP^^UHVSHFWLYHO\^^^^),*^^34D) 2D small-angle x-ray scattering patterns, and (FIG. 34E) azimuthal distributions derived from 2D small-angle x-ray scattering patterns of (i) pre-gelled, (ii) 1X PB, (iii) 3X PB, and (iv) 5X PB hydrogels. The full width at half maximum for the largest peak is indicated underneath the curve. [0082] FIGS. 35A & 35B show the bulk geometry of MDP Hydrogels formed in various gelation bath compositions. Hydrogel diameter measurements of K2 hydrogels fabricated through (FIG. 35A) 100-^^^^^^/^^Q^ ^^^VDPSOHV^^PHDQ^^^VWDQGDUG^GHYLDWLRQ^^^3^^^ 0.05, ****P < 0.0001 by one-way ANOVA and Dunnett’s multiple comparisons test) and (FIG. 35B) 0.1-^^^^/^SLSHWWH^WLSV^^Q^ ^^^VDPSOHV^^PHDQ^^^VWDQGDUG^GHYLDWLRQ^^^3^^^^^^^^^^^3^ < 0.01, ****P < 0.0001 by one-way ANOVA and Dunnett’s multiple comparisons test). The 19 4863-6540-6909, v.1 expected diameters for the large and small hydrogels are indicated in orange and are 860 and ^^^^ ^P^^ UHVSHFWLYHO\^ PB: Phosphate Buffer; 1X PB: 10 mM phosphate buffer/ 140 mM NaCl, pH 7; 3X PB: 30 mM phosphate buffer/ 140 mM NaCl, pH 7; 5X PB: 50 mM phosphate buffer/ 140 mM NaCl, pH 7. [0083] FIGS. 36A-36G show the mechanical characterization of aligned MDP hydrogels. (FIG. 36A) Pre-gelled, (FIG. 36B) 1X PB, (FIG. 36C) 3X PB, and (FIG. 36D) 5X PB hydrogels (i) structural optical coherence tomography representations and (ii) mechanical wave snapshots at t = 4.5 ms, (iii) t = 5 ms, and (iv) 6.5 ms following a 1 kHz microtapping excitation. The color bar to the right indicates the particle velocity spectrum. (FIG. 36E) Brillouin shift profile for 5X PB hydrogel. The color bar to the right indicates the Brillouin shift (GHz). (FIG. 36F) Comparison of average wave speeds calculated using particle velocity profiles for each hydrogel type (n = 3 samples per condition; line at mean; *P < 0.05, **P < 0.01 by multiple Student’s t tests). (FIG. 36G) Comparison of Brillouin shift along the z-D[LV^IRU^^;^3%^K\GURJHOV^^Q^ ^^^VDPSOHV^ZLWK^GDWD^SRROHG^LQWR^^^^^P^JURXSLQJV^^PHDQ^^^ standard deviation; **P < 0.01 by Student’s t test). [0084] FIGS. 37A-37D illustrate the stiffness of aligned K2 hydrogels. Optical Coherence Elastography elastic wave speed maps for (FIG. 37A) pre-gelled, (FIG. 37B) 1X PB, (FIG. 37C) 3X PB, and (FIG. 37D) 5X PB hydrogels. The color bar to the right indicates wave speed (m/s). [0085] FIGS. 38A-38C show Brillouin shift profiles for 5X PB Hydrogels. (FIGS. 38A-38C) Brillouin frequency shift maps of three 5X PB hydrogels. The color bar to the right indicates the Brillouin shift (GHz). [0086] FIGS. 39A-39D show VIC Cell Viability on K2 Hydrogels. (FIG. 39A) Day 1, (FIG. 39B) Day 3, and (FIG. 39C) Day 7 confocal microscopy images of VICs on (I) pre- gelled, (II) 1X PB, (III) 3X PB, and (IV) 5X PB hydrogels (Calcein AM = green, Ethidium KRPRGLPHU^ ^UHG^^VFDOH^EDU^ ^^^^^^P^^^$OO^LPDJHV^DUH^PD[LPXP^LQWHQVLW\^SURMHFWLRQV^RI^=- stacks. (FIG. 39D) Cell viability calculations derived from confocal microscopy images (n = 3 images per time point per group; mean ± standard deviation). [0087] FIGS. 40A-40H show valvular interstitial cell spreading on aligned K2 hydrogels. (FIG. 40A) Day 1, (FIG. 40B) Day 3, and (FIG. 40C) Day 7 confocal microscopy images of VICs each on (i) pre-gelled, (ii) 1X PB, (iii) 3X PB, and (iv) 5X PB hydrogels (DAPI = blue, F-DFWLQ^ ^ JUHHQ^^ 6FDOH^ EDUV^ ^ ^^^^ ^P^^^$OO^ LPDJHV^ DUH^PD[LPXP^ LQWHQVLW\^ projections of Z-stacks that have been cropped and rotated so the direction of K2 fibrous 20 4863-6540-6909, v.1 alignment is horizontal. Polar histograms to the right of each image display the Fourier gradient structure tensor calculated using the F-actin channels, where the direction of K2 fibrous alignment is 0°. (FIG. 40D) Day 1, (FIG. 40E) Day 3, and (FIG. 40F) Day 7 comparisons of (i) resultant vector lengths calculated from Fourier gradient structure tensors (n = 3 images; line at mean; *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Dunnett’s multiple comparisons test) and (ii) nuclear angle distributions with respect to the angle of K2 fibrous alignment (n = 3 images with between 294 and 1854 pooled nuclei; center-line, box bounds, and whiskers indicate the median, first and third quartiles, and 10 and 90 percentiles, respectively; **P < 0.01, ***P < 0.001, ****P < 0.0001 by Kolmogorov- Smirnov test). (FIG. 40G) Day 3 and (FIG. 40H) Day 7 confocal microscopy image stitches of VICs on 1X PB hydrogels (DAPI = blue, F-DFWLQ^ ^JUHHQ^^6FDOH^EDU^ ^^^^^^P^^ [0088] FIGS 41A-41D show C2C12 Cell Viability on K2 Hydrogels. (FIG. 41A) Day 1, (FIG. 41B) Day 3, and (FIG. 41C) Day 7 confocal microscopy images of C2C12 cells on (I) pre-gelled, (II) 1X PB, (III) 3X PB, and (IV)5X PB hydrogels (Calcein AM = green, (WKLGLXP^ KRPRGLPHU^ ^ UHG^^ VFDOH^ EDU^ ^ ^^^^ ^P^^^ $OO^ LPDJHV^ DUH^ PD[LPXP^ LQWHQVLW\^ projections of Z-stacks. (FIG. 41D) Cell viability calculations derived from confocal microscopy images (n = 3 images per time point per group; mean ± standard deviation). [0089] FIGS. 42A-42F show C2C12 Cell Spreading on K2 Hydrogels. (FIG. 42A) Day 1, (FIG. 42B) Day 3, and (FIG. 42C) Day 7 confocal microscopy images of C2C12 cells each on (I) pre-gelled, (II) 1X PB, (III) 3X PB, and (IV) 5X PB hydrogels (DAPI = blue, F- DFWLQ^ ^JUHHQ^^VFDOH^EDU^ ^^^^^^P^^^$OO^LPDJHV^DUH^PD[LPXP^LQWHQVLW\^SURMHFWLRQV^RI^=-stacks that have been cropped and rotated so the direction of K2 fibrous alignment is horizontal. Polar histograms to the right of each image display the Fourier gradient structure tensor calculated using the F-actin channels, where the direction of K2 fibrous alignment is 0°. (FIG. 42D) Day 1, (FIG. 42E) Day 3, and (FIG. 42F) Day 7 comparisons of (I) resultant vectors lengths calculated from Fourier gradient structure tensors (n = 3 images; line at mean; **P < 0.01, ***P < 0.001 by one-way ANOVA and Dunnett’s multiple comparisons test) and (II) nuclear angle distributions with respect to the angle of K2 fibrous alignment (n = 3 images with between 242 and 2827 pooled nuclei; center-line, box bounds, and whiskers indicate the median, first and third quartiles, and 10 and 90 percentiles, respectively; ***P < 0.001, ****P < 0.0001 by Kolmogorov-Smirnov test). [0090] FIGS 43A-43D show C2C12 Cell Differentiation on K2 Hydrogels. Confocal microscopy images of C2C12 cells on (FIG. 43A) pre-gelled, (FIG. 43B) 1X PB, (FIG. 43C) 21 4863-6540-6909, v.1 3X PB, and (FIG. 43D) 5X PB hydrogels (DAPI = blue, Myosin heavy chain = green; scale EDU^ ^ ^^^^ ^P^^^$OO^ LPDJHV^ DUH^PD[LPXP^ LQWHQVLW\^ SURMHFWLRQV^ RI^ =-stacks that have been cropped and rotated so the direction of K2 fibrous alignment is horizontal. [0091] FIGS. 44A-44F show VIC Cell Spreading on Small Diameter K2 Hydrogels. (FIG. 44A) Day 1, (FIG. 44B) Day 3, and (FIG. 44C) Day 7 confocal microscopy images of VICs on (I) pre-gelled, (II) 1X PB, (III) 3X PB, and (IV) 5X PB hydrogels (DAPI = blue, F- actin = JUHHQ^^ VFDOH^EDU^ ^^^^^^P^^ IDEULFDWHG^XVLQJ^^^^-10 µL pipette tips. All images are maximum intensity projections of Z-stacks that have been cropped and rotated so the direction of K2 fibrous alignment is horizontal. Polar histograms to the right of each image display the Fourier gradient structure tensor calculated using the F-actin channels, where the direction of K2 fibrous alignment is 0°. (FIG. 44D) Day 1, (FIG. 44E) Day 3, and (FIG. 44F) Day 7 comparisons of (I) resultant vectors lengths calculated from Fourier gradient structure tensors (n = 3 images; line at mean; *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Dunnett’s multiple comparisons test) and (II) nuclear angle distributions with respect to the angle of K2 fibrous alignment (n = 3 images with between 113 and 1416 pooled nuclei; center-line, box bounds, and whiskers indicate the median, first and third quartiles, and 10 and 90 percentiles, respectively; *P < 0.05, **P < 0.01, ****P < 0.0001 by Kolmogorov-Smirnov test). [0092] FIGS. 45A-45F show C2C12 Cell Spreading on Small Diameter K2 Hydrogels. (FIG. 45A) Day 1, (FIG. 45B) Day 3, and (FIG. 45C) Day 7 confocal microscopy images of C2C12 cells on (I) pre-gelled, (II) 1X PB, (III) 3X PB, and (IV) 5X PB hydrogels (DAPI = blue, F-actin = JUHHQ^^VFDOH^EDU^ ^^^^^^P^^IDEULFDWHG^XVLQJ^^^^-10 µL pipette tips. All images are maximum intensity projections of Z-stacks that have been cropped and rotated so the direction of K2 fibrous alignment is horizontal. Polar histograms to the right of each image display the Fourier gradient structure tensor calculated using the F-actin channels, where the direction of K2 fibrous alignment is 0°. (FIG. 45D) Day 1, (FIG. 45E) Day 3, and (FIG. 45F) Day 7 comparisons of (I) resultant vectors lengths calculated from Fourier gradient structure tensors (n = 3 images; line at mean; *P < 0.05, **P < 0.01 by one-way ANOVA and Dunnett’s multiple comparisons test) and (II) nuclear angle distributions with respect to the angle of K2 fibrous alignment (n = 3 images with between 128 and 2437 pooled nuclei; center-line, box bounds, and whiskers indicate the median, first and third quartiles, and 10 and 90 percentiles, respectively; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Kolmogorov-Smirnov test). 22 4863-6540-6909, v.1 [0093] FIGS. 46A & 46B show K2 hydrogel swelling. Hydrogel diameter measurements of K2 hydrogels fabricated through (FIG. 46A) 100-^^^^^ ^/^ ^Q^ ^ ^^ - 6 samples per time point; mean ± standard deviation; ****P < 0.0001 by two-way ANOVA and Dunnett’s multiple comparisons test) and (FIG. 46B) 0.1-^^^^/^SLSHWWH^ WLSV^ ^Q^ ^^^ - 6 samples per time point; mean ± standard deviation; ****P < 0.0001 by two-way ANOVA and Dunnett’s multiple comparisons test). The expected diameters for the large and small K\GURJHOV^DUH^^^^^DQG^^^^^^P^^UHVSHFWLYHO\^ [0094] FIGS. 47A-47C show cell-matrix interactions on aligned K2 scaffolds. (FIG. 47A) 1X PB, (FIG. 47B) 3X PB, and (FIG. 47C) 5X PB hydrogel matrices with (i) C2C12 DQG^ ^LL^^ 9,&V^ REVHUYHG^ YLD^ 6(0^ ^6FDOH^ EDUV^ ^ ^^ ^P^^^ &^&^^^ DQG^ 9,&^ FHOOV^ DUH^ IDOVHO\^ colored red and blue, respectively. [0095] FIGS. 48A-48D show additional K2 Ink characterization for Example 4. FIG. 48A) Attenuated total reflectance Fourier transform infrared spectroscopy between 1500 and 1750 cm^-1, FIG. 48B) cryo-transmission electron microscopy (scale bar = 200nm), FIG. 48C) shear sweeps from 0.01 – 100 s^-1, and d) strain sweeps from 0.1 – 100% of 4wt% K2 in Milli- Q water. [0096] FIGS. 49A & 49B show data relating to the assessment of support bath candidates. FIG. 49A) Shear sweeps from 0.01 – 100 s^-1 of K2 ink compared to the various support bath candidates. FIG. 49B) 2x2 log pile I) as designed, II) printed into FRESH v1.0, and III) printed into agarose support bath (Scale bars = 3 mm). [0097] FIGS. 50A-50D provide data relating to 3D printing optimization. (FIG. 50A) Calibration lines example print. Printhead speed increases from bottom to top (Scale bar = 2 mm). (FIG. 50B) Calibration curve for K2 ink into agarose support bath. (FIG. 50C) Optimized I) 2x2 log pile and II) 1x1 log pile printed into agarose support baths (Scale bars = 2 mm). (FIG. 50D) Scanning electron micrographs of prints into I) agarose with 1X PBS and II) agarose with 5X PBS (Scale bars = 1 µm). [0098] FIGS. 51A & 51B illustrate myoblast spreading on anisotropic hydrogels. Confocal maximum intensity projection of immunofluorescently stained C2C12 cells after FIG. 51A) three days and FIG. 51B) seven days in culture (DAPI = blue, F=actin = green; Scale bar = 200µm). 23 4863-6540-6909, v.1 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [0099] The present methods provide for the formation of an ink comprising a multidomain peptide (MDP). The ink may be useful or suitable for printing, including extrusion printing, 3-dimensional hydrogel structures. In some embodiments, the hydrogel structures have aligned nanofibers. In some embodiments, the degree of nanofiber alignment is tunable. The hydrogel structures comprising MDPs as disclosed herein may be useful or suitable for tissue engineering or other biomedical applications. Further details on these aspects and more are provided above and in the sections that follow. Multidomain Peptides (MDPs) [00100] Multidomain peptides (MDPs) are a class of self-assembling peptides (SAPs) that have an amphiphilic core and charged residues at either terminus (Moore, & Hartgerink, 2017; Dong et al., 2007). Without being bound by theory, these structural elements make MDPs self-DVVHPEOH^ LQWR^ ȕ-sheets and form a nanofibrous hydrogel under physiological conditions. MDPs have been shown to support cell survival and growth. [00101] The present disclosure provides methods for printing multidomain peptides (MDPs), a class of self-assembling peptides that rely, without being bound by theory, solely on supramolecular mechanisms to form a nanofibrous hydrogel at low concentrations. Distinct inks corresponding to MDPs with different charge functionalities are disclosed herein and are used to create complex three-dimensional structures, including for example structures comprising multiple types of MDPs. Additionally, methods of the present disclosure may be useful to print MDP constructs that can be used to demonstrate charge- dependent differences in cellular behavior in vitro. In some embodiments, the methods disclosed herein facilitate the printing with self-assembling peptides of layered structures with overhangs and internal porosity. [00102] The work presented here can facilitate research to gain an understanding of the interplay between mechanical properties, charge, and their combined effects on cells. MDPs are favorable in that they are synthesized one amino acid at a time, are tunable, and can easily be modified with bioactive peptide sequences to influence cell fate and behavior. Complex hydrogel geometries and hydrogel structures comprising a variety of cell types are contemplated by the present disclosure. 24 4863-6540-6909, v.1 [00103] MDPs are shown in the present disclosure to possess the necessary rheological properties useful for extrusion 3D printing. Complex, overhanging hydrogel structures and/or multi-material hydrogel structures may be prepared according to the methods disclosed herein. The present disclosure represents an advance in 3D printing involving self-assembling peptides and surpasses the level of printing complexity that is known in the art. [00104] In some embodiments, the present methods involve forming a precursor solution comprising an MDP according to the present disclosure and a solvent that mediates the self-assembly of the MDP. In some embodiments, the precursor solution has properties that are favorable for the formation of the hydrogel structure. For example, in some embodiments the precursor solution has less than about 20% change in storage modulus G` between a temperature of about 4C and about 37C at a strain of about 1% and at a frequency of about 1 rad/s. In some embodiments, the precursor solution has less than about 20% change, less than about 19% change, less than about 18% change, less than about 17% change, less than about 16% change, less than about 15% change, less than about 14% change, less than about 13% change, less than about 12% change, less than about 10% change, less than about 9% change, less than about 8% change, less than about 7% change, less than about 6% change, less than about 5% change, less than about 4% change, less than about 3% change, less than about 2% change, less than about 1% in storage modulus G` between a temperature of about 4C and about 37C at a strain of about 1% and at a frequency of about 1 rad/s, or any range derivable therein. In some embodiments, the precursor solution has less than about 5% change in storage modulus G` between a temperature of about 4C and about 37C at a strain of about 1% and at a frequency of about 1 rad/s. In some embodiments, the precursor solution has less than about 5% change, less than about 4% change, less than about 3% change, less than about 2% change, or less than about 1% in storage modulus G` between a temperature of about 4C and about 37C at a strain of about 1% and at a frequency of about 1 rad/s. [00105] In some embodiments, the precursor solution is characterized by a storage modulus G` greater than about 1000 Pa at a strain of about 1% and at a frequency between about 0.01 rad/s and about 100 rad/s. In some embodiments, the precursor solution is characterized by a storage modulus G` greater than about 1000 Pa, greater than about 1250 Pa, greater than about 1500 Pa, greater than about 1750 Pa, greater than about 2000 Pa, 25 4863-6540-6909, v.1 greater than about 2250 Pa, greater than about 2500 Pa, greater than about 2750 Pa, greater than about 3000 Pa, greater than about 3250 Pa, greater than about 3500 Pa, greater than about 3750 Pa, greater than about 4000 Pa, greater than about 4250 Pa, greater than about 4500 Pa, greater than about 4750 Pa, or greater than about 5000 Pa, or any range derivable therein, at a strain of about 1% and at a frequency between about 0.01 rad/s and about 100 rad/s. [00106] The present methods provide for the formation of hydrogels possessing aligned nanofibers. Hydrogels formed according to the presently disclosed methods may be useful for directing cell growth and spreading, thereby facilitating tissue growth from individual cells. The present disclosure provides methods for creating hydrogel structures that have varying, controllable degrees of nanofibrillar alignment. The hydrogels formed according to the present disclosure also are observed to have varying, controllable mechanical properties. The present methods also allow for cell growth in different patterns, including but not limited to longitudinally aligned cells, randomly oriented cells, and circumferentially aligned cells. The present methods may be useful for facilitating the production of muscle and nerve tissues that can be used for pharmaceutical drug testing and to treat people who suffer from severe muscle and nerve injuries. By fabricating 3D printed tissue in vitro, the presently disclosed methods may provide a platform that is more cost effective, predictive, and ethical compared to current methods used during early-stage drug development by pharmaceutical companies. In addition, these tissues can be used to treat severe muscle and nerve injuries (such as volumetric muscle loss and peripheral nerve transection injuries), which have been drawing increased attention due to a lack of effective therapies. [00107] As mentioned above, the present disclosure provides methods for fabricating hydrogels that possess a controlled degree of nanofibrillar alignment. The methods disclosed herein disclose the formation of such hydrogels from multidomain peptides (MDPs). MDPs are a class of self-assembling peptides that form a nanofibrous hydrogel and have been demonstrated to support cell survival and growth. The novel alignment process presented herein involves extruding MDP precursor solutions into a gelation bath that causes nanofibrillar alignment and hydrogel gelation. By varying the composition of the gelation bath, the degree of alignment can be controlled, which has a direct effect on the hydrogels’ mechanical properties and drives differences in cell behavior. 26 4863-6540-6909, v.1 [00108] In some embodiments, the gelation bath comprises a buffering agent, an additive, a crosslinker, an inorganic salt, or any combination thereof. In some embodiments, the gelation bath comprises PBS, HBSS, cell culture medium, or a biological fluid, or any combination thereof. In some embodiments, the gelation bath comprises between 5 mM phosphate buffer and 150 mM phosphate buffer. In some embodiments, the gelation buffer comprises about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM phosphate buffer or any range derivable therein. In some embodiments, the gelation bath comprises 10 mM phosphate buffer. In some embodiments, the gelation bath comprises 30 mM phosphate buffer. In some embodiments, the gelation bath comprises 50 mM phosphate buffer. In some embodiments, the gelation bath comprises NaCl, for example 140 mM or 1400 nM NaCl. [00109] By varying the degree of MDP fiber alignment, a novel pattern of cellular behavior can be controlled: Below an alignment threshold, MDP nanofibrillar alignment causes longitudinal cellular alignment, but above an alignment threshold, MDP nanofibrillar alignment causes circumferential cellular alignment. The fabrication of aligned MDP hydrogels can be performed with an extrusion 3D printer to create multilayer structures that possess nanofibrillar alignment. This process is biologically friendly as it only involves short peptides and buffers that are commonly used in cell culture. Overall, the 3D printing of aligned MDPs can be used to fabricate hydrogel scaffolds that mirror the structure of aligned tissues such as muscle and nerve and may serve as a regenerative medicine therapy. Further details are provided above and in the sections that follow. [00110] The present disclosure provides certain compositions, such as precursor solutions and multidomain peptide-ink compositions as detailed in the sections that follow, that comprise a peptide having a multidomain peptide sequence as defined elsewhere in this application. Nonlimiting examples of multidomain peptide sequences that may be used in the presently disclosed methods or may form a part of a presently disclosed composition are provided in Table 1 below. The sequences of the multidomain peptides disclosed herein may optionally comprise additional domains; nonlimiting example sequences for optional 27 4863-6540-6909, v.1 domains are also provided in Table 1. Details of MDPs and their further use in the presently disclosed methods are provided above and in the sections that follow. Table 1: Exemplary domain sequences of the present disclosure.

