Supramolecular polymer putty for bone / tissue regeneration

Abstract

Spinal fusion is a surgical procedure for treating debilitating back and neck pain in which two adjacent spinal vertebrae are fused by inducing bone growth between them. Iliac crest autograft bone has been the gold standard for spinal fusion due to its low cost and reliable fusion results, but the morbidity and limited volume of the harvest site have led to the search for alternatives. Provided herein are compositions comprising a peptide amphiphile, a soft covalent polymer, and a ceramic material. A composite putty-like material is provided for medical applications, particularly for repairing bone / tissue injuries / defects and regenerating bone or other tissues.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This invention claims the benefit of U.S. Provisional Patent Application No. 63 / 399,467, filed August 19, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The text of the computer-readable sequence listing submitted herewith, entitled "41195-601_SEQUENCE_LISTING," created on August 17, 2023, and having a file size of 31,731 bytes, is hereby incorporated by reference in its entirety. [Background technology]

[0003] Provided herein are compositions comprising a peptide amphiphile, a soft covalent polymer, and a ceramic material. A composite putty-like material is provided for medical applications, particularly for repair of bone / tissue damage / defects and regeneration of bone or other tissue. Summary of the Invention [Problem to be solved by the invention]

[0004] Spinal fusion is a surgical procedure for treating debilitating back and neck pain in which two adjacent spinal vertebrae are fused by inducing bone growth between them. Iliac crest autograft bone has been the gold standard for spinal fusion due to its low cost and reliable fusion results, but the morbidity and limited volume of the harvest site have led to the search for alternatives. Spinal biologics such as bone morphogenetic protein 2 (BMP-2) are popular among surgeons because they offer a synthetic, off-the-shelf implant that requires a single surgical site and has consistent clinical outcomes. However, due to significant safety concerns about supraphysiological doses of BMP-2, its approval is limited to use in the lumbar region of the spine, and the high production costs of the recombinant protein have made it prohibitive for some hospitals / institutions.

[0005] Provided herein are compositions comprising a peptide amphiphile, a soft covalent polymer, and a ceramic material. A composite putty-like material is provided for medical applications, particularly for repair of bone / tissue damage / defects and regeneration of bone or other tissue. [Means for solving the problem]

[0006] In some embodiments, provided herein are compositions comprising a composite material comprising: (a) a peptide amphiphile nanofiber; (b) a polymeric component; and (c) a ceramic component.

[0007] In some embodiments, the peptide amphiphile nanofibers comprise supramolecular assemblies of peptide amphiphiles, the peptide amphiphiles comprising (i) hydrophobic non-peptidic segments, (ii) structural peptide segments, and (iii) charged segments. In some embodiments, the polymer component comprises polyethylene glycol (PEG).

[0008] In some embodiments, the polymer component comprises a PEG selected from PEG200, PEG300, PEG400, PEG500, PEG550, PEG600, PEG700, PEG800, PEG900, PEG1000, PEG1450, PEG3350, PEG4500, PEG8000, or combinations thereof. In some embodiments, the polymer component comprises PEG600 and PEG1450.

[0009] In some embodiments, the ceramic component comprises hydroxyapatite (HA), tricalcium phosphate (TCP), bioglass, or calcium sulfate. In some embodiments, the ceramic component comprises TCP. In some embodiments, the composite further comprises a bioactive factor.

[0010] In some embodiments, the composite material comprises less than 5 wt% peptide amphiphile nanofibers, 60-80 wt% polymer component, and 20-40 wt% ceramic component. In some embodiments, the composite material comprises less than 1 wt% peptide amphiphile nanofibers, 65-75 wt% polymer component, and 25-35 wt% ceramic component. In some embodiments, the composite material comprises less than 1 wt% peptide amphiphile nanofibers, about 69 wt% polymer component, and about 30 wt% ceramic component.

[0011] In some embodiments, all or a portion of the peptide amphiphile further comprises a bioactive peptide segment. In some embodiments, the bioactive peptide segment mimics the biological function of or is capable of binding to a bioactive factor and / or cellular component. In some embodiments, the bioactive factor and / or cellular component is selected from bone morphogenic proteins, transforming growth factor, epidermal growth factor, growth differentiation factor, human endothelial growth factor, granulocyte-macrophage colony-stimulating factor, nerve growth factor, vascular endothelial growth factor, fibroblast growth factor, insulin-like growth factor, cartilage-derived morphogenetic protein, platelet-rich plasma, platelet-derived growth factor, insulin growth factor 1, and platelet-derived growth factor.

[0012] In some embodiments, the peptide amphiphile nanofiber comprises a supramolecular assembly of (i) a bioactive peptide amphiphile comprising (A) a hydrophobic nonpeptidic segment, (B) a structural peptide segment, (C) a charged segment, and (D) a bioactive peptide; and (ii) a dilute peptide amphiphile comprising (A) a hydrophobic nonpeptidic segment, (B) a structural peptide segment, and (C) a charged segment. In some embodiments, (i) and (ii) are present in a ratio of 1:10 to 10:1. In some embodiments, (i) and (ii) are present in a ratio of 1:2 to 2:1. In some embodiments, the hydrophobic nonpeptidic segment comprises an acyl chain. In some embodiments, the acyl chain is a C6-C 20 In some embodiments, the hydrophobic non-peptidic segment of the diluting peptide amphiphile and the hydrophobic non-peptidic segment of the bioactive peptide amphiphile are the same length. In some embodiments, the hydrophobic non-peptidic segment of the diluting peptide amphiphile and the hydrophobic non-peptidic segment of the bioactive peptide amphiphile are different lengths. In some embodiments, the hydrophobic non-peptidic segment of the diluting peptide amphiphile is C 16 and the hydrophobic non-peptidic segment of the bioactive peptide amphiphile is C 12In some embodiments, the structural peptide segment is an alanine- and valine-rich peptide segment. In some embodiments, the alanine- and valine-rich peptide segment comprises AAVV (SEQ ID NO: 2), AAAVVV (SEQ ID NO: 3), VVAA (SEQ ID NO: 4), or VVVAAA (SEQ ID NO: 5). In some embodiments, the charged peptide segment is a glutamic acid and / or aspartic acid-rich segment. In some embodiments, the glutamic acid and / or aspartic acid-rich segment comprises 2-7 amino acids in length, with 50% or more of the amino acids selected from Glu (E) and / or Asp (D) residues. In some embodiments, the glutamic acid and / or aspartic acid-rich segment comprises EE or EEE. In some embodiments, the bioactive peptide is a BMP-2 binding peptide. In some embodiments, the bioactive peptide comprises at least 50% sequence identity to TSPHVPYGGGS (SEQ ID NO: 1). In some embodiments, the binding sequence comprises TSPHVPYGGGS (SEQ ID NO: 1).

