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WO2024233707A1 - Impression 3d d'hydrogels peptidiques à domaines multiples à auto-assemblage et fabrication d'hydrogels peptidiques à auto-assemblage nanofibreux alignés - Google Patents

Impression 3d d'hydrogels peptidiques à domaines multiples à auto-assemblage et fabrication d'hydrogels peptidiques à auto-assemblage nanofibreux alignés Download PDF

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Publication number
WO2024233707A1
WO2024233707A1 PCT/US2024/028436 US2024028436W WO2024233707A1 WO 2024233707 A1 WO2024233707 A1 WO 2024233707A1 US 2024028436 W US2024028436 W US 2024028436W WO 2024233707 A1 WO2024233707 A1 WO 2024233707A1
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Prior art keywords
mdp
hydrogels
hydrogel
amino acid
cells
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Inventor
Jeffrey D. Hartgerink
Adam C. FARSHEED
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William Marsh Rice University
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William Marsh Rice University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • Multidomain Peptides 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 ⁇ -sheets and elongate into nanofibers. Although the assembly mechanism is well understood, control of the directionality of the nanofibers that form remains elusive.
  • 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.
  • 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.
  • the present disclosure relates to methods of printing hydrogel structures with self-assembling multidomain peptides (MDPs).
  • MDPs self-assembling multidomain peptides
  • the present disclosure also relates to methods of forming hydrogels with aligned nanofibers.
  • the present disclosure relates to methods for producing hydrogels with aligned nanofibers.
  • 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.
  • 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.
  • MDP multidomain peptide
  • 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.
  • 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.
  • the MDP further comprises an enzymatic cleavage signaling sequence domain, such as leucine-arginine-glycine.
  • the MDP further comprises a spacer domain.
  • the spacer domain is a sequence of five or fewer independently selected glycine or serine residues.
  • the MDP further comprises a bioactive peptide sequence domain.
  • the bioactive peptide sequence domain has at least 95% sequence identity with SEQ ID NO: 8.
  • the bioactive peptide sequence domain is SEQ ID NO: 8.
  • 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- ⁇ RU ⁇ %03 ⁇
  • 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
  • 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.
  • m is 2, 3, or 4.
  • n 4, 5, 6, 7, or 8.
  • 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.
  • the solvent of the present methods is suitable for cell culture.
  • 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.
  • the solvent is Hank’s Balanced Salt Solution (HBSS).
  • the solvent is water.
  • the solvent further comprises an additive, a crosslinker, a stiffener, a synthetic material, an inorganic salt, a gelling agent, or any combination thereof.
  • the solvent further comprises cells, such as myoblasts.
  • the precursor solution is between 0.1%-10% MDP by weight.
  • 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.
  • 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.
  • the hydrogel structures further comprise layers of a second MDP comprising at least a second domain.
  • the second domain has at least 95% sequence identity with SEQ ID NO: 1.
  • the second 6 4863-6540-6909, v.1 domain is SEQ ID NO: 1.
  • the second domain has at least 95% sequence identity with SEQ ID NO: 2.
  • the second domain is SEQ ID NO: 2.
  • the printing comprises 3D printing.
  • the present methods further comprise contacting the printed hydrogel structure with one or more cells in vitro.
  • the present application discloses hydrogel structures made according to the methods disclosed herein.
  • 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.
  • the presently disclosed methods comprise seeding and growing cells on the hydrogel structure in vitro.
  • the present methods further comprise using the seeded and grown cells in vivo.
  • the cells are administered to animals.
  • the cells are administered to humans.
  • 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.
  • 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.
  • MDP multidomain peptide
  • the sequence of the multidomain peptide comprises a sequence of the formula Xm(YZ)nXm.
  • X is selected from the group consisting of glutamic acid, aspartic acid, arginine, histidine, and lysine.
  • X is selected from the group consisting of lysine, arginine, and histidine.
  • X is selected from the group consisting of glutamic acid and aspartic acid.
  • X is lysine.
  • X is arginine.
  • X is histidine.
  • X is glutamic acid.
  • X is aspartic acid.
  • the hydrophobic amino acid is selected from the group consisting of alanine, leucine, glycine, isoleucine, tryptophan, phenylalanine, proline, methionine, and cysteine
  • the hydrophilic amino acid is selected from the group consisting of serine, tyrosine, threonine, asparagine, and glutamine.