28 4863-6540-6909, v.1

I. Definitions [00111] The term “substantial identity” in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide. Thus, a polypeptide is identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. 29 4863-6540-6909, v.1 [00112] The use of the word “a” or “an,” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” [00113] Unless otherwise indicated, all numbers expressing quantities of ingredients, concentrations properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques [00114] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or patients. Unless otherwise noted, the term “about” is used to indicate a value of ±10% of the reported value, preferably a value of ±5% of the reported value. It is to be understood that, whenever the term “about” is used, a specific reference to the exact numerical value indicated is also included.” [00115] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. [00116] The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, inhibit, reduce, or decrease an undesired physiological change or disorder, such as the development, progression or worsening of the disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilizing a (i.e., not worsening or progressing) symptom or adverse effect 30 4863-6540-6909, v.1 of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed (e.g., as determined by a genetic assay). [00117] The above definitions supersede any conflicting definition in any reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention. IV. Examples [00118] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. [00119] The present disclosure provides for the use of multidomain peptides (MDPs) which comprise at least a first domain, such as those shown in Table 1. The MDPs disclosed herein may optionally comprise additional domains, example sequences for which are also provided in Table 1. Details of MDPs and their further use in the presently disclosed methods are provided in the sections that follow. A. Example 1 Multidomain Peptide Characterization [00120] The cationic MDP used in the present disclosure, named K2, has the sequence K2(SL)6K2 (SEQ ID NO: 1) whereas the anionic MDP, named E2, has the sequence E2(SL)6E2 (SEQ ID NO: 2) (FIG. 2) (Aulisa et al., 2009; Bakota et al., 2011; Lopez-Silva et al., 2020). Both K2 and E2 are N-terminally acetylated and C-terminally amidated. They 31 4863-6540-6909, v.1 were synthesized via solid phase peptide synthesis and confirmed with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (FIG. 3). The peptides were dissolved in Hank’s Balanced Salt Solution (HBSS) at 1 wt% to analyze the secondary structure and macroscopic assembly. HBSS was used as the solvent throughout the study to create a hydrogel that contains the necessary osmotic pressure, salt content, glucose concentration, and pH to optimize cellular survival in vitro. These peptides have previously been characterized at lower concentrations of peptide and ionic strength, so recharacterization was performed to ensure that changing these variables, which could be necessary for 3D printing, did not alter the secondary structure of the MDPs. Circular dichroism on K2 and E2 showed a FKDUDFWHULVWLF^ ȕ-sheet secondary structure, with a maximum around 198 nm and minimum DURXQG^^^^^QP^ ^),*^^^$^^^.^^ IRUPV^ D^ VOLJKWO\^ VWURQJHU^ȕ-sheet as indicated by the higher maximum and lower minimum, which can be attributed to differences between the amino group on lysine and the carboxyl group on glutamic acid. Attenuated total reflectance Fourier transform infrared spectroscopy on K2 and E2 revealed a peak at around 1620 and 1695 cm^í1^^ZKLFK^FRUUHVSRQG^WR^DQ^DPLGH^,^SHDN^DQG^WKH^IRUPDWLRQ^RI^DQ^DQWLSDUDOOHO^ȕ-sheet (FIG. 4B). [00121] Scanning electron microscopy was used to observe the fibrous network that forms in both K2 and E2 hydrogels (FIGS. 4C-4F). At low magnification, an expansive, dense network of fibers are seen in both MDPs (FIG. 4C and FIG. 4E), whereas at higher magnification, MDP nanofibers are visualized creating a web-like structure (FIG. 4D and FIG. 4F). [00122] Rheological testing was performed on both K2 and E2 to assess their potential use as extrudable inks. 3D printing has been shown using inks with a range of storage moduli, so a frequency sweep from 0.1 to 100 rad s^í1 at 1% strain was performed on 1, 2, 3, and 4 wt% K2 and E2 to understand how increasing peptide concentration affected the resulting gel storage modulus (Gƍ) (FIG. 5A and FIG. 5E). 4 wt% was chosen as the upper peptide concentration limit because dissolving MDPs at higher concentrations was difficult to DFFRPSOLVK^^$W^^^UDG^Ví^^^^^^^^^^^^DQG^^^ZW^^.^^KDYH^*ƍ^YDOXHV^RI^^^^^^^^^^^^^^^^^^DQG^^^^^^ 3D^^ UHVSHFWLYHO\^^ ,Q^ FRQWUDVW^^ ^^^ ^^^ ^^^ DQG^ ^^ ZW^^ (^^ KDV^ *ƍ^ YDOXHV^ RI^ ^^^^^ ^^^^^ ^^^^^ DQG^ 1040 Pa, respectively. Thus, K2 gels have storage moduli that are on average 4.2X greater than E2 gels at equal concentrations, although both MDPs demonstrate around a 2X increase in storage modulus for each additional wt% (2.4X for K2 and 1.8X for E2). 32 4863-6540-6909, v.1 [00123] 4 wt% K2 and E2, which had the highest Gƍ values, were subjected to additional rheological testing to ensure they were shear thinning, shear yielding, and rapidly self-healing, which are essential properties for a 3D printable ink (Paxton et al., 2017; Chen et al., 2017). A shear sweep from 0.01 to 100 s^í1 was performed on 4 wt% K2 and E2 (FIG. 5B and FIG. 5F). At shear rates of 0.1, 1, and 10 s^í1, K2 has viscosity values of 577, 63, and 7.7 Pa s. At the same shear rates, E2 has viscosity values of 336, 70, and 6.4 Pa s. Importantly, both 4 wt% MDPs had a negative slope, which means that they are both shear thinning. [00124] A strain sweep from 0.1 to 100% strain at 1 rad s^í1 was also performed on both 4 wt% MDPs (FIG. 5C and FIG. 5G). Throughout the linear viscoelastic region, the ratio between the storage and loss modulus for both K2 and E2 were very similar (16 and 14 at 1% strain, respectively). In contrast, the storage modulus of K2 (4810 Pa) was 4.5X that of E2 (1080 Pa) at 1% strain, which corroborates the results of the frequency sweeps. Both K2 and E2 exhibited shear yielding behavior at high strains, although the storage/loss modulus crossover was observed to occur at around 40% strain for K2, compared to 20% strain for E2. [00125] To assess the self-recovery of K2 and E2, a series of low (1%) and high (500%) strains was applied to each gel as the storage and loss moduli were measured (FIG. 5D and FIG. 5H). Both MDPs exhibited an inversion of the moduli during high strain, indicating liquid-like behavior, followed by rapid recovery during low strain. K2 and E2 both exhibited a recovery of storage modulus to 86% of the pre-strain value within 1 min of low shear conditions. This rapid recovery of the storage modulus following liquification is necessary, but not sufficient, for fiber formation during 3D printing. Multidomain Peptide Ink Printing Optimization [00126] Here and elsewhere throughout the application, the term “MDP ink” and “precursor solution” are used equivalently and interchangeably to signify a composition comprising a multidomain peptide (MDP) and a solvent. In some embodiments, the solvent mediates the self-assembly of the MDP. [00127] MDP inks were created by dissolving peptides in dye supplemented HBSS. Before making large batches of precursor solution (>1 mL), preliminary extrusion testing was performed to determine the minimum storage modulus required to form a self- supporting filament when extruded from a needle. It was empirically concluded that only 33 4863-6540-6909, v.1 MDP precursor solution with storage moduli greater than 1 KPa were able to form robust fibers (FIG. 6A and FIG. 6B). Based on this result and the previous rheological findings, larger batches of MDP precursor solution were made at 4% K2, 4% E2, and 3% K2. [00128] Batch-to-batch variation possibly due, without being bound by theory, to the dynamic state of the MDP nanofibers was evaluated along with the effect of temperature and time on rheological properties. Frequency sweeps on 3 replicates of each ink showed reproducible rheological properties that matched closely to initial rheological testing (FIG> 5I). In addition, the MDP precursor solution VKRZHG^VWDEOH^*ƍ^YDOXHV^EHWZHHQ^^^DQG^^^^ ^&^^ZLWK^WKH^WZR^^^^LQNV^DYHUDJLQJ^MXVW^D^§^^^FKDQJH^RYHU^WKH^WHPSHUDWXUH^VZHHS^^),*^^^-^^^ Thus, without being bound by theory, MDP inks avoid the need for temperature control during 3D printing, which is a necessary complication for many 3D printing inks that have temperature-dependent properties. In addition, the introduction of cells in vitro or implantation of printed constructs in vivo will, without being bound by theory, necessitate gel stability at 37 °C, which is demonstrated here for MDP inks. The MDP inks also showed long term stability when stored at 4 °C (FIG. 5K). Frequency sweeps on the different inks after being stored at 4 °C for up to 2.5 months revealed a negligible change in their rheological properties. In contrast to a time sensitive ink, MDP inks offer much more flexibility and the ability for on-demand use, such as what might be necessary in a clinical setting. Taken together, these tests revealed a high level of stability for the supramolecular MDP inks. [00129] Fiber formation tests were performed on 4% K2 and 4% E2 inks paired with 25G, 27G, and 30G needles to determine the finest needle that could be used to form uniform fibers for 3D printing (FIG. 6A and FIG. 6B). 4% K2 inks formed uniform fibers when extruded through 25G, 27G, and 30G needles. 4% E2 inks only formed uniform fibers when extruded through a 25G needle, and fiber “flaring” was evident when extruded from 27G and 30G needles. Thus, 25G needles were used for the remainder of the study to have an equal comparison between the K2 and E2 inks. [00130] 3D printing of MDPs was performed on an Allevi 3 bioprinter. The same print optimization process was completed for the 4% K2, 4% E2, and 3% K2 inks. First, the minimum pressure needed for ink extrusion through a 25G needle was determined by increasing pressure in 0.5 PSI increments until filament flow occurred. Using this pressure, a series of calibration lines at varying printhead speeds was then printed. These lines were designed in a U shape to ensure that initial under or over extrusion did not influence the 34 4863-6540-6909, v.1 width of the lines measured. The width of calibration lines was measured as a function of printhead speed (FIG. 7A). The calibration curves for the three inks followed an exponential decay curve with R2 values of 0.96, 0.79, and 0.81 for 4% K2, 4% E2, and 3% K2, respectively. At 300 mm min^í1, 4% K2, 4% E2, and 3% K2 had an average line width of 543, 426, and 590 µm, respectively. A filament extruded from a 25G needle has an ideal height and width of 250 µm, or the inner diameter of the needle. Gravity, without being bound by theory, deforms a printed fiber and leads to one with a smaller height than width (Ouyang et al., 2016). Because of this, MDP filaments were not observed to have widths equal to 250 ^P^^LQVWHDG^^ILEHUV^IUDFWXUHG^DW^KLJK^SULQW^VSHHGV^^$^SULQW^VSHHG^RI^^^^^PP^PLQí^^ZDV^XVHG^ for the remainder of the study, as this was the first point on each calibration curve where the data started to form an asymptote and had heights approximately equal to 250 µm; instead, fibers fractured at high print speeds. An “overhang test” was performed as previously described (Ribeiro et al., 2017) to demonstrate the printability of all three MDP inks. The overhang test structure was designed with overhang lengths of 1, 2, 4, 8, and 16 mm and itself was 3D printed on a Form 3 printer. Using optimized pressures and print speeds, all three inks were successfully printed over the entire overhang length (FIG. 7B). Minor deflection of the filament was only observed at overhangs of 8 and 16 mm. These represent the first time SAPs have been printed into an overhanging structure with the use of a monoaxial printing strategy. In addition, it shows that noncovalent assemblies can be strong enough to self-support, avoiding the need for covalent crosslinking. [00131] To extend the proof-of-concept test prints, a series of 3D structures with increasing complexity was printed using MDPs. 4% K2 was found to be the best performing ink and therefore was used as the primary ink. G-code was manually written ignoring the thickness of printed filaments, so the X/Y dimensions of printed constructs were expected, without being bound by theory, to be offset from the designed dimensions by the width of the filaments. In addition, the layer height of all constructs was set as 250 µm (the inner diameter of a 25G needle). First, a 10-layer tall cylinder was designed to have a diameter of 6 mm and height of 2.5 mm. Although a cylinder is a relatively simple stacking structure without overhangs, it is one of the most difficult 3D designs that has been successfully printed with SAPs in previous publications (Susapto et al., 2021; Sather et al., 2021). A cylinder was successfully printed with 4% K2 with smooth sides and no observable defects (FIG. 7C). To increase complexity, the log pile design was used, which has been used in multiple 3D printability studies and is difficult to print due to having overhangs at each 35 4863-6540-6909, v.1 layer and internal porosity (Hull et al., 2021; Ouyang et al., 2016). A 10-layer tall log pile was designed to have a length and width of 10 mm, a height of 2.5 mm, and to contain 2 × 2 mm pores. The 2 × 2 log pile was successfully printed with 4% K2 and had all 25 pores unobstructed (FIG. 7D). To assess print quality, the designed pore dimensions were compared to those of the printed construct. Since the G-code contained 2 × 2 mm pores, the expected pore dimensions when accounting for the filament width was 1457 × 1457 µm (2000–543 µm from the 4% K2 calibration curve). The inner 9 pores were measured to have an average length and width of 1528 µm, which represents just a 4.9% error between design and print. [00132] Following this success, a 10-layer tall log pile was designed to have a length and width of 5 mm, a height of 2.5 mm, and to contain 1 × 1 mm pores. The 1 × 1 log pile was printed with 4% K2 and had all 9 inner pores unobstructed (FIG. 7E). The expected pore length and width for this design is 457 × 457 µm (1000–543 µm from the 4% K2 calibration curve). The inner 9 pores were measured to have an average length and width of 595 µm, which represents a 30.1% error between design and print. This high error is attributed, without being bound by theory, to hardware limitations as opposed to a limitation of the MDP ink, as the printer had a slight delay in movement during turns that caused over extrusion and negatively impacted the accuracy of the 1 × 1 log pile. [00133] In addition to printing with 4% K2 alone, a multi-MDP structure was successfully fabricated. A 1 × 1 log pile was printed alternating between 4% K2 and 4% E2 at each layer (FIG. 7F). The pore dimensions were measured separately, with pore width being used to assess K2 print quality and pore length being used to assess E2 print quality. The expected pore width was 457 µm (1000–543 µm from the 4% K2 calibration curve) and the expected pore length was 574 µm (1000–426 µm from the 4% E2 calibration curve). The measured pore width and length were an average of 517 and 644 µm, and represented errors of 13.1% and 12.2%, respectively. Together, there was a 12.7% error in the K2/E2 combined 1 × 1 pore print. [00134] To better visualize that overhangs could be printed within a complex structure, a modified 2 × 2 log pile that alternated layer direction every 2 layers was designed. The structure was successfully printed with the 4% K2 ink and overhangs throughout this structure were clearly visible (FIG. 7G and FIG. 8). 