[0013] In some embodiments, provided herein are methods that include administering to a subject a composite material described herein, hi some embodiments, the composite material is administered to repair bone or tissue damage or defects.

[0014] In some embodiments, provided herein are compositions comprising: (a) (i) a bioactive peptide amphiphile comprising (A) a hydrophobic non-peptidic segment, (B) a structural peptide segment, (C) a charged segment, and (D) a BMP-2-binding peptide; and (ii) a peptide amphiphile nanofiber comprising a supramolecular assembly with a diluent peptide amphiphile comprising (A) a hydrophobic non-peptidic segment, (B) a structural peptide segment, and (C) a charged segment; (b) a PEG-containing polymer component; (c) a ceramic component comprising TCP or HA; and (d) BMP-2.

[0015] In some embodiments, provided herein are compositions comprising: (a) (i) a bioactive peptide amphiphile comprising (A) a hydrophobic non-peptidic segment, (B) a structural peptide segment, (C) a charged segment, and (D) a BMP-2-binding peptide; and (ii) a peptide amphiphile nanofiber comprising a supramolecular assembly with a diluent peptide amphiphile comprising (A) a hydrophobic non-peptidic segment, (B) a structural peptide segment, and (C) a charged segment; (b) a PEG-containing polymer component; and (c) a ceramic component comprising TCP or HA, wherein the composition does not comprise BMP-2.

[0016] In some embodiments, provided herein are methods of promoting bone formation, the methods comprising administering to a subject a composite material described herein.

[0017] In some embodiments, provided herein is a method of repairing a bone injury or defect in a subject, the method comprising administering a composite material described herein.

[0018] In some embodiments, provided herein are methods of promoting arthrodesis, the methods comprising administering to a subject a composite material described herein.

[0019] In some embodiments, provided herein are methods of promoting spinal fusion, the methods comprising administering to a subject a composite material described herein. [Brief explanation of the drawings]

[0020] [Figure 1] Chemical structures of PA molecules containing supramolecular polymers to bind rhBMP-2 protein and enhance its signal: dilutable PA (top) and bioactive PA (bottom) are shown. [Figure 2]1 shows the storage modulus of different carriers for bioactive supramolecular polymers (all containing supramolecular polymers). The collagen carrier is described in U.S. Patent Application Publication No. 20170106120 (incorporated by reference in its entirety) and the putty carrier is described herein. [Figure 3] Figure 1 shows rat spine fusion rates for putty with supramolecular polymer, with small or large TCP particles as indicated, and without and with rhBMP-2 as indicated. Three blinded raters assigned scores to rat spines, with 0 meaning no fusion, 1 meaning fusion on one side of the spine, and 2 meaning fusion on both sides. Animals with an average score of 1 or greater were considered to have successful fusion for the calculation of fusion rates. [Figure 4] Figure 1 shows the storage modulus of different putties containing supramolecular polymers with the indicated weight percentage of large TCP particles (250-1000 µm). [Figure 5] Figure 1 shows rat spine fusion scores and percentages for putty with supramolecular polymer, with 10% or 30% TCP by weight as indicated. Scores are the average of three blinded assessors, with 0 meaning no fusion, 1 meaning unilateral fusion of the spine, and 2 meaning bilateral fusion. Animals with an average score of 1 or greater are considered fused for the calculation of fusion percentage. [Figure 6A-C]Characterization of PA putty. (Figure 6A) Fourier transform infrared spectrograph (FTIR) of a putty containing PA. The peak near 1630 cm-1 indicates the presence of β-sheet structure. Confocal micrographs of a PA putty containing PEG tagged with fluorescein (FITC), PA tagged with rhodamine (TAMRA), and bovine serum albumin tagged with Alexa Fluor 647 (a far-red dye) are shown. Individual channels and an overlay are plotted. Enzyme-linked immunosorbent assay (ELISA) showing BMP-2 release from the collagen ACS carrier (current clinical product) and from the putty are shown. To start, all materials were loaded with 100 ng of BMP-2. (Figure 6B) Fourier transform infrared spectrograph (FTIR) of a putty containing PA. The peak near 1630 cm-1 indicates the presence of β-sheet structure. Figure 6C shows a confocal micrograph of a PA putty in which PEG was tagged with fluorescein (FITC), PA was tagged with rhodamine (TAMRA), and bovine serum albumin was tagged with Alexa Fluor 647 (a far-red dye). Individual channels and an overlay are plotted. An enzyme-linked immunosorbent assay (ELISA) is shown demonstrating BMP-2 release from the collagen ACS carrier (current clinical product) and from the putty. To begin, all materials were loaded with 100 ng of BMP-2. (Figure 6D) Fourier transform infrared spectroscopy (FTIR) of the PA-containing putty is shown. The peak near 1630 cm indicates the presence of β-sheet structure. Figure 6E shows a confocal micrograph of a PA putty in which PEG was tagged with fluorescein (FITC), PA was tagged with rhodamine (TAMRA), and bovine serum albumin was tagged with Alexa Fluor 647 (a far-red dye). Individual channels and an overlay are plotted. 1 shows an enzyme-linked immunosorbent assay (ELISA) demonstrating BMP-2 release from the collagen ACS carrier (current clinical product) and from the putty. To start, all materials were loaded with 100 ng of BMP-2. [Figure 7]Figure 1 shows the rabbit spine fusion rate for putty with supramolecular polymer containing the indicated BMP-2 doses. Three blinded evaluators assigned scores to the rabbit spines, with 0 representing no fusion, 1 representing fusion on one side of the spine, and 2 representing fusion on both sides. Animals with an average score of 1 or greater were considered to have successfully fused for the calculation of fusion rate. DETAILED DESCRIPTION OF THE INVENTION

[0021] definition Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, certain preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it should be understood that this invention is not limited to the particular molecules, compositions, methodologies, or protocols described herein, as these may vary according to routine experimentation and optimization. It should also be understood that the terminology used herein is for the purpose of describing particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will prevail. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide amphiphile" is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth.