  • Y or Z is serine.
  • Y or Z is leucine.
  • m is 2, 3, or 4.
  • m is 2.
  • n is 6, 7, 8, 9, or 10.
  • n is 6.
  • 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.
  • the multidomain peptide comprises a spacer domain.
  • 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.
  • the multidomain peptide comprises a bioactive peptide sequence domain.
  • 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.
  • 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.
  • the bioactive peptide sequence is derived from fibronectin, vitronectin, laminin, collagen, VEGF, FGFs, PDGF, HGF, TGF- ⁇ and BMP.
  • 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).
  • 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.
  • 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.
  • 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.
  • the gelation bath comprises PBS, HBSS, cell culture medium, or a biological fluid, or any combination thereof.
  • the gelation bath comprises PBS.
  • the gelation bath comprises between about 10 mM Phosphate buffer and about 100 mM Phosphate buffer and about 140 mM NaCl.
  • the gelation bath comprises about 10mM Phosphate buffer and about 140mM NaCl.
  • the gelation bath comprises about 30mM Phosphate buffer and about 140mM NaCl.
  • the gelation bath comprises about 50mM Phosphate buffer and about 140mM NaCl.
  • the precursor solution is extruded into the gelation bath with a 3D printer.
  • the hydrogel is a multilayer structure.
  • the method does not comprise heating the precursor solution.
  • the precursor solution further comprises cells.
  • the method further comprises seeding or growing one or more cells on the hydrogel with aligned nanofibers.
  • 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.
  • the cells grow longitudinally along the aligned nanofibers.
  • the cells grow circumferentially about the aligned nanofibers.
  • 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.
  • the cells are muscle cells.
  • the cells are nerve cells.
  • the cells are fibroblast cells.
  • the cells are endothelial cells.
  • the cells are cardiac cells.
  • the cells are bone cells.
  • the cells are fat cells.
  • the cells are epithelial cells.
  • 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.
  • the hydrogel remains intact at physiological pH.
  • the present disclosure provides pharmaceutical compositions comprising: (a) a hydrogel as provided above; and (b) a therapeutic or prophylactic molecule.
  • the molecule is a protein.
  • the protein is an antibody.
  • 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.
  • the patient is a mammal.
  • the patient is a human.
  • the disease or disorder is a cancer.
  • 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.
  • the patient is a mammal.
  • the patient is a human.
  • the disease or disorder is a muscle injury, such as volumetric muscle loss.
  • the disease or disorder is a nerve injury, such as peripheral nerve transection.
  • FIGS. 1A & 1B show a schematic of MDP assembly and 3D printing.
  • FIGS. 2A-2B show structures of the peptides used in certain embodiments of the present disclosure.
  • 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 ⁇ 1 .
  • Scale bars 5 ⁇ m in FIG. 4C, FIG. 4E and 1 ⁇ m in FIG. 4D and FIG. 4F. 13 4863-6540-6909, v.1 [0052]
  • FIGS. 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 ⁇ 1
  • 5A-5K show the rheology of MDP gels and MDP inks.
  • Frequency sweeps from 0.1 to 100 rad s ⁇ 1 on 1, 2, 3, 4 wt% K2 (FIG. 5A) and E2(FIG. 5E).
  • Shear sweeps from 0.01 to 100 s ⁇ 1 on 4 wt% K2 (FIG. 5B) and E2 (FIG. 5F).
  • White regions represent 1% strain and grey regions represent 500% strain.
  • FIG. 5A-5K show the rheology of MDP gels and MDP inks.
  • 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.
  • FIGS. 7A-7H show MDP 3D printing optimization and printed structures.
  • FIG. 7C Cylinder
  • FIG. 7D 2 ⁇ 2 log pile
  • FIG. 7E 1 ⁇ 1 log pile
  • FIG. 7F multimaterial 1 ⁇ 1 log pile.
  • FIG. 7B Overhang tests for MDP inks, where orange, blue, and yellow correspond to 4%
  • FIG. 7H Top view images of 2 ⁇ 2 log pile and 1 ⁇ 1 log pile after being incubated in HBSS for 1 day.
  • FIG. 10 shows the 4% K2 overhang test (left) before and (right) after drying. [0058] FIG.
  • FIGS. 13A-13F show the in vitro characterization of 3D printed MDP log pile hydrogels with differing charge.