36 4863-6540-6909, v.1 [00135] Attempts to print log pile structures with 4% E2 alone were not as successful as those created with 4% K2 alone or the combined K2/E2 constructs. Although 4% E2 filaments were robust enough to self-support, excessive stacking, such as what is required to print 10-layer log piles, led to sagging of the bulk structure and failed prints (FIG. 9). Because the height of subsequent layers did not match with where previous layers had been deposited, we observed various printing errors. [00136] Structure drying was not observed to be a significant issue, as all the constructs printed were relatively small and had short print times. Still, multiple overhang test lines were left out to dry to observe the potential effects. Within 15 min, the hydrogel fibers were observed drying into straight, rigid fibers (FIG. 10). While interesting, the long-term stability and swelling of MDP constructs in a hydrated environment was more relevant for in vitro use. Thus, all complex hydrogel constructs were incubated in HBSS directly after printing. The successfully printed 2 × 2 and 1 × 1 log piles were stored in HBSS for 24 h and maintained their structure and internal porosity (FIG. 7H). There were no statistically significant changes in the pore dimensions in either the 1 × 1 or 2 × 2 log pile structures for up to 10 days (FIG. 11, FIG. 12). In addition, the constructs could be manipulated with a spatula without causing damage. [00137] The present methods disclose the first use of an SAP to 3D print structures containing overhangs and porosity. Using MDPs, log pile structures were fabricated with pores as small as 600 µm. In addition, the present disclosure allows for production of high quality, multimaterial prints accomplished using SAPs. The ability to print layered structures without any covalent crosslinks, and the stability of these constructs over time in HBSS both serve as a evidence, without being bound by theory, that MDPs are excellent hydrogel candidates for 3D printing. Frequently used crosslinking chemistries can lead to the foreign body response in vivo, so non-covalent self-assembly chemistries such as MDPs may lend to better biocompatibility (Delgado et al., 2015). In addition, the ability to print with multiple MDPs allows for chemical complexity to be coupled with the 3D complexity achieved through printing. Because MDP inks primarily consist of HBSS (>95%), they are inherently cell-friendly in terms of osmolarity and pH. In addition, incubation in cell friendly solutions (such as HBSS) does not negatively affect the structural integrity of 3D printed MDP constructs, showing their viability for long-term in vitro use. 37 4863-6540-6909, v.1 In Vitro Studies [00138] In vitro studies were conducted to assess how the charge of 3D printed MDPs affected C2C12 cells, a myoblast cell line frequently used in studies of skeletal muscle (Gilbert-Honick et al., 2020; Kim et al., 2020a). The ability to 3D print MDPs allowed for the creation of structurally similar, yet oppositely charged 3D constructs with macroscopic porosity. 2-layer tall 1 ǘ 1 log pile patterns were printed with only 4% K2, only 4% E2, and a combination where the first layer was K2 and the second was E2. Cells were then seeded onto the constructs, and cell viability and morphology were analyzed over time. Live/dead staining at 1 day post seeding revealed that cells adhered to all three constructs and that few dead cells were observed across groups (FIG. 13A). After 1 day of culture, K2, E2, and the combined structure supported 97%, 92%, and 95% cell viability, respectively (FIG. 13C). At days 3, 5, and 10, the viabilities remained high for K2 (89%, 77%, 79%), E2 (88%, 74%, 79%), and K2/ E2 (81%, 90%, 95%), and there were no statistically significant changes in viability within inks from day 3 through 10. Although MDP charge did not significantly influence viability, more cells adhered to the cationic K2 MDP compared to the anionic E2 MDP (FIG. 13D). At days 1, 3, and 5, the average number of cells observed was 79, 143, and 377 cells/field on K2 and 12, 24, and 27 cells/field on E2. In addition to higher seeding, K2 led to faster cell proliferation compared to E2. Using the average number of viable cells measured at day 1 and 5, the doubling times on K2 and E2 were calculated to be 1.8 and 3.4 days, respectively. Regardless, both MDPs supported a significantly higher number of cells by day 5 of culture compared to the number observed on day 1, which suggests, without being bound by theory, that the poorly adhesive nature of E2 was not enough to stop proliferation, but only slow it down. Combined K2/E2 constructs shared high cell viability up to 10 days in culture, which confirms, without being bound by theory, that combining these oppositely charged MDPs does not cause cytotoxicity. Cells were qualitatively observed to adopt different morphologies on K2 compared to E2, which was most clearly seen on day 10 of culture (FIG. 13B). Differences in cell spreading were also visible on K2/E2 combined constructs, where the inner K2 region had high confluency of extended cells, whereas the outer E2 regions showed less confluent and more spherical cells (FIG. 13A and FIG. 13B). To quantify this phenomenon, the length of the longest axis of each cell volume was calculated and compared between constructs, which is a metric that has been previously used to quantify cell spreading (Chaudhuri et al., 2016). Cells adhered to K2 were observed to spread significantly more than those adhered to E2 after 10 days in culture, with average 38 4863-6540-6909, v.1 OHQJWKV^ RI^ ^^^ DQG^ ^^^ ^P^^ UHVSHFWLYHO\^ ^),*^^13E). Immunostaining of actin filaments was performed to obtain a higher quality view of cell morphology (FIG. 13F). Myoblasts were observed fusing into myotubes on the K2 gels and extending in multiple directions. In contrast, cells on the E2 constructs were much smaller in size and very few were seen expanding actin filaments at all. Interestingly, cells on the K2/ E2 constructs seemed to, without being bound by theory, adopt a morphology dictated by which gel they were directly on, while also being influenced by the oppositely charged peptide in close proximity. The red dashed lines represent regions where K2 was printed, while the white dashed lines represent where E2 gel was printed over the K2 (FIG. 13F, bottom right). These images are maximum intensity projections through the full thickness of the constructs. The actin filaments of cells within the K2 region appeared spread out and noticeable nuclei clumping was observed. In contrast, cells on the E2 region were primarily spherical, though some actin filaments could be observed protruding outward from these cells. This feature was not observed in the E2 only constructs. Preliminary printing of cell laden MDPs was demonstrated (FIG. 14), but their use as a bioink has not been fully characterized. Materials and Methods Materials [00139] Low loading rink amide MBHA resin and FMOC protected amino acids were purchased from EMD Millipore (Burlington, MA). O-(7- Azabenzotriazol-1-yl)- N,N,Nƍ,Nƍ-tetramethyluronium hexafluorophosphate (HATU) was purchased from P3 BioSystems (Louisville, KY). Dichloromethane (DCM), N,N-dimethylformamide (DMF), acetic anhydride, and diethyl ether were purchased from Thermo Fisher Scientific (Waltham, MA). Dimethyl sulfoxide (DMSO), piperidine, N,Ndiisopropylethylamine (DiEA), trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and anisole were purchased from Sigma-Aldrich (St. Louis, MO). Peptide Synthesis [00140] All peptides were synthesized via solid phase peptide synthesis. For each FMOC deprotection, 25% piperidine in 50% DMF/ 50% DMSO was added to the reaction vessel for 10 min. For each coupling, 4 equivalents of amino acid, 4 equivalents of HATU, and 6 equivalents of DiEA were dissolved in 50% DMF/ 50% DMSO and mixed in the reaction vessel for 20 min. Acetylation of the N-terminus was completed with an excess 39 4863-6540-6909, v.1 of DiEA and acetic anhydride in DCM. Peptides were cleaved from the resin with TFA for 3 h in the presence of Milli-Q water, TIPS, and anisole, which acted as scavengers. TFA was then evaporated with nitrogen, and the crude peptide was triturated in cold diethyl ether. Centrifugation and resuspension of the crude peptide was repeated 3 times to dissolve cleavage scavengers, before the crude peptide was left to dry overnight. Peptide Purification [00141] Crude peptides were dissolved at 10 mg mL^í1 and dialyzed for 4 days against Milli-Q water in 100–500 Dalton Spectra/Por Biotech Cellulose Ester Dialysis Membranes (Spectrum Laboratories Inc. Rancho Dominguez, CA). Peptides were then SDVVHG^ WKURXJK^^^^^^P^FHOOXORVH^DFHWDWH^ VWHULOH^ V\ULQJH^ ILOWHUV^ ^9:5^,QWHUQDWLRQDO^^5DGQRU^^ 3$^^ XQGHU^ VWHULOH^ FRQGLWLRQV^^ IUR]HQ^^ O\RSKLOL]HG^^ DQG^ VWRUHG^ DW^ í^^^ ^&^^ 3HSWLGH^PDVV^ ZDV^ confirmed to be correct using a Bruker AutoFlex Speed MALDI ToF (Bruker Instruments, Billerica, MA). Circular Dichroism [00142] ^^^^/^RI^SHSWLGH^LQ^+%66^^&RUQLQJ^,QF^^^&RUQLQJ^^1<^^DW^^^^PJ^P/^í1 was pipetted into a 0.01 mm cuvette and loaded into a Jasco J-810 spectropolarimeter (JASCO Corporation, Tokyo, Japan). A wavelength scan from 180 to 250 nm was performed for 5 accumulations with a pitch of 0.1 nm and a scanning speed of 50 nm min^í1. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy [00143] ^^^^/^RI^SHSWLGH^LQ^+%66^DW^^^^PJ^P/^í1 was pipetted onto a Golden Gate diamond window within a Nicolet iS20 FT/IR spectrometer (Thermo Scientific, Waltham, MA) and dried with nitrogen. An infrared scan was performed for 30 accumulations at a resolution of 4 cm^í1 and background subtraction was performed on the spectra. Scanning Electron Microscopy [00144] Samples were immersed in a series of ethanol dilutions (30%, 50%, 60%, 70%, 80%, 90%, 2 × 100%) for 10 min each to dehydrate the gels and then dried using a Leica EM CPD300 Critical Point Dryer (Leica Biosystems, Deer Park, IL). Samples were 40 4863-6540-6909, v.1 then transferred to a Denton Desk V Sputter System (Denton Vacuum, Moorestown, NJ) and coated with 10 nm of gold. Samples were then imaged on a Helios NanoLab 660 Scanning Electron Microscope (FEI Company, Hillsboro, OR) at 1 kV and 25 pA. Rheology [00145] Rheology tests were performed on an AR-G2 rheometer (TA Instruments, New Castle, DE) with a 12 mm parallel SODWH^^^^^^^/^RI^SHSWLGH^ZDV^H[WUXGHG^ RQWR^ WKH^ VWDJH^DQG^ WKH^SDUDOOHO^SODWH^ZDV^ ORZHUHG^ WR^D^JDS^RI^^^^^^P^^$Q\^H[FHVV^JHO^ZDV^ VFUDSHG^ DZD\^^ EHIRUH^ WKH^ JDS^ ZDV^ ORZHUHG^ WR^ ^^^^ ^P^ DQG^ RLO^ ZDV^ DSSOLHG^ WR^ SUHYHQW^ evaporation during testing. Before each test, an hour-long equilibration period was performed at 1 rad s^í1 and 1% strain. The parameters for each test were as follows: Frequency sweep: 0.1 to 100 rad s^í1 at 1% strain. Shear sweep: 0.01 to 100 s^í1. Strain sweep: 0.1 to 100% strain at 1 rad s^í1. Shear recovery: A minute each at high (500%), low (1%), high (500%), low (1%) strain at 1 rad s^í1. Temperature sweep: 4 to 39 °C at 5 °C increments, with 5 min at 1 rad s^í1 and 1% strain at each temperature increment. Ink Preparation [00146] Pure peptides were dissolved in HBSS at either 30 or 40 mg mL^í1. Sonication was used to dissolve all the peptide, while centrifugation was used to remove any bubbles. For the 3 wt% K2, 4 wt% K2, and 4 wt% E2 peptide bioinks, Tartrazine, Allura red, and Brilliant green (Sigma-Aldrich, St. Louis, MO) were dissolved in HBSS at 0.1, 0.1, and 0.01 mg mL^í1, respectively, prior to the addition of peptide. After being fully dissolved, the peptide bioinks were drawn into a 3 mL syringe, centrifuged to remove any bubbles, and then loaded into an Allevi plastic syringe (Allevi by 3D Systems, Philadelphia, PA) via a female- to-female syringe coupler. All procedures were done under sterile conditions. 