[0024] The term "amino acid," unless otherwise indicated, refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers, provided that their structure permits such stereoisomeric forms. Embodiments herein refer to various amino acid abbreviations (one-letter or three-letter abbreviations) that will be understood by those of skill in the art. All amino acid abbreviations not defined herein refer to their art-accepted meanings.

[0025] The term "proteinogenic amino acids" refers to the 20 amino acids encoded in the human genome, including alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). Selenocysteine ​​and pyrolysine may also be considered proteinogenic amino acids.

[0026] The term "non-proteinogenic amino acid" refers to an amino acid that is not naturally encoded or found in the genetic code and is not biosynthetically incorporated into proteins during translation. A non-proteinogenic amino acid can be a "non-natural amino acid" (a non-naturally occurring amino acid) or a "naturally occurring non-proteinogenic amino acid" (e.g., norvaline, ornithine, homocysteine, etc.). Examples of non-proteinogenic amino acids include azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, Non-proteinogenic amino acids include hydroxylysine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-alkylglycines including N-methylglycine, N-methylisoleucine, N-alkylpentylglycines including N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine ("Norleu"), octylglycine, ornithine, pentylglycine, pipecolic acid, thioproline, homolysine, and homoarginine. Non-proteinogenic amino acids include D-amino acid forms of any of the amino acids herein, as well as non-alpha amino acid forms of any of the amino acids herein (such as β-amino acids, γ-amino acids, δ-amino acids, etc.), all of which are within the scope of the present invention and may be included in the peptides herein.

[0027] The term "amino acid analog" refers to a natural or unnatural amino acid in which one or more of the C-terminal carboxy group, the N-terminal amino group, and side chain functional groups are reversibly or irreversibly chemically blocked or modified to another functional group. For example, aspartic acid-(β-methyl ester) is an amino acid analog of aspartic acid, N-ethylglycine is an amino acid analog of glycine, or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, and S-(carboxymethyl)-cysteine ​​sulfone.

[0028] As used herein, the term "peptide" refers to a short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are about 50 amino acids or less in length. Peptides may contain natural amino acids, unnatural amino acids, amino acid analogs, and / or modified amino acids. Peptides may be subsequences of naturally occurring proteins or unnatural (artificial) sequences.

[0029] As used herein, the term "artificial" refers to compositions and systems that are designed or prepared by humans and do not occur in nature. For example, an artificial peptide or nucleic acid is one that contains a non-naturally occurring sequence (e.g., a peptide that does not have 100% identity to a naturally occurring protein or fragment thereof).

[0030] As used herein, a "conservative" amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. In this disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) alanine (A) and glycine (G); 2) aspartic acid (D) and glutamic acid (E); 3) asparagine (N) and glutamine (Q); 4) arginine (R) and lysine (K); 5) isoleucine (I), leucine (L), methionine (M), and valine (V); 6) phenylalanine (F), tyrosine (Y), and tryptophan (W); 7) serine (S) and threonine (T); and 8) cysteine ​​(C) and methionine (M).

[0031] Naturally occurring residues can be divided into classes based on shared side chain properties, e.g., polar positive (histidine (H), lysine (K), and arginine (R)); polar negative (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); nonpolar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); nonpolar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a "semi-conservative" amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0032] In some embodiments, unless otherwise specified, conservative or semi-conservative amino acid substitutions may also include non-naturally occurring amino acid residues that have similar chemical properties to the natural residues. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0033] Non-conservative substitutions may involve the exchange of a member of one class for a member from another class.

[0034] As used herein, the term "sequence identity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequence composition of monomer subunits. The term "sequence similarity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) differ only by conservative and / or semi-conservative amino acid substitutions. "Percent sequence identity" (or "percent sequence similarity") is calculated by (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., the same amino acid is present in both sequences, similar amino acids are present in both sequences) to obtain the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to obtain the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids long and have identical amino acids at all but one position, then peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., both are acidic), then peptides A and B have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some amino acids in peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity over the optimal comparison window of peptide C. For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is treated as a mismatch at that position.

[0035] Any peptide described herein as having a particular percent sequence identity or similarity (e.g., at least 70%) with a reference sequence can also be expressed as having a maximum number of substitutions (or terminal deletions) relative to that reference sequence. For example, a sequence "having at least 70% sequence identity to SEQ ID NO:X" can have up to three substitutions with SEQ ID NO:X (if SEQ ID NO:X is 10 amino acids in length), and therefore can also be expressed as "having no more than three substitutions with SEQ ID NO:X." Furthermore, a sequence "having at least 80% sequence similarity with SEQ ID NO:X" can have zero, one, or two non-conservative substitutions with SEQ ID NO:X, and therefore can also be expressed as "having no more than two non-conservative substitutions with SEQ ID NO:X."

[0036] As used herein, the term "nanofiber" refers to an elongated or thread-like (e.g., having a length dimension significantly greater than its width or diameter) filament typically having a diameter of less than 100 nanometers (e.g., 10 nm).

[0037] As used herein, the term "supramolecule" (e.g., "supramolecular complex," "supramolecular interaction," "supramolecular fiber," "supramolecular polymer," etc.) refers to non-covalent interactions between molecules (e.g., polymers, marcomolecules, etc.) and the resulting multi-component assemblies, complexes, systems, and / or fibers.

[0038] As used herein, the term "physiological conditions" refers to the range of conditions of temperature, pH and osmolality normally encountered within tissues in the living human body.