  • FIG. 13A 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. 13A 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 ⁇ LPD
  • FIGS. 14A-14B show MDP bioprinting.
  • FIG. 14A Live/dead staining 1 day after printing within K2 and E2.
  • 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 ⁇
  • FIG. 16 illustrates the effect of gelation bath composition on alignment.
  • 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.
  • 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.
  • FIG. 19 shows the effect of MDP precursor solution pH on alignment.
  • PS Precursor Solution
  • GB Gelation Bath
  • PB 3KRVSKDWH ⁇ %XIIHU ⁇ 6FDOH ⁇ EDU ⁇ ⁇ P ⁇
  • FIG. 20 illustrates the effect of MDP precursor solution concentration on alignment.
  • FIG. 21 shows the effect of MDP precursor solution concentration and sequence on alignment.
  • FIG. 22 illustrates the effect of MDP precursor solution concentration, sequence, and solvent on alignment.
  • 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.
  • FIG. 24 illustrates aligned multidomain peptide (MDP) hydrogels fabricated using a 3D printer in conjunction with various gelation-support baths.
  • MDP MDP
  • Milli-Q water MDP precursor solution (3wt% K2)
  • MDP precursor solution 3wt% K2
  • support baths listed above images
  • 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.
  • 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]
  • FIGS. 29A-29D illustrate a concentration screen of extruded MDP precursor solution. Polarized light microscopy of (FIG. 29A) 1wt%, (FIG.
  • FIGS. 30A-30D relate to equilibration of precursor solutions described herein.
  • FIG. 42A Rheology shear sweep between 0.1 and 10 s-1,
  • FIG. 42A Rheology shear sweep between 0.1 and 10 s-1,
  • FIGS. 31A-31X illustrate the effect of additional gelation bath compositions on alignment. Gelation Bath Composition Screen.
  • 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. 31R 50 mM PB/ 140 mM NaCl.
  • 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.
  • FIGS. 33A-33J show the results of a gelation bath phosphate buffer screen. Polarized light microscopy of (FIG. 33A, FIG. 33F) pregelled, (FIG.
  • FIG. 34A-34E show the tunable alignment of nanofibrous MDP hydrogels.
  • FIG. 34C VFDQQLQJ ⁇ HOHFWURQ ⁇ PLFURJUDSKV ⁇ VFDOH ⁇ EDUV ⁇ ⁇ P ⁇ DQG ⁇ QP ⁇ UHVSHFWLYHO ⁇ ),* ⁇ 34D) 2D small-angle x-ray scattering patterns, and (FIG.
  • 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).
  • FIGS. 36A-36G show the mechanical characterization of aligned MDP hydrogels.
  • FIG. 36A Pre-gelled
  • FIG. 36B 1X PB
  • FIGS. 37A-37D illustrate the stiffness of aligned K2 hydrogels.
  • FIGS. 38A-38C show Brillouin shift profiles for 5X PB Hydrogels.
  • FIGGS. 38A-38C Brillouin frequency shift maps of three 5X PB hydrogels.
  • the color bar to the right indicates the Brillouin shift (GHz).
  • FIGS. 39A-39D show VIC Cell Viability on K2 Hydrogels.
  • FIGG. 39A Day 1,
  • FIGG. 39B Day 3, and
  • FIG. 39A-39D show VIC Cell Viability on K2 Hydrogels.
  • FIG. 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 3 Day 3
  • FIGS 41A-41D show C2C12 Cell Viability on K2 Hydrogels.
  • FIG. 41A Day 1,
  • FIG. 41B Day 3, and
  • FIG. 42A-42F show C2C12 Cell Spreading on K2 Hydrogels.
  • FIG. 42A Day 1,
  • FIG. 42B Day 3
  • 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 3 Day 3
  • 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.
  • FIGS. 44A-44F show VIC Cell Spreading on Small Diameter K2 Hydrogels.
  • FIG. 44A Day 1, (FIG. 44B) Day 3, and (FIG.
  • FIGS. 45A-45F show C2C12 Cell Spreading on Small Diameter K2 Hydrogels.
  • FIG. 45A Day 1,
  • FIG. 45B Day 3
  • 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 ⁇ L 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. 45E) Day 3 and (FIG.
  • 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).
  • FIGS. 47A-47C show cell-matrix interactions on aligned K2 scaffolds.
  • FIG. 47A 1X PB
  • FIG. 47B 3X PB
  • 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.