3D Printing [00147] 3D printing was performed on an Allevi 3 (Allevi by 3D Systems, Philadelphia, PA). Repetier-Host (Hot-World GmbH & Co. KG.) was used to manually write G-Code, which was then uploaded to Bioprint Essential (Allevi by 3D Systems, Philadelphia, PA) to be read by the 3D printer. The calibration line print was designed to have three U- shaped patterns, each with a length of 8 mm and width of 4 mm. Only the final line segment of the U was used for measurements to account for any differences in preflow. Each print 41 4863-6540-6909, v.1 contained 3 U shapes, each being printed at 50 mm min^í1 greater than the previous one. The filament collapse test platform STL file was downloaded from a previous publication and contained overhangs of 1, 2, 4, 8, and 16 mm (Ribeiro et al., 2017). The platform was printed using a Form 3+ (Formlabs, Boston, MA) and glued to a standard glass slide. Constructs were imaged using an OMAX 18 MP USB 3.0 Digital Camera (OMAX Microscope) and analyzed with ToupView (ToupTek Photonics, Hangzhou, China). Cell Seeding [00148] C2C12 myoblasts (ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA) supplemented with high glucose (4500 mg L^í1), L-glutamine (4 mm), pyruvate (1 mm), penicillin-streptomycin (1%), and fetal bovine serum (10%). Nunc non-treated 6-well plates (Thermo Fisher Scientific, Waltham, MA) were loaded on the Allevi 3 stage and printed onto directly. Printed gels were briefly submerged in HBSS to prevent evaporation, which was subsequently aspirated off prior to cell seeding. Cells were trypsinized, resuspended in medium, and 100,000 cells were pipetted onto each printed gel. 2 mL of medium was added to each well and changed daily for the duration of the experiment. Live/Dead staining was performed using Calcein AM and Ethidium homodimer (Thermo Fisher Scientific, Waltham, MA) according to manufacturer protocols. For immunostaining, gels were washed with PBS, fixed in 4% paraformaldehyde (Thermo Fisher Scientific, Waltham, MA) for 30 min, washed with PBS, quenched with 100 mm glycine (Thermo Fisher Scientific, Waltham, MA), permeabilized with 0.2% Triton X- 100 (Thermo Fisher Scientific, Waltham, MA) in PBS for 30 min, blocked with 1% BSA (Genetex, Irvine, CA) in 0.2% Triton X in PBS for 1 h, incubated with Alexa Fluor 488 Phalloidin (1:20) (Thermo Fisher Scientific, Waltham, MA) for 1–2 h, washed with PBS, counterstained with Dapi (1:500) (Invitrogen, Carlsbad, CA) for 10 min, washed with PBS and cleared in 88% glycerol for at least 30 min prior to imaging. All imaging was performed on a Nikon A1 Confocal Laser Microscope (Nikon Corporation, Tokyo, Japan) and all LPDJHV^ ZHUH^ ^^^^ ^P^ ]-stacks and shown visually as maximum intensity projections. Quantification for live/dead staining and cell spreading were performed in image J using the “3D objects counter” plugin. Doubling time was calculated using the following formula: [00149] 42 4863-6540-6909, v.1 Statistical Analysis [00150] Data were not preprocessed in any way. All results were graphed as the mean +/- standard deviation aside from FIG. 13E, which contains box and whiskers representing min and max values. Sample sizes were noted in figure captions along with the statistical tests applied. GraphPad Prism 9 was used to carry out analyses. Significance was represented as: *= p < 0.05, **= p < 0.01, ***= p < 0.001, ****= p < 0.0001. B. Example 2 Gelation Bath Composition Effects on MDP Alignment [00151] In some embodiments, formation of aligned hydrogels according to the present invention requires the following steps: 1) creation of an MDP precursor solution which entails dissolving an MDP into Milli-Q water, or a similar solvent that does not cause gelation. 2) extrusion of the MDP precursor solution into a secondary “gelation bath” that triggers MDP gelation. This two-step process results in the formation of a MDP hydrogel that possesses nanofibrillar alignment (FIG. 15). Fabrication conditions, including properties of both the precursor solution and the gelation bath, affect the degree of MDP nanofibrillar alignment, as shown in the Figures and described below. [00152] FIGS. 15- 24 depict polarized light microscopy images, which is an optical method used to analyze if a structure is anisotropic. In the Figures provided herein, illuminated (also termed birefringent) MDP hydrogels possess some degree of nanofibrillar alignment. In contrast, randomly oriented MDP hydrogels do not illuminate under a polarized light microscope. A dye is commonly mixed into MDP precursor solutions throughout this work to better visualize the hydrogels, which results in a colored illumination. The brightness intensity within a polarized light microscopy image can be correlated with the degree of nanofibrillar alignment within MDP hydrogels, and this technique is used as a screening method to analyze the degree of MDP hydrogel alignment that results from the presently disclosed methods. FIGS. 25 – 28 are scanning electron microscopy images, which allow for the direct observation of MDP nanofibers. These images may be used to corroborate observations from polarized light microscopy and to allow for improved understanding of the MDP nanostructure that are formed according to the presently disclosed methods. [00153] To investigate how the gelation bath composition affects MDP nanofibrillar alignment, a cationic MDP K2 (SEQ ID NO: 1) which has been previously characterized as an isotropic hydrogel was used as a model MDP. 3wt% K2 in Milli-Q water 43 4863-6540-6909, v.1 was prepared as a precursor solution and extruded into a series of gelation baths. The effects of buffer identity (HEPES, phosphate buffer, HBSS), buffer strength (10mM, 100mM), and ionic strength or NaCl concentration (0mM, 140mM, 1400mM) of the gelation bath were probed using a combination of polarized light microscopy and a mechanical test that involved attempting to lift the MDP hydrogel out of solution (FIG. 16). A standard 1-^^^/^SLSHWWH^WLS^ was used as the extrusion nozzle to fabricate the MDP hydrogels. Out of the conditions tested, phosphate buffer concentration was found to be the largest contributor to the birefringence of the resulting hydrogel, and therefore had the greatest effect on nanofibrillar alignment of the formed hydrogel. In addition, an increase in buffer strength and ionic strength were found to be positively correlated with the alignment of the resulting MDP hydrogels. Differences in fiber alignment were determined related to the mechanical properties of the gels, with more aligned gels being more robust (FIG. 16 checkmarks). Differences between K2 hydrogels prepared into 10mM and 100mM phosphate buffer gelation baths demonstrated, surprisingly, that phosphate buffer concentration could be used to control hydrogel properties. Other embodiments of the present invention may have the same or different properties under the gelation bath conditions described herein according to the sequence of the MDP or the gelation bath composition. The gelation bath compositions described in these examples may be altered for any of the variables described herein to achieve a desired property, such as degree of alignment. [00154] To confirm that the MDP alignment as well as the mechanical properties were stable over both time and transfer to a secondary solution, 3wt% K2 was again used as a precursor solution and extruded into a series of gelation baths. The resulting aligned K2 hydrogels were transferred to Hank’s Balanced Salt Solution (HBSS), a solution commonly used for cell culture applications that has physiological pH and osmotic strength (FIG. 17). The K2 hydrogels maintained the properties imparted to them during the fabrication process after 2.5 hours of being incubated in HBSS. This confirmed that the aligned MDP hydrogels were stable over time and after being transferred to a new solution. [00155] To get a deeper understanding of how phosphate buffer concentration affected MDP alignment, 3wt% K2 precursor solutions were extruded into 10mM, 30mM, and 50mM phosphate buffer gelation baths, each having 140mM NaCl, and the resulting hydrogels were analyzed via scanning electron microscopy (FIG. 25 – FIG. 28). The scanning electron microscopy images indicate that higher phosphate buffer concentration of the gelation bath was associated with increased alignment of the resulting MDP hydrogel nanofibers. In addition, the higher alignment was associated with more nanofiber bundling. 44 4863-6540-6909, v.1 The altered nanostructure was also evident while handing the hydrogels, as those created in the 10mM phosphate buffer could not be removed from solution without fracturing, whereas the more aligned hydrogel structures that were formed using 30 and 50mM phosphate buffer were able to be lifted from solution without damage. Phosphate buffer concentration is therefore, without being bound by theory, an effective gelation bath variable that can be modified to tune the degree of alignment of a hydrogel formed from a 3wt% K2 precursor solution. The present disclosure therefore provides methods which may be tuned to achieve hydrogels with a desired degree of alignment. Precursor Solution Effects on MDP Alignment [00156] The precursor solution of the present methods may also be utilized to tune MDP nanofibrillar alignment. 3wt% K2 precursor solutions were prepared in either 1mM, 5mM, or 10mM phosphate buffer. These precursor solutions were then used to fabricate aligned MDP hydrogels into a 50mM phosphate buffer, 140mM NaCl gelation bath. As shown in FIG. 18, higher concentration of phosphate buffer in the precursor solution is associated with less birefringence, and therefore less alignment, in the resulting MDP hydrogels. Without being bound by theory, higher phosphate buffer concentration in the precursor solution may facilitate “assembly” of the MDP fibers within the precursor solution, the “assembled” MDP fibers may exhibit less alignment upon extrusion into the gelation bath, and thus the resulting MDP hydrogels are less aligned. Accordingly, in some embodiments the present disclosure provides methods for forming aligned hydrogels from precursor solutions which comprise various concentrations phosphate buffer. [00157] The effect of MDP precursor solution pH on nanofibrillar alignment was also examined. 3wt% K2 precursor solutions were prepared in 10mM phosphate buffer with a pH between 4.7 and 9.0 and extruded into a 100mM phosphate buffer gelation bath with a pH of 7.4 (FIG. 19). Since K2 is cationic, the more acidic the solution it is dissolved in, the less assembled it was observed to be, and vice versa. MDP precursor solutions with pH values of 4.7 and 5.8 led to K2 gels that were birefringent and could be picked up out of solution, whereas those with pH values of 8.0 and 9.0 led to K2 gels that were not birefringent and fractured easily. Other embodiments of the present invention may have the same or different properties under the precursor solution conditions described herein according to the sequence of the MDP. The properties of the precursor solutions used herein may be altered for any of the variables described herein to achieve a desired property, such as degree of alignment. This data further supports, without being bound by theory, that less 45 4863-6540-6909, v.1 assembly of MDP nanofibers in the precursor solution of the present methods is associated with higher alignment in a hydrogel formed according to the presently disclosed methods. Accordingly, in some embodiments the present disclosure provides methods for forming aligned hydrogels from precursor solutions which comprise various pH values. In some embodiments, the optimal precursor solution pH for hydrogel formation may depend on the identity of the peptide. [00158] The effect of MDP concentration within the precursor solution on the alignment of the hydrogel was also examined. 1, 2, 3, and 4wt% K2 precursor solutions in Milli-Q water were prepared and extruded into a 50mM phosphate buffer, 140mM NaCl gelation bath (FIG. 20). It was found that below 3wt%, K2 hydrogels were not birefringent or did not form when extruded into the gelation bath. Above this threshold concentration, MDP hydrogels that formed were birefringent and further, that there was a correlation between precursor solution concentration and the birefringence and robustness of the resulting hydrogel. Accordingly, in some embodiments the present disclosure provides methods for forming aligned hydrogels from precursor solutions which comprise various concentrations of peptide. [00159] The methods disclosed herein may be used to form hydrogels from other MDPs. The anionic MDP E2 (SEQ ID NO: 2) was investigated to probe hydrogel formation in negatively charged MDPs. Precursor solutions that consisted of 2 and 3wt% of K2 and E2 were dissolved in Milli-Q water independently, and extruded into a 50mM phosphate buffer, 140mM NaCl gelation bath (FIG. 21). The hydrogels that formed from 3wt% K2 and E2 were birefringent and therefore possessed nanofibrillar alignment, whereas the one that formed from 2wt% K2 was not birefringent and the one that formed from 2wt% E2 was slightly birefringent. Therefore, the present methods may be used to form aligned hydrogels is generalizable to different MDPs. To interrogate further, the test was repeated with brightfield and polarized light microscopy, alongside an additional 2wt% K2 dissolved in HBSS precursor solution (FIG. 22). Consistent with the data from FIG. 18 and FIG. 19, the hydrogel that formed from the 2wt% K2 in HBSS precursor solution was not birefringent and was therefore unaligned. The 2wt% K2 hydrogel did not possess birefringence and did not form cylindrical fibers, whereas the 2wt% E2 hydrogel did possess slight birefringence and did form cylindrical fibers. Accordingly, in some embodiments the present disclosure provides methods for forming aligned hydrogels from precursor solutions of a variety of MDPs. 46 4863-6540-6909, v.1 [00160] As mentioned previously, the process used to fabricate aligned MDP hydrogels described above involved manually extruding the MDP precursor solution into a gelation bath using a standard 1-^^^/^ SLSHWWH^ WLS^^ ZKLFK^ KDV^ DQ^ LQQHU^ GLDPHWHU^ RI^ DSSUR[LPDWHO\^^^^^P^^7R^GHWHUPLQH^WKH^HIIHFW^^LI^DQ\^^RI^WKH^GLDPHWHU^RI^WKH^WLS^RQ^K\GURJHO^ formation, the same process was performed using standard 20-^^^^/^DQG^^^^-^^^^^/^SLSHWWH^ WLSV^^ZKLFK^SRVVHVV^LQQHU^GLDPHWHUV^RI^DSSUR[LPDWHO\^^^^^P^DQG^^^^^P^^UHVSHFWLYHO\^^),*^^ 23). The MDP hydrogels resulting from all examined pipette tips possess birefringence (FIG. 23) and have similar looking nanofibrous organization and alignment (FIG. 25, FIG. 26, FIG. 28). Accordingly, in some embodiments the present disclosure provides methods for forming aligned hydrogels by extruding a precursor solution through any diameter of needle. [00161] The presently disclosed hydrogels may also be extruded using an extrusion 3D printer. Use of an extrusion 3D printer also provides, according to the current methods, formation of multi-layer aligned hydrogels. The layers of the multi-layer aligned hydrogels may have the same degree of alignment or different degrees of alignment. 