[0039] As used herein, the terms "self-assemble" and "self-assembly" refer to the formation of discrete, non-random aggregate structures from component parts, where the assembly occurs spontaneously through the random movement of the components (e.g., molecules) due solely to the intrinsic chemical or structural properties and attractive forces of those components.

[0040] As used herein, the term "peptide amphiphile" refers to a molecule that contains, at a minimum, a non-peptide lipophilic (hydrophobic) segment, a structural peptide segment, and, optionally, a functional peptide segment. Peptide amphiphiles can carry a net charge at physiological pH, i.e., either a net positive or net negative charge, or can be zwitterionic (i.e., possessing both positive and negative charges). Certain peptide amphiphiles consist of or comprise: (1) a hydrophobic, non-peptidic segment (e.g., containing an acyl group of six or more carbons), (2) a structural or β-sheet-forming peptide segment; (3) a carboxyl-rich peptide segment, and (4) a biologically active moiety (e.g., a BMP-2 binding moiety).

[0041] As used herein and in the appended claims, the terms "lipophilic moiety" or "hydrophobic moiety" refer to the portion (e.g., acyl moiety) located at the N-terminus of a peptide amphiphile, which may be referred to herein and elsewhere as a lipophilic or hydrophobic segment or moiety. The hydrophobic moiety should be of sufficient length to provide amphiphilic behavior and micelle (or nanosphere or nanofiber) formation in water or another polar solvent system. Thus, in the context of the embodiments described herein, the hydrophobic moiety preferably has the formula: C n-1 H 2n-1 They contain a single linear acyl chain of C(O)-- (where n=6-22). In some embodiments, the linear acyl chain is the lipophilic group palmitic acid. However, other small lipophilic groups can be used in place of the acyl chain.

[0042] As used herein, the term "structural peptide" or "β-sheet-forming peptide" refers to an intermediate amino acid sequence of a peptide amphiphile molecule that lies between the hydrophobic segment and the charged peptide segment of the peptide amphiphile. This "structural peptide" or "β-sheet-forming peptide" generally consists of 3 to 10 amino acid residues with nonpolar, uncharged side chains selected for their tendency to form β-sheet secondary structure. Examples of suitable amino acid residues selected from the 20 naturally occurring amino acids include Met (M), Val (V), Ile (I), Cys (C), Tyr (Y), Phe (F), Gln (Q), Leu (L), Thr (T), Ala (A), and Gly (G) (listed in order of their tendency to form β-sheets). However, similar non-naturally occurring amino acids with a tendency to form β-sheets may also be used. Peptide segments that can interact to form a β-sheet and / or have a tendency to form a β-sheet are understood to be peptide segments (see, e.g., Mayo et al., Protein Science (1996), 5:1301-1315; incorporated herein by reference in its entirety). In a preferred embodiment, the N-terminus of the structural peptide segment is covalently bonded to an oxygen of the lipophilic segment, and the C-terminus of the structural peptide segment is covalently bonded to the N-terminus of the charged peptide segment.

[0043] As used herein, the terms "carboxy-rich peptide segment," "acidic peptide segment," and "negatively charged peptide segment" refer to a peptide sequence that is either (i) located intermediately between a structural peptide segment (β-sheet-forming segment) and a bioactive peptide (BMP-2-binding segment) or (ii) the C-terminal segment of a PA (e.g., a diluent PA) that does not have a bioactive peptide. In some embodiments, a carboxy-rich peptide segment comprises two or more amino acid residues (e.g., Glu (E), Asp (D), or unnatural amino acids) that have side chains exhibiting a carboxylic acid side chain. A carboxy-rich peptide segment can optionally contain one or more additional (e.g., non-acidic) amino acid residues. As will be apparent to one of skill in the art, unnatural amino acid residues with acidic side chains can be used. There can be from about 2 to about 7 amino acids, and / or about 3 or 4 amino acids in this segment.

[0044] As used herein, the term "bioactive peptide" refers to an amino acid sequence that mediates the action of the sequence, molecule, or supramolecular complex to which it is linked. Peptide amphiphiles and structures (e.g., nanofibers) bearing bioactive peptides (e.g., BMP-2 binding peptides) exhibit the functionality of functional peptides.

[0045] Detailed Description Provided herein are compositions comprising a peptide amphiphile, a soft covalent polymer, and a ceramic material. Composite putty-like materials are provided for medical applications, particularly for repair of bone / tissue injuries / defects and regeneration of bone or other tissues. In some embodiments, the composition further comprises a bioactive factor (e.g., that can be bound by the bioactive peptide of the peptide amphiphile). In other embodiments, the peptide amphiphile comprises a bioactive peptide capable of binding a bioactive factor (e.g., BMP-2), but the composition does not comprise an exogenous bioactive factor (e.g., the composition is configured to interact with an endogenous bioactive factor (e.g., BMP-2) upon administration to a treatment site).

[0046] In some embodiments, the composite materials herein comprise peptide amphiphile nanofiber components. Peptide amphiphile molecules have been demonstrated to be useful as building blocks for creating biomaterials, for example, in regenerative medicine. PAs have been designed to self-assemble in aqueous conditions into high-aspect-ratio nanofibers with diameters of approximately 10 nanometers and lengths of several microns. Their formation is primarily driven by the collapse of hydrophobic molecular segments away from the aqueous environment and secondary interactions, such as hydrogen bonding, between peptide segments, resulting in a β-sheet secondary structure (Hartgerink, E. Beniash, S. Stupp, Science 2001, 294, 1684; incorporated by reference in its entirety). These supramolecular nanofibers can be designed to exhibit a high surface density of bioactive peptides capable of diverse functions. Various PA nanofibers have been demonstrated to be useful in, among other things, central nervous system and cartilage repair, vascularization of ischemic cardiac tissue, enamel growth, and bone repair (Tysseling-Mattiace et al. Journal of Neuroscience 2008, 28, 3814; Shah et al. Proceedings of the National Academy of Sciences 2010, 107, 3293; Webber et al. Proceedings of the National Academy of Sciences 2011, 108, 13438; Huang et al. Biomaterials 2010, 31, 9202; Mata et al. Biomaterials 2010, 31, 6004; Sargeant et al. Biomaterials 2008, 29, 161; incorporated by reference in their entireties).