  • 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. 48C shear sweeps from 0.01 – 100 s -1
  • d strain sweeps from 0.1 – 100% of 4wt% K2 in Milli- Q water.
  • 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. 49A Shear sweeps from 0.01 – 100 s -1 of K2 ink compared to the various support bath candidates.
  • FIGS. 50A-50D provide data relating to 3D printing optimization.
  • FIG. 50B Calibration curve for K2 ink into agarose support bath.
  • MDP multidomain peptide
  • the ink may be useful or suitable for printing, including extrusion printing, 3-dimensional hydrogel structures.
  • the hydrogel structures have aligned nanofibers.
  • 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
  • SAPs self-assembling peptides
  • MDPs self-DVVHPEOH ⁇ LQWR ⁇ ⁇ -sheets and form a nanofibrous hydrogel under physiological conditions.
  • MDPs have been shown to support cell survival and growth.
  • 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.
  • 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.
  • the methods disclosed herein facilitate the printing with self-assembling peptides of layered structures with overhangs and internal porosity.
  • 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.
  • 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.
  • 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.
  • the precursor solution has properties that are favorable for the formation of the hydrogel structure.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the gelation bath comprises a buffering agent, an additive, a crosslinker, an inorganic salt, or any combination thereof.
  • the gelation bath comprises PBS, HBSS, cell culture medium, or a biological fluid, or any combination thereof.
  • the gelation bath comprises between 5 mM phosphate buffer and 150 mM phosphate buffer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.”
  • 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.
  • 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.
  • 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.
  • MDPs multidomain peptides
  • 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.
  • HBSS Hank’s Balanced Salt Solution
  • 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.
  • 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).
  • a shear sweep from 0.01 to 100 s ⁇ 1 was performed on 4 wt% K2 and E2 (FIG. 5B and FIG. 5F).
  • K2 has viscosity values of 577, 63, and 7.7 Pa s.
  • E2 has viscosity values of 336, 70, and 6.4 Pa s.
  • both 4 wt% MDPs had a negative slope, which means that they are both shear thinning.
  • a strain sweep from 0.1 to 100% strain at 1 rad s ⁇ 1 was also performed on both 4 wt% MDPs (FIG. 5C and FIG. 5G).
  • MDP ink and “precursor solution” are used equivalently and interchangeably to signify a composition comprising a multidomain peptide (MDP) and a solvent.
  • the solvent mediates the self-assembly of the MDP.
  • 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.
  • 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.
  • MDP inks show long term stability when stored at 4 °C (FIG. 5K).
  • 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.
  • 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.
  • 25G needles were used for the remainder of the study to have an equal comparison between the K2 and E2 inks.
  • MDP filaments were not observed to have widths equal to 250 ⁇ P ⁇ LQVWHDG ⁇ ILEHUV ⁇ IUDFWXUHG ⁇ DW ⁇ KLJK ⁇ SULQW ⁇ VSHHGV ⁇ $ ⁇ SULQW ⁇ VSHHG ⁇ RI ⁇ PP ⁇ PLQ ⁇ 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 ⁇ m; 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.
  • the layer height of all constructs was set as 250 ⁇ m (the inner diameter of a 25G needle).
  • 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).
  • 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).
  • 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 ⁇ m (2000–543 ⁇ m from the 4% K2 calibration curve). The inner 9 pores were measured to have an average length and width of 1528 ⁇ m, 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 ⁇ m (1000–543 ⁇ m from the 4% K2 calibration curve).
  • the inner 9 pores were measured to have an average length and width of 595 ⁇ m, 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.
  • 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 ⁇ m (1000–543 ⁇ m from the 4% K2 calibration curve) and the expected pore length was 574 ⁇ m (1000–426 ⁇ m from the 4% E2 calibration curve).
  • the measured pore width and length were an average of 517 and 644 ⁇ m, 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 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 ⁇ m. 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.
  • 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.
  • Circular Dichroism [00142] ⁇ / ⁇ RI ⁇ SHSWLGH ⁇ LQ ⁇ +%66 ⁇ &RUQLQJ ⁇ ,QF ⁇ &RUQLQJ ⁇ 1 ⁇ DW ⁇ PJ ⁇ P/ ⁇ 1 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 ⁇ 1 .
  • Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy [00143] ⁇ / ⁇ RI ⁇ SHSWLGH ⁇ LQ ⁇ +%66 ⁇ DW ⁇ PJ ⁇ P/ ⁇ 1 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 ⁇ 1 and background subtraction was performed on the spectra.