3D printing methods, including some embodiments of the present disclosure, may comprise use of a support bath for temporarily stabilizing soft structures during the printing process (Hinton et al., 2015). In some embodiments, gelation baths of the present methods may be combined with support baths to form combination gelation-support baths. For example, gelation-support baths were formed using a 50mM phosphate buffer, 140mM NaCl gelation bath in combination with gelatin microparticles (Fresh 1.0), Pluronic F-127, and 300,000 M.W. Poly(ethylene oxide) (PEO). A 3wt% K2 precursor solution was formed, and calibration lines were printed with the resulting precursor solution into various gelation- support baths (FIG. 24) using an Allevi 3 extrusion 3D printer. The printed hydrogels were aligned as demonstrated by their birefringence. Accordingly, in some embodiments the present disclosure provides methods for 3D printing precursor solutions, optionally using gelation-support baths as described above, to form aligned hydrogels. Materials and Methods Peptide synthesis and purification [00162] Peptides were synthesized via solid phase peptide synthesis and purified via dialysis following previously published procedures (Farsheed et al., 2023). Briefly, deprotections were performed with 25% piperidine for 10 mins and couplings were performed with 4 equivalents of amino acids, 4 equivalents of HATU, and 6 equivalents of 47 4863-6540-6909, v.1 DiEA for 20 mins. Peptides were acetylated before being cleaved from resin with TFA. Following evaporating the TFA, peptides were triturated with cold diethyl ether, before being left overnight to dry. Peptides were then redissolved and dialyzed against Milli-Q water for 4 days, before being sterile filtered, frozen, and lyophilized. Polarized Light Microscopy [00163] Polarized light microscopy was performed using a Nikon Eclipse E400 microscope (Nikon Corporation, Tokyo, Japan) and a Nikon D7000 camera was used to take all images. Images were transferred to Adobe Illustrator (Adobe Inc, San Jose, CA) for the addition of scale bars. Scanning Electron Microscopy [00164] Samples were dehydrated using a series of ethanol dilutions then transferred to a Leica EM CPD300 Critical Point Dryer (Leica Biosystems, Deer Park, IL). Following this process, 5nm of gold were coated onto samples using a Denton Desk V Sputter System (Denton Vacuum, Moorestown, NJ). Imaging was performed on a Helios NanoLab 660 Scanning Electron Microscope (FEI Company, Hillsboro, OR) with settings between 1 – 2 kV and 25 pA. Peptide Precursor Solution Preparation [00165] Allure Red AC, Brilliant Green, or Crystal Violet (Sigma-Aldrich, St. Louis, MO) were dissolved in Milli-Q at 0.1 wt% prior to the addition of peptide. Then, lyophilized peptides were added to this solution at the appropriate concentration, followed by repeating cycles of sonication and centrifugation until fully dissolved. When testing the effect of precursor solution pH or ionic strength on peptide nanofibrillar alignment, other buffering solutions were used in place of Milli-Q water. Gelation Solution Preparation [00166] Gelation baths were prepared by dissolving the appropriate components in Milli-Q water. To demonstrate that a range of gelation-support baths compatible with 3D printing could be used, 175 g Bloom, Type A Gelatin from porcine skin (Sigma Aldrich, St. Louis, MO), Pluronic F-127 (Sigma Aldrich, St. Louis, MO), or 300,000 M.W. Poly(ethylene oxide) (Thermo Scientific Chemicals, Waltham, MA) were used. For 48 4863-6540-6909, v.1 gelatin support baths, the procedure published by Hinton et al. was followed (Hinton et al., 2015). Briefly, gelatin was dissolved at 4.5wt% in 150mL of gelation bath solution overnight at 4C. The following day, 350mL of cold PBS was added and blended at pulse speed for 1 – 3 minutes on an Oster 6812 Core 16-Speed Blender (Sunbeam-Oster co, Fort Lauderdale, FL). The gelatin slurry was then loaded into 50mL conical vials, centrifuged, and resuspended until there were no bubbles. For Pluronic support baths, cold gelation bath solution was added to Pluronic to a concentration of 23 wt% and allowed to dissolve overnight on a shaker at 4C. For PEO support baths, gelation bath solution was added to PEO to a concentration of 8 wt% and allowed to dissolved overnight on a shaker at room temperature. Hydrogel Fabrication and 3D Printing [00167] Aligned MDP hydrogels were manually fabricated by pipetting MDP precursor solutions through standard pipette tips into various gelation baths. Specifically, VPDOO^ ^^^^^P^^^PHGLXP^ ^^^^^P^^^ DQG^ ODUJH^ ^^^^^P^^pipette tips were used as nozzles for extrusion. 3D printing with MDP precursor solutions into gelation-support baths was performed on an Allevi 3 (Allevi by 3D Systems, Philadelphia, PA). Before printing, MDP precursor solutions were drawn into a 3mL syringe, capped, centrifuged to remove bubbles, and transferred to an Allevi plastic syringe. G-code for printing was manually written using Repetier-Host (Hot-World GmbH & Co. KG.). Cell Work [00168] C2C12 myoblasts (ATCC, Manassas, VA) were cultured in DMEM, high glucose, pyruvate (Thermo Fisher Scientific) supplemented with 1% penicillin- streptomycin and 10% Fetal Bovine Serum. For cell seeding experiments, aligned MDP hydrogels were fabricated in µ-Slide 8 Well High Bioinert chambers (Ibidi GmbH, Grafelfing, Germany). Cells were then trypsinized, resuspended, and pipette onto the aligned MDP hydrogels. Cell media was replaced every 2 days prior to imaging. Calcein AM and Ethidium homodimer (Thermo Fisher Scientific, Waltham, MA) staining was used to assess cell viability following manufacturer protocols. Immunostaining was performed following a previously published procedure (Farsheed et al., 2023). Briefly, this process consisted of fixation, quenching, permeabilization, blocking, staining with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific, Waltham, MA), staining with Dapi, and clearing in glycerol. Imaging was either performed on a Nikon A1 Confocal Laser Microscope (Nikon 49 4863-6540-6909, v.1 Corporation, Tokyo, Japan) or a Zeiss LSM 800 (Zeiss Group, Oberkochen, Germany). Presented images are maximum intensity projections of acquired z-stacks. C. Example 3 - Investigation of Alignment Parameters [00169] In some embodiments, the precursor solution’s MDP concentration influences the properties of the resulting hydrogel. Using a standard 100-^^^^^^/^SLSHWWH^WLS^ and a phosphate-buffered saline (PBS) gelation bath, polarized light microscopy revealed that no hydrogel formed using a 1wt% K2 precursor solution and that an unstable amorphous hydrogel with slight birefringence formed using a 2wt% K2 precursor solution (FIG. 29A, FIG. 29B). In contrast, use of 3 or 4wt% K2 precursor solutions resulted in cylindrical, birefringent hydrogels (FIG. 29C, FIG. 29D). These data suggest, without being bound by theory, that K2 must be above a concentration threshold to form a robust, birefringent hydrogel, which is consistent with previously reported liquid crystalline behavior in other self-assembling peptide systems (Yuan et al., 2019). [00170] While performing this study using a 3wt% K2 precursor solution, an unexpected increase in viscosity over time was observed upon storage at 4°C (FIG. 30A). Cryogenic transmission electron microscopy (Cryo-TEM) revealed that K2 nanofiber density increased after 1 day at 4°C, suggesting that the supramolecular equilibration in this system is on the order of days (FIG. 30D). Further supporting these findings, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) showed an increase in the amide I band at 1616 cm^-1^^FRUUHVSRQGLQJ^WR^& 2^VWUHWFKLQJ^ZLWKLQ^WKH^ȕ-sheet (FIG. 30B). In addition, dynamic light scattering (DLS) showed a significant increase in particle size between day 0 and day 1 (FIG. 30C). Further analysis of the 3wt% K2 precursor solution at day 21 revealed that an equilibrium had been reached by day 7 (FIGS. 24A-C). Therefore, 3wt% K2 that had been allowed to equilibrate at 4° C for >7 days as the precursor solution as used in the rest of this Example. [00171] Next, the effect of gelation bath composition on the properties of the resulting hydrogel was examined. Using a standard 0.1-^^^^/^SLSHWWH^WLS^WR^VFUHHQ^D^VHULHV^RI^ pH 7 gelation baths, polarized light microscopy revealed a positive correlation between ionic strength and hydrogel birefringence (FIG. 31). In addition, by manually lifting hydrogels from solution and observing if they fractured, a positive correlation between phosphate buffer concentration and hydrogel strength was found (FIG. 31 checkmarks). Further implicating the importance of gelation bath composition, the precursor solution extrusion rate did not affect the observed birefringence pattern within the same gelation bath (FIG. 32). 50 4863-6540-6909, v.1 [00172] Differences between the 10 mM phosphate buffer/ 140 mM NaCl gelation bath, henceforth referred to as 1X PB, and the 50 mM phosphate buffer/ 140 mM NaCl gelation bath, henceforth referred to as 5X PB, warranted further investigation due to observed differences in hydrogel birefringence, strength, and similarity in composition to phosphate-buffered saline (PBS) commonly used in cell culture. 3X PB (30 mM phosphate buffer/ 140 mM NaCl, pH 7) and 10X PB (100 mM phosphate buffer/ 140 mM NaCl, pH 7) gelation baths were also tested in order to analyze a spectrum of phosphate buffer concentrations. In addition, a “pre-gelled” 3wt% K2 unaligned control was included, in which 3wt% K2 was dissolved in Hank’s Balanced Salt Solution (HBSS) to form a hydrogel (Farsheed et al., 2023) and then extruded into 1X PB. Polarized light microscopy revealed a positive correlation between PB concentration and hydrogel birefringence at multiple extrusion diameters, although minor differences between groups were difficult to discern (FIG. 33). Scanning electron microscopy (SEM) and 2D small-angle x-ray scattering (2D SAXS) were used to carry out a more robust analysis of hydrogels extruded through a standard 100-^^^^^ ^/^ SLSHWWH^ WLS^ ^),*^^ 34). Regardless of alignment parameters, an expansive network of nanofibers was observed, while phosphate buffer concentration was qualitatively found to positively correlate with an increase in fiber alignment and packing (FIG. 34B, FIG. 34C), consistent with earlier experiments (FIGS. 25-28). In addition, close observation revealed an increase in nanofiber bundling as a function of PB concentration (FIG. 34C). 2D SAXS quantitatively confirmed the trends observed via PLM and SEM, as the full width at half maximum (FWHM) of the peak within the azimuthal distribution decreased from 71° to 61° to 53° between 1X, 3X, and 5X PB hydrogels, respectively (FIG. 34D, FIG. 34E). Thus, the present invention discloses a self-assembling peptide system by which modest changes in phosphate buffer can be used to tune hierarchical nanofibrillar alignment. [00173] The bulk geometry of hydrogels formed according to the current methods is affected by the composition of the gelation bath used in the method. It was expected that hydrogel diameters would match the inner diameters of the tips used for H[WUXVLRQ^^^^^^DQG^^^^^^P^IRU^^^^-1000 and 0.1-^^^^/^SLSHWWH^WLSV^^UHVSHFWLYHO\^^^7KLV^ZDV^ true for pre-JHOOHG^ K\GURJHOV^ ^^^^^ DQG^ ^^^^ ^P^^^ EXW^ WKH^ ^;^^ 3X, and 5X PB hydrogels followed an unexpected trend (FIG. 35): 1X PB hydrogels were significantly larger (1156 and ^^^^ ^P^^^ ^;^ 3%^ K\GURJHOV^ ZHUH^ VLPLODU^ ^^^^^ DQG^ ^^^^ ^P^^^ DQG^ ^;^ 3%^ K\GURJHOV^ ZHUH^ VLJQLILFDQWO\^VPDOOHU^^^^^^DQG^^^^^^P^^ZKHQ^FRPSDUHG^WR^SUH-gelled hydrogels. 51 4863-6540-6909, v.1 [00174] These data help elucidate a diffusion-mediated mechanism underlying the positive correlation between PB concentration and K2 nanofibrillar alignment. Specifically, it helps in understanding what occurs between when the liquid K2 extrudate leaves the pipette tip and when ions within the gelation bath physically crosslink enough K2 nanofibers to initiate bulk gelation. Without being bound by any particular theory, there are at least two competing diffusive interactions within the liquid extrudate during this time: 1) the outward diffusion of high-concentration K2 monomers toward the gelation bath; 2) the outward diffusion of water molecules toward the high-ionic strength gelation bath (dehydration). Macroscopically, the former causes an increase in hydrogel diameter whereas the latter causes a decrease in hydrogel diameter. Thus, as PB concentration increases, the relative rate of dehydration increases, which explains why hydrogels prepared in 1X (outward diffusion of K2 monomers > outward diffusion of water), 3X (outward diffusion of K2 monomers = outward diffusion of water), and 5X PB (outward diffusion of K2 monomers < outward diffusion of water) gelation baths have declining diameters. In addition, again without being bound by theory, this mechanism is consistent with a diameter of 5X PB hydrogels that is smaller than the pipette tip inner diameter. Considering the possible explanations regarding the observed differences in nanoscale geometry PB concentration leads to, the greater water diffusion compared to diffusion of MDP monomers, the higher nanofiber bundling and alignment is expected. This is in agreement, again without being bound by theory, with the mechanism for thermally-mediated peptide amphiphile bundling and alignment, (Zhang et al., 2010) which also implicates dehydration as the primary driver. Thus, although the shear forces present during extrusion are consistent regardless of PB concentration, higher alignment is achieved for higher PB concentrations. In total, the present disclosure provides a self-assembly pathway to tune the macroscopic alignment of self- assembling peptide nanofibers. Mechanical Characterization of Aligned Hydrogels [00175] In some embodiments, differences in fibrillar nanostructure affect the mechanical properties of MDP hydrogels. It was hypothesized, without being bound by theory, that greater nanofibrillar alignment and packing would correspond to stiffer MDP hydrogels. As MDP hydrogels are lifted from their gelation bath, hydrophilic cohesive forces counteract the hydrogel exiting the solution, and a downward tensile force is applied. This phenomenon was utilized to qualitatively compare the strength of K2 hydrogels fabricated into different gelation baths. Pre-gelled (see above) and 1X PB hydrogels cannot be removed from solution without fracturing, whereas 5X PB hydrogels (of large and small diameter) can 52 4863-6540-6909, v.1 be fully removed without fracture. These results were surprising, as only covalently crosslinked K2 hydrogels had previously been found to be strong enough to withstand be removed from solution (Li et al., 2017). In addition to being anisotropic and hydrated, the mechanical property regime of K2 hydrogels makes them challenging to characterize through most standard mechanical testing means. As a result, optical coherence elastography (OCE) (Larin et al., 2017), in which a 1 kHz non-contact microtap excitation was applied to one end of the cylindrical hydrogels, was employed, with the elastic wave speed monitored as it propagated along the direction of nanofibrillar alignment (FIGS. 36A-36D, FIG. 37). Using this method, K2 hydration could be maintained and quantitative differences in stiffness determined between samples. The average wave speed through pre-gelled unaligned hydrogels was measured to be 0.80 m/s (FIG. 36A), which was not significantly different from 1X PB hydrogels, which had an average wave speed of 0.71 m/s (FIG. 36B). In contrast, the average wave speeds through 3X and 5X PB hydrogels were computed to be 1.9 (FIG. 36C) and 5.5 m/s (FIG. 36D), respectively. Together, these data show a positive correlation between PB concentration and wave speed along the direction of nanofibrillar alignment (FIG. 36F). Assuming a constant density and using the Scholte wave model, the Young’s Moduli were calculated to be 2.0, 1.5, 11, and 99 kPa for the pre-gelled, 1X, 3X, and 5X PB hydrogels, respectively. [00176] 5X PB hydrogels were also imaged via Brillouin microscopy, which allows for an understanding of a material’s bulk modulus at a given location (Prevedel et al., 2019). The technique was used to understand how the mechanical properties of presently disclosed aligned hydrogels vary radially approaching the neutral axis (FIG. 36E). The Brillouin shift was found to increase as a function of the Z-axis (FIG. 36E, FIG. 36G and FIG. 38), which suggests, without being bound by theory, that the fiber alignment observed with SEM (FIG. 34B-iv) persists to the core. In addition, it agrees with the understanding, again without being bound by theory, that shear forces during extrusion are highest along the neutral axis. Robust characterization of aligned K2 hydrogels using OCE and Brillouin microscopy is consistent, without being bound, with higher nanofibrillar alignment corresponding with stiffer hierarchical matrices. Cellular Spreading on Aligned Hydrogels [00177] The present disclosure also provides for, in some embodiments, aligned hydrogels wherein cells recognize and align along the direction of K2 nanofibrillar alignment, with higher scaffold alignment better directing cellular alignment. Porcine 53 4863-6540-6909, v.1 valvular interstitial cells (VICs), which lie in aortic valve leaflets and are primarily responsible for secreting ECM, are known to respond to directional cues (Taylor et al., 2003; Puperi et al., 2015; Rutkovskiy et al., 2017). Porcine VICs were seeded them onto pre-gelled, 1X, 3X, and 5X PB hydrogels and observed high cellular viability for up to seven days (FIG. 39). One day after seeding, confocal microscopy qualitatively revealed that VICs on 1X, 3X, and 5X PB hydrogels aligned along the direction of K2 nanofibrillar alignment, unlike the pre-gelled control (FIG. 40A). Quantification of immunofluoresenctly labelled actin filaments (FIG. 40D-i) and cell nuclei (FIG. 40D-ii) corroborated these observations (see methods section below for additional details). VICs maintained this alignment trend at day 3 (FIG. 40B, FIG. 40E), and by day 7, VICs on 1X and 3X PB hydrogels directionally grew to confluency (FIG. 40C, FIG. 40F). Further, spreading patterns of VICs on 1X PB hydrogels at day 3 (FIG. 40G) and day 7 (FIG. 40H) show that these scaffolds promote macroscopic cellular alignment. Surprisingly, VICs on 5X PB hydrogels aligned no better than those on the pre-gelled control at day 7 (FIG. 40C-iv, FIG. 40F). These results suggested, without being bound by theory, that greater nanofibrillar alignment had no benefit, and further, that matrices with extremely high alignments were not able to direct cell spreading. [00178] To corroborate these striking findings, this study was repeated using C2C12 cells, a murine myoblast cell line commonly used for skeletal muscle tissue engineering, which have also been shown to directionally align in response to mechanical cues (Nakayama et al., 2019; Liu et al., 2022; Luo et al., 2022). K2 hydrogels again supported high viability (FIG. 41) and at day 1 after seeding, myoblasts aligned along the direction of K2 nanofibrillar alignment on 1X, 3X, and 5X PB hydrogels (FIG. 42A, FIG. 42D). While C2C12 cells on 1X and 3X PB hydrogels maintained this alignment trend at day 3, cells on 5X PB hydrogels instead exhibited similar spreading patterns to those on pre- gelled unaligned controls (FIG. 42B, FIG. 42E). By day 7, myoblasts on 1X and 3X PB hydrogels directionally grew to confluency (FIG. 42C, FIG. 42F) and staining of myosin heavy chain at day 14 revealed that some myoblasts spontaneously differentiated into aligned myotubes on 1X and 3X PB hydrogels (FIG. 43B, FIG. 43C). In contrast, myoblasts on 5X PB hydrogels spread orthogonally to the direction of K2 nanofibers (that is, circumferentially around the hydrogel fibers) (FIG. 42C-iv, FIG. 42F-ii) and aligned myotubes were not observed at day 14 (FIG. 43D). Together, this data shows that MDP hydrogels prepared under different conditions lead to differences in their degrees of alignment, and that these differences in nanofibrillar alignment are recognized by cells and lead to differing cell spreading patterns. Regardless of MDP alignment, minimal evidence of cell death was 54 4863-6540-6909, v.1 observed. Accordingly, the present methods provide for the directed growth, such as longitudinal growth or circumferential growth, of cells along the nanofibers of aligned hydrogels prepared according to the presently disclosed methods. [00179] To further verify these results, VIC and C2C12 seeding studies were repeated onto K2 hydrogel scaffolds with half the diameter, supposing that hydrogel curvature may be affecting cellular growth. However, the cellular spreading patterns matched the previously observed trend (FIG. 44. FIG. 45). Finally, it was examined whether hydrogel swelling could account for these results, but no differences in diameter were observed for 5X PB hydrogels when incubated in cell culture media for up to a week (FIG. 46). Thus, cells on 1X and 3X PB hydrogels elongated in the direction of nanofibrillar alignment, but higher scaffold anisotropy led to no observable improvements in directing cells spreading, and the highly aligned 5X PB hydrogels did not promote cellular alignment. [00180] Subsequently, cellular spreading patterns were imaged via SEM to directly visualize how cells were interacting with the K2 matrices of varying alignments (FIG. 47). Myoblasts and VICs were observed pulling on and becoming entangled in the 1X PB matrix (FIG. 47A). In addition, both cell types were seen interacting with the 3X PB matrix similarly (FIG. 47B), which was evident due to differences in matrix alignment directly next to, versus far from, cells (FIG. 47B-ii). Cells on the 5X PB matrix had drastically different appearances and were not seen pulling on or disrupting the highly aligned matrix (FIG. 47C). Instead, cells appeared to be spreading as if they were on 2D surface and astoundingly were even found to be spreading orthogonal to the direction of K2 nanofibrillar alignment (FIG. 47C-ii). [00181] Without being bound by any particular theory, direct observation of cell-matrix interactions allows speculation on 1) why cells on 1X and 3X PB K2 hydrogels align similarly and 2) why cells on 5X PB K2 hydrogels do not align along K2 alignment. Because K2 nanofibers are <10 nm in diameter (FIG. 30D), cells are not observed interacting with individual fibers, but rather with the fibrous network. Cells are seen pulling on the 1X and 3X PB matrices similarly (FIG. 47A, FIG. 47B), and differences in alignment and packing do not seem to impede this cellular behavior. Without being bound by theory, this pulling may be a visual representation of mechanical coupling between the cell body and the underlying matrix and is what allows cells to sense and elongate along scaffold anisotropy. Thus, still without being bound by theory, differences in alignment and packing between 1X and 3X PB matrices do not lead to significant differences in cells being able to mechanically couple with the K2 nanofibrous network, and therefore cells can sufficiently align on both 55 4863-6540-6909, v.1 matrices. In contrast, without being bound by theory, K2 nanofibrous packing and bulk stiffness are too high for cells to effectively pull at and mechanically couple with the fibrous network of the 5X PB matrix, and therefore they cannot understand the mechanical alignment cues. Together, the present disclosure adds more evidence towards the previously proposed “fibre recruitment” mechanism (Baker et al., 2015). Methods Peptide Synthesis and Purification [00182] FMOC protected amino acids and Low loading rink amide MBHA resin were purchased from EMD Millipore (Burlington, MA). O-(7-Azabenzotriazol-1-yl)- 1^1^1ƍ^1ƍ-tetramethyluronium hexafluorophosphate (HATU) was purchased from P3 BioSystems (Louisville, KY). N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), piperidine, N,N-diisopropylethylamine (DiEA), acetic anhydride, trifluoroacetic acid (TFA), and diethyl ether were purchased from Fisher Scientific (Pittsburgh, PA). Piperidine, triisopropylsilane (TIPS), and anisole were purchased from Millipore Sigma (Burlington, MA). [00183] K2 (full sequence: KKSLSLSLSLSLSLKK) (SEQ ID NO: 1) was manually synthesized via solid phase peptide synthesis. Each coupling followed the same process: 2, 5-minute deprotections using 25% piperidine in DMF; 5, 30-second DMF washes; a ninhydrin test to confirm successful deprotection; a 20-minute coupling using 4 equivalents of an amino acid and 4 equivalents of HATU dissolved in 50% DMF/ 50% DMSO with 6 equivalents of DiEA; 2, 1-minute DCM washes; 2, 1-minute DMF washes; a ninhydrin test to confirm successful coupling. After coupling the last amino acid, one last deprotection was performed before acetylation of the N-terminus via 2, 45-minute couplings using an excess of DiEA and acetic anhydride in DCM. Following 3, 1-minute DCM washes, a ninhydrin test was used to confirm successful coupling. Peptide cleavage was performed for 3 hours using TFA with Milli-Q water, TIPS, and Anisole in excess as cleavage scavengers. TFA was then evaporated off using Nitrogen gas, followed by trituration of the peptide in cold diethyl ether. Three cycles of centrifugation (10 minutes at 3400 RCF) followed by decanting of the supernatant were used to isolate the crude peptide. After allowing excess diethyl ether to evaporate overnight, the crude peptide was dissolved in Milli-Q water (0.5 – 1 wt%) and dialyzed against Milli-Q water for 4 days in 100–500 Dalton Spectra/Por Biotech Cellulose Ester Dialysis Membranes (Spectrum Laboratories Inc. Rancho Dominguez, CA). Next, the 56 4863-6540-6909, v.1 peptide solutions were pH adjusted to 7 and sterile filtered using 0.2 µm cellulose acetate sterile syringe filters (VWR International, Radnor, PA) under sterile conditions. Finally, peptides were frozen overnight at -80° C and lyophilized using a FreeZone 4.5 Liter Cascade Benchtop Freeze Dry System (Labconco Corporation, Kansas City, MO) for 3 days, before being transferred to a -20° C freezer for long term storage. Peptide synthesis was confirmed (FIG. 2, FIG. 3) using a Bruker AutoFlex Speed MALDI ToF (Bruker Instruments, Billerica, MA). Phosphate Buffer Gelation Baths [00184] For each gelation bath, the masses of monosodium phosphate monohydrate and disodium phosphate heptahydrate were calculated to achieve the desired phosphate buffer concentration at pH 7. The buffer components and NaCl were then dissolved in Milli-Q water and sterile filtered using 0.2 µm cellulose acetate sterile syringe filters (VWR International, Radnor, PA) under sterile conditions. Polarized Light Microscopy (PLM) [00185] PLM was performed on an Eclipse E400 (Nikon Corporation, Tokyo, Japan) equipped with cross polarizers. Images were captured on a mounted D7000 Digital Camera (Nikon Corporation, Tokyo, Japan) using a fixed exposure time and ISO. Samples were imaged on standard glass microscopy slides (Fisher Scientific, Pittsburgh, PA) at a consistent angle. Rheology [00186] Rheology was performed using an AR-G2 rheometer (TA Instruments, New Castle, DE) equipped with a 12 mm parallel plate. 100 µL of 3% K2 in Milli-Q water was added to the stage and the plate was lowered to a gap of 500 µm. Excess solution was removed with a spatula and mineral oil was dripped around the stage to prevent dehydration of the sample during testing. Viscosity measurements were recorded during a shear sweep from 0.1 to 10 s-1. Cryo-Transmission Electron Microscopy (Cryo-TEM) [00187] Cryo-TEM was performed using an FEI Tecnai F20 (FEI Company, Hillsboro, Oregon) equipped with a K2 summit camera (Gatan Inc, Pleasanton, CA). 3 wt% K2 in Milli-Q water was diluted in Milli-Q water to a final concentration of 0.1 wt% and added to glow discharged Quantifoil CUR 1.2/1.3 400 mesh grids (Electron Microscopy 57 4863-6540-6909, v.1 Sciences, Hatfield, PA). Samples were then frozen using a Vitrobot Mark IV Plunge System (Thermo Fisher Scientific, Waltham, MA) and loaded into a 626 Single tilt liquid nitrogen cryo-transfer holder (Gatan Inc, Pleasanton, CA). Cryo-transmission electron micrographs were captured at 200 kV. Brightness and contrast have been consistently modified to aid in visualization. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) [00188] ATR-FTIR was performed using a Nicolet iS20 FT/IR spectrometer (Thermo Scientific, Waltham, MA) with a Golden Gate diamond window. 10 µL of 3 wt% K2 in Milli-Q water was added onto the window and dried using nitrogen. Spectra consisted of 30 accumulations at a resolution of 4