[0047] In some embodiments, peptide amphiphile molecules and compositions of the embodiments described herein are synthesized using preparative techniques well known to those of skill in the art, preferably by standard solid-phase peptide synthesis, with the addition of fatty acids in place of standard amino acids at the N-terminus (or C-terminus) of the peptide to create lipophilic segments. Synthesis typically begins at the C-terminus using either Rink amide resin (which provides an -NH group at the C-terminus of the peptide after cleavage from the resin) or Wang resin (which provides an -OH group at the C-terminus), with amino acids added sequentially to the C-terminus. Thus, embodiments described herein encompass peptide amphiphiles having a C-terminal moiety that can be selected from the group consisting of --H, --OH, --COOH, --CONH, and --NH.

[0048] In some embodiments, peptide amphiphiles comprise a hydrophobic (non-peptide) segment linked to a peptide. In some embodiments, the peptide comprises a structural segment (e.g., a hydrogen-bond-forming segment, a beta-sheet-forming segment, etc.) and a charged segment (e.g., an acidic segment, a basic segment, a zwitterionic segment, etc.). In some embodiments, the peptide further comprises a linker or spacer segment to add solubility, flexibility, distance between segments, etc. In some embodiments, the peptide amphiphile comprises a spacer segment (e.g., a peptide and / or non-peptide spacer) at the end of the peptide opposite the hydrophobic segment. In some embodiments, the spacer segment comprises peptide and / or non-peptide elements. In some embodiments, the spacer segment comprises one or more active functional groups (e.g., an alkene, alkyne, azide, thiol, etc.). In some embodiments, various segments may be connected by a linker segment (e.g., a peptide (e.g., GG) or a non-peptide (e.g., alkyl, OEG, PEG, etc.) linker).

[0049] The lipophilic or hydrophobic segment is typically incorporated into the N- or C-terminus of the peptide after the final amino acid coupling and consists of a fatty acid or other acid linked to the N- or C-terminal amino acid via an acyl bond. In aqueous solution, PA molecules self-assemble (e.g., into cylindrical micelles (also known as nanofibers)) with the lipophilic segment buried in its core and the bioactive peptide displayed on its surface. The structural peptides form intermolecular hydrogen bonds to form β-sheets oriented parallel to the long axis of the micelle.

[0050] In some embodiments, the compositions described herein comprise a PA component comprising, in order, a hydrophobic segment and a peptide segment. In certain embodiments, a hydrophobic (e.g., hydrocarbon and / or alkyl / alkenyl / alkynyl tail, or steroid such as cholesterol) segment of sufficient length (e.g., 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, 20 carbons, 21 carbons, 22 carbons, 23 carbons, 24 carbons, 25 carbons, 26 carbons, 27 carbons, 28 carbons, 29 carbons, 30 carbons, or more, or any range therebetween) is covalently attached to a peptide segment (e.g., a peptide comprising a segment that favors β-strand conformation or other supramolecular interactions) to provide a peptide amphiphile. In some embodiments, multiple such PAs self-assemble into nanostructures (e.g., nanofibers) in water (or aqueous solutions). In various embodiments, the relative lengths of the peptide and hydrophobic segments result in different PA molecular shapes and nanostructural architectures. For example, wider peptide segments and narrower hydrophobic segments result in roughly conical molecular shapes that influence PA assembly (e.g., JN Israelachvili, Intermolecular and surface forces; 2nd ed.; Academic: London San Diego, 1992; incorporated herein by reference in its entirety). Other molecular shapes have similar effects on assembly and nanostructural architecture.

[0051] In some embodiments, to induce self-assembly of an aqueous solution of peptide amphiphiles, the pH of the solution may be changed (raised or lowered), or multivalent ions such as calcium, or charged polymers or other macromolecules may be added to the solution.

[0052] In some embodiments, the hydrophobic segment is a non-peptide segment (e.g., an alkyl / alkenyl / alkynyl group). In some embodiments, the hydrophobic segment comprises a 4-25 carbon (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) alkyl chain (e.g., saturated), a fluorinated segment, a fluorinated alkyl tail, a heterocycle, an aromatic segment, a π-conjugated segment, a cycloalkyl, an oligothiophene, etc. In some embodiments, the hydrophobic segment comprises an acyl / ether chain (e.g., saturated) of 2 to 30 carbons (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).

[0053] In some embodiments, the PA comprises one or more peptide segments. The peptide segments may comprise natural amino acids, modified amino acids, unnatural amino acids, amino acid analogs, peptidomimetics, or combinations thereof. In some embodiments, the peptide segments comprise at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity or similarity (e.g., conservative or semi-conservative) with one or more of the peptide sequences described herein.

[0054] In some embodiments, the peptide amphiphile comprises a charged peptide segment, which can be acidic, basic, or zwitterionic.

[0055] In some embodiments, the peptide amphiphile comprises an acidic peptide segment. For example, in some embodiments, the acidic peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or more) acidic residues (D and / or E) in the sequence. In some embodiments, the acidic peptide segment comprises a length of up to 7 residues and comprises at least 50% acidic residues. In some embodiments, the acidic peptide segment is (Xa) 1-7 and each Xa is independently D or E. In some embodiments, the acidic peptide segment comprises EE or EEE.

[0056] In some embodiments, the peptide amphiphile comprises a basic peptide segment. For example, in some embodiments, the acidic peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or more) basic residues (R, H, and / or K) in the sequence. In some embodiments, the basic peptide segment comprises a length of up to 7 residues and comprises at least 50% basic residues. In some embodiments, the acidic peptide segment comprises (Xb) 1-7, where each Xb is independently R, H, and / or K.

[0057] In some embodiments, peptide amphiphiles comprise structural and / or β-sheet forming segments. In some embodiments, the structural segments are rich in H, I, L, F, V, and A residues. In some embodiments, the structural and / or β-sheet forming segments comprise alanine and valine-rich peptide segments (e.g., AAVV (SEQ ID NO: 2), AAAVVV (SEQ ID NO: 3), VVAA (SEQ ID NO: 4), VVVAAA (SEQ ID NO: 5), or other combinations of V and A residues). In some embodiments, the structural and / or β-sheet peptides comprise four or more consecutive A and / or V residues, or conservative or semi-conservative substitutions therefor. In some embodiments, the structural and / or β-sheet forming peptide segments comprise four or more consecutive non-polar aliphatic residues (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)). In some embodiments, the structural segments and / or beta-sheet forming peptide segments comprise 2-16 amino acids in length and include 4 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or any range therebetween) non-polar aliphatic residues.