  • 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.
  • a Helios NanoLab 660 Scanning Electron Microscope FEI Company, Hillsboro, OR
  • 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.
  • 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 ⁇ 1 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.
  • 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 ⁇ 1 ), L-glutamine (4 mm), pyruvate (1 mm), penicillin-streptomycin (1%), and fetal bovine serum (10%).
  • Dulbecco’s modified Eagle’s medium Thermo Fisher Scientific, Waltham, MA
  • high glucose 4500 mg L ⁇ 1
  • L-glutamine (4 mm)
  • pyruvate (1 mm
  • penicillin-streptomycin 1%
  • fetal bovine serum fetal bovine serum
  • 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.
  • FIGS. 15- 24 depict polarized light microscopy images, which is an optical method used to analyze if a structure is anisotropic.
  • illuminated (also termed birefringent) MDP hydrogels possess some degree of nanofibrillar alignment.
  • randomly oriented MDP hydrogels do not illuminate under a polarized light microscope.
  • 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.
  • 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.
  • buffer identity HEPES, phosphate buffer, HBSS
  • buffer strength (10mM, 100mM)
  • ionic strength or NaCl concentration (0mM, 140mM, 1400mM)
  • 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.
  • the present disclosure provides methods for forming aligned hydrogels from precursor solutions which comprise various concentrations phosphate buffer.
  • precursor solutions which comprise various concentrations phosphate buffer.
  • K2 is cationic, the more acidic the solution it is dissolved in, the less assembled it was observed to be, and vice versa.
  • 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.
  • the present disclosure provides methods for forming aligned hydrogels from precursor solutions which comprise various pH values.
  • the optimal precursor solution pH for hydrogel formation may depend on the identity of the peptide.
  • the present disclosure provides methods for forming aligned hydrogels from precursor solutions which comprise various concentrations of peptide.
  • 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.
  • 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.
  • the present disclosure provides methods for forming aligned hydrogels by extruding a precursor solution through any diameter of needle.
  • 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 may comprise use of a support bath for temporarily stabilizing soft structures during the printing process (Hinton et al., 2015).
  • gelation baths of the present methods may be combined with support baths to form combination gelation-support baths.
  • 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.
  • 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).
  • Peptide Precursor Solution Preparation 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 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).
  • gelatin was dissolved at 4.5wt% in 150mL of gelation bath solution overnight at 4C.
  • 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.
  • 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.
  • 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.
  • aligned MDP hydrogels were fabricated in ⁇ -Slide 8 Well High Bioinert chambers (Ibidi GmbH, Grafelfing, Germany).
  • the precursor solution’s MDP concentration influences the properties of the resulting hydrogel.
  • Attenuated total reflectance Fourier transform infrared spectroscopy showed an increase in the amide I band at 1616 cm -1 ⁇ FRUUHVSRQGLQJ ⁇ WR ⁇ & 2 ⁇ VWUHWFKLQJ ⁇ ZLWKLQ ⁇ WKH ⁇ -sheet (FIG. 30B).
  • dynamic light scattering 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).
  • 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.
  • 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.
  • HBSS Hank’s Balanced Salt Solution
  • 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).
  • 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.
  • 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.
  • 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.
  • VICs v.1 valvular interstitial cells
  • 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).
  • VICs on 1X PB hydrogels at day 3 (FIG. 40G) and day 7 (FIG. 40H) show that these scaffolds promote macroscopic cellular alignment.
  • 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.
  • VIC and C2C12 seeding studies were repeated onto K2 hydrogel scaffolds with half the diameter, supposing that hydrogel curvature may be affecting cellular growth.
  • the cellular spreading patterns matched the previously observed trend (FIG. 44. FIG. 45).
  • 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).
  • 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.
  • 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 ⁇ 1 ⁇ 1 ⁇ -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).
  • K2 full sequence: KKSLSLSLSLSLSLSLKK) (SEQ ID NO: 1) was manually synthesized via solid phase peptide synthesis.
  • 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.
  • PLM Polarized Light Microscopy
  • Rheology was performed using an AR-G2 rheometer (TA Instruments, New Castle, DE) equipped with a 12 mm parallel plate. 100 ⁇ L of 3% K2 in Milli-Q water was added to the stage and the plate was lowered to a gap of 500 ⁇ m. 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).