with background subtraction. To compare relative heights in the amide I band at 1616 cm^í1, spectra were normalized to the TFA peak at 1674 cm^í1. Dynamic Light Scattering (DLS) [00189] DLS was performed using a Malvern Zen 3600 Zetasizer (Malvern Instruments Ltd., Malvern, U.K.). 2 µL of 3 wt% K2 in Milli-Q water was diluted in Milli-Q water to a final concentration of 0.3 wt% and added to a disposable cuvette. Z-average and polydispersity index (PDI) were acquired using the default water parameters for all measurements. Scanning Electron Microscopy (SEM) [00190] SEM was performed using a Helios NanoLab 660 Scanning Electron Microscope (FEI Company, Hillsboro, OR). K2 hydrogel samples were transferred to Porous Spec Pots (Electron Microscopy Sciences, Hatfield, PA) and subjected to a series of ethanol in Milli-Q water dilutions (30%, 50%, 60%, 70%, 80%, 90%, 2 x 100%), each for 10 minutes. Samples that also contained cells were fixed in 4% paraformaldehyde (Thermo Fisher Scientific, Waltham, MA) for 30 minutes prior to the serial dilution process. Next, samples were critical point dried using a Leica EM CPD300 (Leica Biosystems, Deer Park, IL) and coated with 5 nm of gold using a Denton Desk V Sputter System (Denton Vacuum, Moorestown, NJ). Scanning electron micrographs were captured at 1 - 2 kV, 25 pA and were cropped and rotated so the direction of K2 nanofibrillar alignment was horizontal. 2D Small-Angle X-Ray Scattering (2D SAXS) 58 4863-6540-6909, v.1 [00191] 2D SAXS was performed using a Rigaku S-MAX 3000 (Rigaku, Tokyo, Japan) at the University of Houston within the lab of Dr. Megan Robertson. K2 hydrogel samples were placed horizontally onto SpectroMembrane Polyimide 7.5 µm films (Chemplex Industries, Palm City, FL), mounted perpendicular to the x-ray path, and pulled to vacuum. After locating the X/Y location of maximum signal intensity, a 5-minute x-ray exposure was performed on each sample. Following background subtraction, the exposure pattern was integrated over the azimuthal angle using 1° increments, between the Q-range of 0.012 – 0.04 Å-1. Data points corresponding to the following angles were omitted from during plotting as they consistently presented large outliers: 1.5, 44.6, 88.7, 89.7, 90.8, 134.9, 152.9, 153.9, 177.9, 178.9, 180°. Swelling Measurements [00192] After fabrication into their respective gelation baths using standard 100-1000 µL and 0.1-10 µL pipette tips (VWR International, Radnor, PA), K2 hydrogels were transferred to C2C12 growth media within a 12-well plate and stored in a 37° C incubator. Hydrogels were imaged using an OMAX 18 MP USB 3.0 Digital Camera (OMAX Microscope) at each time point and all diameter measurements were performed manually in ImageJ (National Institutes of health, Bethesda, MA). Optical Coherence Elastography (OCE) [00193] Wave excitation was generated using a 7x7x42 mm Piezo Stack Actuator (PiezoDrive, Newcastle, Australia) attached to a needle that was placed in direct contact with the hydrogel surface. Wave propagation was detected using a phase-sensitive Optical Coherence Tomography (PhS - OCT) system with ~9 µm axial resolution (in air), ~8 µm transversal resolution, and 0.28 nm of displacement stability. The A-line acquisition was set to 25 kHz during OCE acquisition. The piezo stack was driven by 5 pulses at 1 KHz, which was generated by a DG4162 Waveform Generator (RIGOL Technologies, Suzhou, China) and amplified by a PDu150 Three Channel 150 V piezo drive (PiezoDrive, Newcastle, Australia). The burst of the signal was synchronized with the OCT frame trigger during the M-B mode scan, to scan 500 points over 2.5 mm laterally with each M-mode scan consisting of 500 A-lines. [00194] Axial particle velocities (^^z) were calculated based on the depth dependent phase (^) difference between two consecutive complex values A-lines, ^z(^, ^) = ¨^(^, ^)^0/(4^ǻ^), using ^ = 1.37 as the refractive index for phosphate buffered saline 59 4863-6540-6909, v.1 gelation baths (Kim et al., 2020b)^^¨^ §^^^^^^ (temporal resolution), and ^0 = 840 ^^ for the central wavelength of the OCT light source. Wave velocities were computed as the slope of the wave propagation on spatiotemporal images. Since K2 hydrogels were immersed in phosphate buffered saline gelation baths, the scan and wave propagation areas were under liquid. Therefore, the Scholte wave model was used to describe the elastic properties of the ^{hydrogels where the shear wave velocity (^s) is related with the Scholte wave speed (^sch) by} _{^sch = 0.846^s (Zevallos-Delgado et al., 2021). The Scholte Young’s modulus (^) was} calculated with the formula: ^ = (3^/0.846²) כ ^sch , assuming a hydrogel density (^) of 1000 kg/m3. All calculations were performed in MATLAB 2020b (Mathworks Inc., Natick, MA). Brillouin Microscopy [00195] The home-built Brillouin microscopy system was based on a two-stage virtually imaged phase array (VIPA) spectrometer. A single-mode 660 nm laser (Torus, Laser Quantum Inc., Fremont, CA) with an incident sample power of ~17 mW was utilized, and a microscope was placed coaxially with the system to align the sample. A 40X water immersion microscope objective with a 0.8 numerical aperture was used to focus the laser EHDP^RQWR^WKH^VDPSOH^^7KH^ODWHUDO^UHVROXWLRQ^ZDV^a^^^^^P^DQG^WKH^D[LDO^UHVROXWLRQ^ZDV^a^^^^ ^P^^%HIRUH^HDFK^H[SHULPHQW^^FDOLEUDWLRQ^ZDV^SHUIRUPHG^XVLQJ^ZDWHU^^DFHWRQH^^DQG^PHWKDQRO^ WR^ DFTXLUH^ WKH^ VSHFWUDO^ SL[HO^ UHVROXWLRQ^ DQG^ WKH^ IUHH^ VSHFWUDO^ UDQJH^^ $^ ^^^^ ^P^ [^ ^^^ ^P^ EULOORXLQ^VFDQ^ZDV^WDNHQ^ZLWK^D^VWHS^VL]H^RI^^^^P^^$Q^HOHFWURQ-multiplying charged coupled device camera (iXon Andor, Belfast, U.K.) with an exposure time of 0.1 s was used to capture the backscattered light from the sample after passing through the VIPA-based spectrometer. A program written in the LabVIEW 20.0.1 Development System (NI, Austin, TX) was used to synchronously control the CCD camera, 3D linear motorized stage, and data acquisition board. The obtained Brillouin spectrum was analysed using a Lorentzian fit to determine the Brillouin frequency shift. During the measurement, the sample was translated in 3D with the prescribed step size. Over a window, the Brillouin spectrum was summed before fitting to enhance the signal-to-noise ratio (Scarcelli et al., 2012). Cell Culture and Seeding [00196] Fresh hearts from male and female young adult porcine (6 - 9 months old) were acquired from a commercial abattoir (Animal Technologies, Tyler, TX) (Balaoing et al., 2014). The aortic valve cusps were dissected, soaked in 2.5% antibiotic/antimycotic solution (ABAM; stock concentration of 10,000 I.U. penicillin, 10,000 ug/mL streptomycin 60 4863-6540-6909, v.1 & 25 ug/mL amphotericin B; Corning, Corning, NY) in phosphate-buffered saline (PBS) and rinsed with PBS. The washing steps were repeated two times before the VICs were harvested following previously described methods (Puperi et al., 2015; Stephens et al., 2007). Briefly, the tissue was first incubated in an enzymatic solution containing 2 mg/mL collagenase II (Worthington Biochemical, Lakewood, NJ) in HyClone Low Glucose Dulbecco’s modified Eagle’s medium (Cytiva, Marlborough, MA) supplemented with 2.5% ABAM for 30 minutes under gentle agitation at 37° C. Next, the cusps were denuded of their endothelium before being minced and placed into an enzymatic mixture of 2 mg/mL collagenase III (Worthington Biochemical, Lakewood, NJ), 0.1 mg/mL hyaluronidase (Worthington Biochemical, Lakewood, NJ) and 2 mg/mL dispase II (STEMCELL Technologies, Vancouver, Canada) for 4 hours under gentle agitation at 37° C. The VICs were isolated from the digested tissue using a 70 µm cell strainer, pelleted at 1500 RCF for 5 minutes, resuspended and grown in 45% HyClone Low Glucose Dulbecco’s modified Eagle’s medium (Cytiva, Marlborough, MA) supplemented with 25 mM HEPES, 44% HyClone Ham’s Nutrient Mixture F12 (Cytiva, Marlborough, MA), 10% Gibco Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA), and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA). Frozen C2C12 myoblasts were purchased from ATCC (Manassas, VA) and were grown in 89% Gibco Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA) supplemented with high glucose, sodium pyruvate, L-glutamine, and phenol red, 10% Gibco Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA), and 1% penicillin- streptomycin (Thermo Fisher Scientific, Waltham, MA). All cells were used between passages 1 and 4. [00197] For cell seeding studies, K2 hydrogels were fabricated under sterile conditions before being transferred to PBS. They were then moved to 8 Well high Bioinert µ- Slides (Ibidi, Gräfelfing, Germany) and placed into an incubator for 10 minutes prior to cell seeding. Cells were passaged to 200,000 cells/mL, PBS was removed from all wells to leave just the K2 hydrogels, and 10,000 cells (100,000 cells for cell viability studies) were added to the bottom of each well. Finally, media was added to each well and the slides were moved back into the incubator. Media was replaced every day for the remainder of the study. Staining + Imaging [00198] Cells on K2 hydrogels were stained with Calcein AM and Ethidium homodimer (Thermo Fisher Scientific, Waltham, MA) following manufacturer protocols for cell viability studies. Imaging was performed using a Zeiss LSM800 Airyscan (Oberkochen, 61 4863-6540-6909, v.1 Germany) and the “3D objects counter” plugin within ImageJ (National Institutes of health, Bethesda, MA) was used to count live and dead cells, before being exported to Microsoft Excel (Microsoft Corporation, Redmond, WA) for viability calculations. [00199] Immunostaining of cells on K2 hydrogels followed the process: 3 PBS washes; 30-minute fixation in 4 wt% paraformaldehyde (Thermo Fisher Scientific, Waltham, MA); 3 PBS washes; 10-minute quenching with 100 mM glycine; 30-minute permeabilization with 0.2% Triton-X (Fisher Scientific, Pittsburgh, PA) in PBS; 1-hour blocking with 1% Bovine Serum Albumin (Genetex, Irvine, CA) / 0.2% Triton-X in PBS; 1- hour staining with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific, Waltham, MA) diluted 1:400 in 1% BSA / 0.2% Triton-X in PBS; 3 PBS washes; 10-minute staining with DAPI (Thermo Fisher Scientific, Waltham, MA) diluted 1:500 in 1% BSA / 0.2% Triton-X in PBS; 3 PBS washes; clearing and storage in 88% Glycerol (Thermo Fisher Scientific, Waltham, MA). For immunostaining of C2C12 cell differentiation, Myosin Heavy Chain (MF 20, DSHB, Iowa City, IA) was diluted 1:10 and used as the primary antibody and Goat Anti-mouse Alexa Fluor Plus 488 (Thermo Fisher Scientific, Waltham, MA) was diluted 1:500 and used as the secondary antibody. All imaging was performed on a Zeiss LSM800 Airyscan (Oberkochen, Germany) and maximum intensity projections of z-stacks were created in ImageJ (National Institutes of health, Bethesda, MA). Collected images were cropped and rotated so the direction of K2 nanofibrillar alignment was always horizontal. Quantification of Actin and Nuclear Alignment [00200] Unprocessed maximum intensity projections of immunofluorescently labelled actin filaments were imported into ImageJ (National Institutes of health, Bethesda, MA) and the plugin OrientationJ46 was used to locally determine the Fourier gradient structure tensor using a 2 pixel local window, a 5% minimum coherency, and a 5% minimum energy. The structure tensor was then imported into MATLAB 2023a (Mathworks Inc., Natick, MA) and the angle corresponding to the maximum value was shifted to be 0°. Next, a polar histogram was generated (which is displayed next to each confocal microscopy image) and the toolbox CircStat47 was used to calculate the mean resultant vector length of the polar distribution, where 0 corresponds to isotropy and 1 to anisotropy, as has been previously published.48 [00201] Unprocessed maximum intensity projections of immunofluoresenctly labelled cell nuclei were imported into ImageJ (National Institutes of health, Bethesda, MA) and the default automatic threshold was applied and converted to a mask. If the automated 62 4863-6540-6909, v.1 process failed, this step was performed manually. Next, the mask was dilated, eroded twice, and a watershed was applied. The Analyze Particle function was then used to find the angle of each ellipse and was exported into MATLAB 2023a (Mathworks Inc., Natick, MA). Finally, the difference between each ellipse angle and angle of K2 nanofibrillar alignment was calculated, similar to previously published methods.13 Statistical Analysis [00202] Prism 10 (GraphPad Software, Boston, MA) was used for all statistical analysis. Specifics about data plotted and statistical tests used are listed in all figure captions. D. Example 4 – 3D Printing Optimization [00203] Multidomain peptide K2 was synthesized via solid phase peptide synthesis and successful synthesis was confirmed via matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (FIG. 2, FIG. 3). 4wt% K2 in Milli-Q water was chosen as the K2 ink formulation due to it leading to anisotropic hydrogels useful for directing cellular growth (Farsheed et al., 2024). Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy of the K2 ink revealed the presence of peaks at 1620 cm-1 and 1695 cm-^^WKDW^FRUUHVSRQG^WR^D^ȕ-sheet secondary structure (FIG. 48A) and cryo-transmission electron microscopy confirmed that this K2 ink contains nanofibers (FIG. 48B). [00204] Next, the viscoelastic properties of the K2 ink were assessed. A shear sweep on the K2 ink revealed a negative slope as a function of shear rate, confirming that the K2 ink is shear thinning (FIG. 48C). In addition, a strain sweep showed that the K2 ink forms a viscoelastic liquid, with storage and loss moduli of 125 and 46 Pa, respectively (FIG. 48D). Together, these data support the characterization of a 4wt% K2 precursor solution as extrusion printable. [00205] Using this peptide ink, a series of support bath candidates were screened. Two support baths that consist of gelatin microparticles (FRESH v1.0 (Hinton et al., 2015) and FRESH v2.0 (Lee et al., 2019)) and a third that consists of agarose (Prendergast et al., 2021) were used. Compared to the K2 ink, both gelatin support baths were more viscous, whereas the agarose support bath was slightly less viscous (FIG. 49A). To assess printability, a 2x2 log pile structure was designed with 2 mm x 2 mm internal pores (FIG. 49B-I). Unoptimized printing into FRESH v1.0 led to hydrogel structures with rough surface morphology (FIG. 49B-II), while those fabricated into agarose were qualitatively 63 4863-6540-6909, v.1 much smoother (FIG. 49B-III). In addition, FRESH v1.0 prints disintegrated upon being liberated, whereas agarose prints maintained structural integrity. Therefore, in some embodiments, gelation support baths comprising agarose as a gelling agent may facilitate the printing of hydrogels with aligned nanofibers. These findings provide evidence that, without being bound by theory, a viscosity match between precursor solution and gelation bath is beneficial for 3D printing of aligned hydrogels formed according to the present disclosure. [00206] Using an agarose support bath, the print parameters to create high fidelity hydrogel structures were optimized. As a first step, a series of optimization lines were designed to increase print speed at each layer (FIG. 50A). Using a 25G needle and extrusion pressure of 7PSI, extruded line width was measured as a function of printhead speed (FIG. 50B). While the inner diameter of a 25G needle is 250um, this optimization yielded lines as thin as 154 µm before lines began to fall apart. Using optimized print conditions, a 2x2 log pile (FIG. 40C-I) and a 1x1 log pile (Figure 40C-II) were then fabricated. To confirm that these hydrogels possessed nanofibrous alignment, scanning electron microscopy was performed following critical point drying. When printing into an agarose support bath supplemented with phosphate-buffered saline (PBS), a moderate amount of fiber alignment is observed (FIG. 50D-I). In contrast, printing into an agarose support bath with five times the phosphate concentration, led to hydrogel constructs with noticeable higher nanofibrous alignment and packing (FIG. 50D-II). Thus, by altering the ionic strengths within similarly prepared agarose support baths, the inventors demonstrate the ability to tune the nanofibrillar alignment of the hydrogels prepared according to the presently disclosed methods. Cell Work [00207] In some embodiments, the aligned macroscopic hydrogels described herein are useful as a template for aligned tissue growth, more particularly skeletal muscle tissue and the C2C12 myoblast cell line. After liberating printed structures, cells were seeded onto the surface of the hydrogels and tissue growth was tracked over time. At day 3 post seeding, cells were observed aligning in the direction of the 3D print, indicated that cells were aligning as expected (FIG. 51A). By day seven, the myoblasts grew to confluency along the print direction (FIG. 51B).Therefore, the present disclosure provides methods and compositions, including hydrogels with aligned nanofibers formed in gelation baths comprising agarose, which facilitate directed aligned spreading of myoblasts. Methods Support Bath Creation 64 4863-6540-6909, v.1 [00208] Creation of FRESH v1.0 baths was adapted from previous literature (Hinton et al., 2015). In brief, powdered porcine gelatin (Type A, Sigma-Aldrich, St. Louis, MO, USA) was dissolved at 4.5 wt% in either 10 or 50 mM phosphate buffered saline and 140 mM NaCl solution. This solution was allowed to gel overnight at 4°C. The gelled solution was then diluted to a 2.7 wt% by addition of the corresponding phosphate buffer solution. Following the dilution, everything was blended on a consumer grade blender (Oster, USA) at “pulse” speed for 2 minutes. The resulting slurry was transferred into 50 mL conical vials and centrifuged at 2000 rcf for 2 minutes. The supernatant was then poured off and replaced with fresh phosphate buffer solution. Then, the slurry was vortexed to resuspend the gelatin microparticles and re centrifuged. This washing was repeated three times, with the supernatant discarded after the final wash. The slurry was then stored at 4°C until use. All FRESH 2.0 support bath was purchased from FluidForm Inc. and prepared according to manufacturer directions. [00209] Agarose support bath preparation was adapted from previous literature (Prendergast et al., 2021). In brief, agarose (Electrophoresis Grade, Sigma-Aldrich, St. Louis, MO, USA) was mixed into either 10 or 50 mM phosphate buffered saline and 140 mM NaCl solution at 0.5 wt%. The resulting suspension was autoclaved at 120°C on the liquid cycle for 1 hour to dissolve the agarose, yielding a clear solution. A stir bar was added, and the solution was sheared at 700 rpm at room temperature overnight. The support bath solution was stored at 4°C until use. 3D Printing [00210] 4 wt% K2 was dissolved in Milli-Q H2O supplemented with 0.01 wt% Allura Red dye to yield the 3D printing ink. After being fully dissolved, the peptide ink was then drawn into a 3 mL syringe and centrifuged to remove bubbles. Then, a female-female syringe coupler was used to transfer the ink from the 3 mL syringe to an Allevi syringe (Allevi by 3D Systems, Philadelphia, PA). All procedures were performed under sterile conditions. [00211] All 3D printing was performed on an Allevi 3 (Allevi by 3D Systems, Philadelphia, PA). G-code was manually written using Repetier-Host (Hot-World GmbH & Co. KG) and was uploaded to Bioprint Essential (Allevi by 3D Systems, Philadelphia, PA). 10 mL of support bath was loaded into each well of a 6 well plate (Corning, Glendale, AZ) which was placed on the stage of the Allevi 3. All log pile structures consisted of 4 layers unless otherwise stated and had crosshatch dimensions of either 2 mm x 2 mm or 1 mm x 1 65 4863-6540-6909, v.1 mm. Constructs were imaged using an OMAX 18 MP CMOS Digital Camera (OMAX Microscope) and analyzed using ToupView (ToupTek Photonics, Hangzhou, China). [00212] FRESH v1.0 and v2.0 support bath prints were liberated by incubating the 6-well plate at 37°C until the gelatin had melted. Agarose support bath prints were liberated by continuously diluting the agarose with Milli-Q water. Specifically, 1 mL of water was pipetted into the support bath then 1 mL of the diluted mixture was removed. This was repeated until the print was freely floating in clear water. 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Claims