[0058] In some embodiments, the peptide amphiphile comprises a non-peptide spacer or linker segment. In some embodiments, the non-peptide spacer or linker segment is located at the opposite end of the peptide from the hydrophobic segment. In some embodiments, the spacer or linker segment provides an attachment site for a biologically active group. In some embodiments, the spacer or linker segment provides a reactive group (e.g., alkene, alkyne, azide, thiol, maleimide, etc.) for functionalization of the PA. In some embodiments, the spacer or linker is a substantially linear chain of CH, O, (CH)O, O(CH), NH, and C=O groups (e.g., CH(O(CH))NH, CH(O(CH))NHCO(CH)CCH, etc.). In some embodiments, the spacer or linker further comprises additional biologically active groups, substituents, branches, etc.

[0059] Suitable peptide amphiphiles, PA segments, PA nanostructures, and related reagents and methods are described, for example, in U.S. Patent Nos. 11,066,444; 10,792,327; 10,752,656; 10,738,294; 10,689,252; 10,316,432; 10,316,18 No. 0; No. 9,926,195; No. 9,650,421; No. 9,556,232; No. 9,517,275; No. 9,512,404; No. 9,1 No. 69,294; No. 8,940,858; No. 8,834,840; No. 8,772,228; No. 8,748,569; No. 8,850,923; Same No. 8,512,693; Same No. 8,450,271; Same No. 8,138,140; Same No. 8,124,583; Same No. 8,114,835; Same No. 8,114, No. 834; No. 8,080,262; No. 8,063,014; No. 7,851,445; No. 7,838,491; No. 7,745,708; No. 7 ,683,025; 7,554,021; 7,544,661; 7,534,761; 7,491,690; 7,452,679; 7,390,526; 7,371,719; and 6,890,654, which are incorporated herein by reference in their entireties.

[0060] The characteristics of PA supramolecular structures (e.g., shape, rigidity, hydrophilicity, etc.) depend on the identity of the peptide amphiphile components (e.g., lipophilic segments, acidic segments, structural segments, bioactive segments, etc.). For example, by adjusting the identity of the PA component moieties, nanofibers, nanospheres, intermediate shapes, and other supramolecular structures are achieved. In some embodiments, the characteristics of PA supramolecular nanostructures are altered by post-assembly manipulations (e.g., heating / cooling, stretching, etc.).

[0061] In some embodiments, the peptide amphiphile comprises: (a) a hydrophobic tail comprising an alkyl chain of 8 to 24 carbons; (b) a structural segment comprising or consisting of 4 to 8 V and A residues; and (c) a charged segment (e.g., comprising EE, EEE, etc.). In some embodiments, any PA within the ranges described herein or within the skill of one of ordinary skill in the art may be used, including the components described herein.

[0062] In some embodiments, the peptide amphiphile comprises a biologically active moiety. In certain embodiments, the biologically active moiety is the most C- or N-terminal segment of the PA (e.g., the end opposite the hydrophobic tail). In some embodiments, the biologically active moiety is attached to the end of a charged segment. In some embodiments, the biologically active moiety is exposed on the surface of the assembled PA structure (e.g., nanofiber). The biologically active moiety is typically, but not limited to, a peptide. The examples detailed herein utilize a peptide sequence that binds BMP-2 as the biologically active moiety. In some embodiments, the biologically active peptide is a therapeutic peptide. Bioactive peptides and other moieties to achieve functionality will be understood. In some embodiments, biologically active moieties are provided that have a binding affinity for a target protein of less than 10 μM, less than 100 μM, less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, etc.

[0063] In some embodiments, the bioactive peptide is a binding peptide capable of binding to a bioactive factor (e.g., a protein or other bioactive molecule) associated with the intended purpose of the material, hi some embodiments, binding of the bioactive peptide to the bioactive factor results in the bioactive factor being associated with the PA nanofiber.

[0064] In some embodiments, the bioactive peptide is a peptidomimetic of a bioactive factor (e.g., a protein or other bioactive molecule) associated with the intended purpose of the material. In such embodiments, the bioactive peptide mimics the function of the bioactive factor rather than recruiting the bioactive factor to the nanofibers.

[0065] In some embodiments, the PA herein presents a bioactive peptide capable of binding to or mimicking the function of a bioactive factor selected from protein / polypeptide agents such as enzymes, receptors, channel proteins, hormones, cytokines, growth factors, and antibody drugs. In some embodiments, the bioactive factor is used in repair of tissue / bone defects and / or tissue / bone generation / regeneration. Suitable bioactive factors include those that stimulate bone formation (osteogenesis), bone regeneration, ... morphogenic proteins (e.g., BMP-1, BMP-2, BMP-4, BMP-6, and BMP-7); members of the transforming growth factor beta (TGF-β) superfamily, including, but not limited to, TGF-β1, TGF-β2, and TGF-β3; epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), growth differentiation factors (GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, myostatin / GDF8, GDF9, GDF10, GDF11, and GDF15); human endothelial growth factor (ECGF); granulocyte-macrophage colony-stimulating factor (GM-CSF); nerve growth factor (NGF); vascular endothelial growth factor (VEGF); fibroblast growth factor (FGF); insulin-like growth factor (IGF); cartilage-derived morphogenetic protein (CDMP); platelet-rich plasma (PRP); platelet-derived growth factor (PDGF); insulin growth factor 1 (IGF-I); or any combination thereof.