  • ATR-FTIR Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy
  • SEM Scanning Electron Microscopy
  • FEI Company Helios NanoLab 660 Scanning Electron Microscope
  • 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.
  • Milli-Q water dilutions 30%, 50%, 60%, 70%, 80%, 90%, 2 x 100%
  • Samples that also contained cells were fixed in 4% paraformaldehyde (Thermo Fisher Scientific, Waltham, MA) for 30 minutes prior to the serial dilution process.
  • K2 hydrogel samples were placed horizontally onto SpectroMembrane Polyimide 7.5 ⁇ m 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 ⁇ -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°.
  • Optical Coherence Elastography [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 ⁇ m axial resolution (in air), ⁇ 8 ⁇ m transversal resolution, and 0.28 nm of displacement stability. The A-line acquisition was set to 25 kHz during OCE acquisition.
  • PhS - OCT phase-sensitive Optical Coherence Tomography
  • 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.
  • the home-built Brillouin microscopy system was based on a two-stage virtually imaged phase array (VIPA) spectrometer.
  • VIPA virtually imaged phase array
  • a single-mode 660 nm laser Torus, Laser Quantum Inc., Fremont, CA
  • an incident sample power of ⁇ 17 mW was utilized, and a microscope was placed coaxially with the system to align the sample.
  • 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.
  • ABAM antibiotic/antimycotic solution
  • PBS phosphate-buffered saline
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • 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.
  • K2 ink formulation 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 ⁇ -sheet secondary structure (FIG. 48A) and cryo-transmission electron microscopy confirmed that this K2 ink contains nanofibers (FIG. 48B).
  • ATR-FTIR Attenuated total reflectance Fourier transform infrared
  • 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).
  • 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).
  • gelation support baths comprising agarose as a gelling agent may facilitate the printing of hydrogels with aligned nanofibers.
  • 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.
  • tissue growth was tracked over time.
  • cells were observed aligning in the direction of the 3D print, indicated that cells were aligning as expected (FIG. 51A).
  • the myoblasts grew to confluency along the print direction (FIG.
  • 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.
  • 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. All liberation was done under sterile conditions.
  • 66 4863-6540-6909, v.1 REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference: Ahrens et al., Adv.

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  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Botany (AREA)
  • Zoology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des procédés d'impression de structures d'hydrogel comprenant des peptides multidomaines (MDP). L'invention concerne également des structures d'hydrogel formées selon de tels procédés, ainsi que des procédés d'utilisation de celles-ci et des kits comprenant les MDP présentement décrits. La présente invention concerne également de nouveaux procédés de formation de structures d'hydrogel avec des nanofibres alignées et des structures d'hydrogel formées selon de tels procédés, ainsi que leurs utilisations.
PCT/US2024/028436 2023-05-09 2024-05-08 Impression 3d d'hydrogels peptidiques à domaines multiples à auto-assemblage et fabrication d'hydrogels peptidiques à auto-assemblage nanofibreux alignés Pending WO2024233707A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363501071P 2023-05-09 2023-05-09
US63/501,071 2023-05-09
US202363510818P 2023-06-28 2023-06-28
US63/510,818 2023-06-28

Publications (1)

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WO2024233707A1 true WO2024233707A1 (fr) 2024-11-14

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PCT/US2024/028436 Pending WO2024233707A1 (fr) 2023-05-09 2024-05-08 Impression 3d d'hydrogels peptidiques à domaines multiples à auto-assemblage et fabrication d'hydrogels peptidiques à auto-assemblage nanofibreux alignés

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WO (1) WO2024233707A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160375177A1 (en) * 2013-11-30 2016-12-29 Agency For Science, Technology And Research Self-assembling peptides, peptidomimetics and peptidic conjugates as building blocks for biofabrication and printing
US20200000875A1 (en) * 2018-06-15 2020-01-02 New Jersey Institute Of Technology Injectable Self-assembling Antibacterial Peptide Hydrogels

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160375177A1 (en) * 2013-11-30 2016-12-29 Agency For Science, Technology And Research Self-assembling peptides, peptidomimetics and peptidic conjugates as building blocks for biofabrication and printing
US20200000875A1 (en) * 2018-06-15 2020-01-02 New Jersey Institute Of Technology Injectable Self-assembling Antibacterial Peptide Hydrogels

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