WHAT IS CLAIMED: 1. A method for extrusion printing of a hydrogel structure wherein the method comprises: a) forming a precursor solution comprising a multidomain peptide (MDP) comprising a first domain of the formula (X)_m(YZ)_n(X)_m, wherein: X is a positively charged amino acid at pH 7 or a negatively charged amino acid at pH 7; Y is a hydrophilic amino acid and Z is a hydrophobic amino acid or Y is a hydrophobic amino acid and Z is a hydrophilic amino acid m is 1, 2, 3, 4, or 5; and n is 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the precursor solution comprises a solvent that mediates the self- assembly of the MDP; and b) printing a hydrogel structure with the precursor solution.

2. The method of claim 1, wherein X is selected from among lysine, arginine, glutamic acid, or aspartic acid.

3. The method according to either claim 1 or claim 2, wherein the hydrophobic amino acid is selected from among alanine, leucine, glycine, isoleucine, tryptophan, phenylalanine, proline, methionine, and cysteine and the hydrophilic amino acid is selected from among serine, tyrosine, threonine, asparagine, and glutamine.

4. The method according to any one of claims 1-3, wherein the MDP further comprises one or more enzymatic cleavage signaling sequence domain, spacer domain, or bioactive peptide sequence domain.

5. The method according to any one of claims 1-4, wherein the first domain has at least 95% sequence identity with SEQ ID NO: 1.

6. The method according to any one of claims 1-5, wherein the solvent is suitable for cell culture. 70 4863-6540-6909, v.1

7. The method according to any one of claims 1-6, wherein the solvent further comprises cells.

8. The method according to any one of claims 1-7, wherein the precursor solution has less than about a 5% change in storage modulus G` between a temperature of about 4C and about 37C at a strain of about 1% and at a frequency of about 1 rad/s.

9. The method according to any one of claims 1-8, wherein the precursor solution is characterized by a storage modulus G` greater than about 1000 Pa at a strain of about 1% and at a frequency between about 0.01 rad/s and about 100 rad/s.

10. A method of using a hydrogel made according to any one of claims 1-9 wherein the hydrogel contacts one or more cells in vitro.

11. A method of forming a hydrogel with aligned nanofibers comprising: a) obtaining a multidomain peptide (MDP) with a sequence comprising: a first domain of the formula X_m; a second domain of the formula (YZ)n; and a third domain of the formula Xm; wherein: X is a negatively or positively charged amino acid, Y is a hydrophilic amino acid and Z is a hydrophobic amino acid or Y is a hydrophobic amino acid and Z is a hydrophilic amino acid; m is 1, 2, 3, or 4; and n is 2, 3, 4, 5, 6, 7, 8, 9 or 10; b) dissolving the multidomain peptide in a solvent to form a precursor solution; and c) extruding the precursor solution into a gelation bath to form a hydrogel with aligned nanofibers.

12. The method according to claim 11, wherein X is selected from the group consisting of glutamic acid, aspartic acid, arginine, histidine, and lysine and wherein the hydrophobic amino acid is selected from the group consisting of alanine, leucine, 71 4863-6540-6909, v.1 glycine, isoleucine, tryptophan, phenylalanine, proline, methionine, and cysteine, and the hydrophilic amino acid is selected from the group consisting of serine, tyrosine, threonine, asparagine, and glutamine.

13. The method according to any one of claims 11-12, wherein the sequence of the multidomain peptide comprises SEQ ID NO: 1.

14. The method according to any one of claims 11-13, wherein the solvent is water.

15. The method according to any one of claims 11-14, wherein the gelation bath comprises a gelling agent.

16. The method according to any one of claims 11-15, wherein the gelling agent is agarose.

17. The method according to any one of claims 11-16, wherein the gelation bath comprises between 10 mM and 100 mM phosphate buffer and 140 mM NaCl.

18. The method according to any one of claims 11-17, wherein the method further comprises seeding or growing one or more cells on the hydrogel with aligned nanofibers.

19. A pharmaceutical composition comprising: (a) a hydrogel with aligned nanofibers according to any one of claims 11-18; and (b) a therapeutic or prophylactic molecule.

20. A method of treating or preventing a disease, disorder, or injury in a patient in need thereof comprising administering a hydrogel with aligned nanofibers according any one of claims 11-18 or the pharmaceutical composition according to claim 19. 72 4863-6540-6909, v.1