[0066] In certain embodiments exemplified herein, the bioactive factor is BMP-2, and the PA nanofibers of the composite material display a BMP-2-binding peptide. In some embodiments, the BMP-2-binding peptide is of the sequence TSPHVPYGGGS (SEQ ID NO: 1). In some embodiments, other bioactive peptides that can mimic and / or bind to bioactive factors are understood and have proven useful when displayed on PA nanofibers. The embodiments herein are not limited to use with BMP-2-binding peptides. In some embodiments, the bioactive peptide binds BMP-2 and has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or ranges therebetween) sequence identity to that of SEQ ID NO: 1 (TSPHVPYGGGS). In some embodiments, the bioactive peptide binds BMP-2 and has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or any range therebetween) sequence similarity (e.g., conservative or semi-conservative) with SEQ ID NO:1 (TSPHVPYGGGS). In some embodiments, the bioactive peptide binds BMP-2 and has four or fewer (e.g., 4, <4, 3, <3, 2, <2, 1, 0) substitutions relative to SEQ ID NO:1 (TSPHVPYGGGS). In some embodiments, the bioactive peptide binds BMP-2 and has four or fewer (e.g., 4, <4, 3, <3, 2, <2, 1, 0) non-conservative substitutions relative to SEQ ID NO:1 (TSPHVPYGGGS). In some embodiments, the bioactive peptide binds BMP-2 and has four or fewer (e.g., 4, <4, 3, <3, 2, <2, 1, 0) semi-conservative substitutions relative to SEQ ID NO: 1. In some embodiments, the bioactive peptide binds BMP-2 and has four or fewer (e.g., 4, <4, 3, <3, 2, <2, 1, 0) conservative substitutions relative to SEQ ID NO: 1 (TSPHVPYGGGS).

[0067] In some embodiments, the bioactive peptide amphiphile comprises: (a) a hydrophobic tail comprising an 8-24 carbon alkyl chain; (b) an alanine- and valine-rich peptide segment (e.g., AAVV (SEQ ID NO: 2), AAAVVV (SEQ ID NO: 3), VVAA (SEQ ID NO: 4), VVVAAA (SEQ ID NO: 5), or other combinations of V and A residues); (c) a charged segment (e.g., comprising EE or EEE), and (d) a bioactive peptide (e.g., a BMP-2 binding peptide (e.g., SEQ ID NO: 1 (TSPHVPYGGGS))).

[0068] In some embodiments, the diluent peptide amphiphile comprises: (a) a hydrophobic tail comprising an 8-24 carbon alkyl chain; (b) an alanine- and valine-rich peptide segment (e.g., AAVV (SEQ ID NO: 2), AAAVVV (SEQ ID NO: 3), VVAA (SEQ ID NO: 4), VVVAAA (SEQ ID NO: 5), or other combinations of V and A residues); and (c) a charged segment (e.g., comprising EE or EEE).

[0069] In some embodiments, the bioactive or dilutable PA further comprises a linking segment or residue (e.g., K) for attaching a hydrophobic tail to the peptide portion of the PA. In some embodiments, the hydrophobic tail is attached to a lysine side chain. In other embodiments, the hydrophobic tail is attached directly to the terminal amino acid of the structural peptide segment.

[0070] In some embodiments, the bioactive peptide amphiphile comprises (e.g., from C-terminus to N-terminus or N-terminus to C-terminus): a bioactive peptide (e.g., a BMP-2 binding peptide); a charged segment (e.g., including EE, EEE, etc.); a structural segment (e.g., including VVAA (SEQ ID NO: 4), AAVV (SEQ ID NO: 2), VA, AV, etc.); and a hydrophobic tail (e.g., including an alkyl chain of 8 to 24 carbons).

[0071] In some embodiments, the bioactive peptide amphiphile comprises (e.g., from C-terminus to N-terminus or N-terminus to C-terminus): a bioactive peptide (e.g., a BMP-2 binding peptide); a charged segment (e.g., including EE, EEE, etc.); a structural segment (e.g., including VVAA (SEQ ID NO: 4), AAVV (SEQ ID NO: 2), VA, AV, etc.); a linking segment or peptide (e.g., K); and a hydrophobic tail (e.g., including an alkyl chain of 8 to 24 carbons).

[0072] In some embodiments, the bioactive peptide amphiphile has the structure (e.g., C-terminus to N-terminus or N-terminus to C-terminus): TSPHVPYGGSEEEAAVVVK-C 12 (SEQ ID NO: 6).

[0073] In some embodiments, the diluent peptide amphiphile comprises (e.g., from C-terminus to N-terminus or N-terminus to C-terminus): a charged segment (e.g., comprising EE, EEE, etc.); a structural segment (e.g., comprising VVAA (SEQ ID NO: 4), AAVV (SEQ ID NO: 6), VA, AV, etc.); and a hydrophobic tail (e.g., comprising an alkyl chain of 8 to 24 carbons).

[0074] In some embodiments, the diluent peptide amphiphile comprises (e.g., from C-terminus to N-terminus or N-terminus to C-terminus): a charged segment (e.g., including EE, EEE, etc.); a structural segment (e.g., including VVAA (SEQ ID NO: 4), AAVV (SEQ ID NO: 6), VA, AV, etc.); a linking segment or peptide (e.g., K); and a hydrophobic tail (e.g., including an alkyl chain of 8 to 24 carbons).

[0075] In some embodiments, the diluent peptide amphiphile has the structure (e.g., from C-terminus to N-terminus or N-terminus to C-terminus): EEEAAAVVV--C 16 (SEQ ID NO: 7).

[0076] In some embodiments, provided herein are nanofibers and nanostructures assembled from peptide amphiphiles described herein. In some embodiments, the nanofibers are prepared by self-assembly of PAs described herein. In some embodiments, the nanofibers comprise or consist of PAs displaying bioactive peptides. In some embodiments, the bioactive peptides are displayed on the surface of the nanofibers. In some embodiments, a dilutable PA is included in the nanofiber in addition to the PA displaying the bioactive peptide. In some embodiments, the dilutable PA is a peptide amphiphile described herein (e.g., structural segment, charged segment, hydrophobic segment, etc.) but lacks the bioactive peptide. In some embodiments, the dilutable PA and the bioactive PA self-assemble into nanofibers comprising both types of PA. In some embodiments, provided herein are nanostructures (e.g., nanofibers) assembled from peptide amphiphiles described herein.

[0077] In some embodiments, the nanostructures are assembled from (1) PAs bearing a biologically active moiety (e.g., a BMP-2 binding moiety) and (2) loaded PAs (e.g., unlabeled PAs or PAs that do not display a biologically active moiety). In some embodiments, the nanostructures (e.g., nanofibers) comprise (i) less than 50% (e.g., 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or any range therebetween) of PAs bearing a biologically active moiety (e.g., a BMP-2 binding moiety). In some embodiments, the nanostructures (e.g., nanofibers) comprise at least 2% (e.g., 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or any range therebetween) PA having a biologically active moiety (e.g., BMP-2 binding moiety). In some embodiments, the nanofibers comprise at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or any range therebetween) loaded peptide amphiphile.

[0078] In some embodiments, the ratio of PA with bioactive moieties to loaded PA determines the density of bioactive moieties (eg, BMP-2 binding moieties) displayed on the nanostructure surface.

[0079] In some embodiments where a PA or PA nanofiber capable of binding a bioactive factor (e.g., nanofibers displaying a BMP-2 binding peptide) is provided, the PA or PA nanofiber is provided in a composition (e.g., a composite) comprising such a bioactive factor. As noted above, bioactive factors that may be used in the compositions herein (e.g., with a bioactive PA capable of binding a bioactive factor) include bone formation inhibitors, bone graft ... morphogenic proteins (e.g., BMP-1, BMP-2, BMP-4, BMP-6, and BMP-7); members of the transforming growth factor beta (TGF-β) superfamily, including, but not limited to, TGF-β1, TGF-β2, and TGF-β3; epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), growth differentiation factors (GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, myostatin / GDF8, GDF9, GDF10, GDF11, and GDF15); human endothelial growth factor (ECGF); granulocyte-macrophage colony-stimulating factor (GM-CSF); nerve growth factor (NGF); vascular endothelial growth factor (VEGF); fibroblast growth factor (FGF); insulin-like growth factor (IGF); cartilage-derived morphogenetic protein (CDMP); platelet-rich plasma (PRP); insulin growth factor 1 (IGF-I); platelet-derived growth factor (PDGF); or any combination thereof. The embodiments herein are not limited to only these bioactive factors.

[0080] In other embodiments, where PA or PA nanofibers capable of binding bioactive factors (e.g., nanofibers displaying BMP-2 binding peptides) are provided, the PA or PA nanofibers are provided in compositions (e.g., composites) lacking exogenous bioactive factors. In some embodiments, PA or PA nanofibers are provided that are capable of binding endogenous bioactive factors and exerting therapeutic benefit, and thus are provided without exogenous bioactive factors.

[0081] In some embodiments, the compositions (e.g., composite materials) herein comprise one or more polymeric materials. In some embodiments, the polymer is provided to allow the material to be molded into a desired shape. In some embodiments, the polymer imparts structural integrity to the composition. Suitable polymers for use in certain embodiments herein may include hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose and salts thereof, Carbopol, poly(hydroxyethyl methacrylate), poly(methoxyethyl methacrylate), poly(methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), methyl methacrylate (MMA), gelatin, polyvinyl alcohol, propylene glycol, PEG 200, PEG 300, PEG 400, PEG 500, PEG 550, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1450, PEG 3350, PEG 4500, PEG 8000, or combinations thereof.

[0082] In certain embodiments, the compositions (e.g., composite materials) herein comprise one or more polyethylene glycols (e.g., PEG1450, PEG600, etc.). In some embodiments, the compositions (e.g., composite materials) herein comprise PEG1450 and PEG600.

[0083] In some embodiments, the polymer component(s) comprise up to 95% by weight of the components of the composition (e.g., 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, or ranges therebetween).

[0084] In some embodiments, the compositions (e.g., composite materials) herein comprise one or more ceramic materials. In some embodiments, the ceramic component(s) impart compression resistance to the material. Suitable ceramics include hydroxyapatite (HA), tricalcium phosphate (TCP), bioglass, calcium sulfate, and the like. In some embodiments, the composition comprises HA and / or TCP. In some embodiments, the ceramic is provided as a particulate material. In some embodiments, the ceramic is uniformly distributed throughout the material. In some embodiments, the ceramic (e.g., HA, TCP) has a particle size (e.g., average diameter of the particles in a population) of 10 to 2000 μm (e.g., 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1500 μm, 2000 μm, or ranges therebetween).

[0085] In some embodiments, the ceramic component(s) comprise 5-50% by weight (e.g., 5%, 10%, 25%, 30%, 35%, 40%, 45%, 50%, or any range therebetween) of the components of the composition.

[0086] Provided herein are compositions comprising composite materials, particularly for medical applications. In some embodiments, the composite materials comprise peptide amphiphilic nanostructures, a polymer component, and a ceramic component.

[0087] In some embodiments, the composite material comprises 5% or less by weight (e.g., 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% or less, or ranges therebetween) of peptide amphiphile nanofibers. In some embodiments, the composite material comprises 50% and 90% by weight (e.g., 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, or ranges therebetween) of the polymer component(s) (e.g., PEG1450 and PEG600 combined). In some embodiments, the composite material comprises 35% and 55% by weight (e.g., 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, or any range therebetween) of the first polymer component (e.g., PEG 1450). In some embodiments, the composite material comprises 15% and 35% by weight (e.g., 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, or any range therebetween) of the second polymer component (e.g., PEG 600). In some embodiments, the composite material comprises between 15% and 45% by weight (e.g., 15%, 20%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, or ranges therebetween) of ceramic component(s) (e.g., HA or TCP). In some embodiments, the composite material comprises 5% by weight or less (e.g., 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% or less, or ranges therebetween) of a bioactive factor (e.g., BMP-2).

[0088] An exemplary composite material comprises <1 wt% dilutable PA, <1 wt% bioactive PA, 46 wt% PEG1450, 23% PEG600, 30 wt% TCP, and <1 wt% BMP-2.

[0089] In some embodiments, the composite material is free of water (e.g., <0.1 wt%, <0.05 wt%, <0.02 wt%, <0.01 wt%, <0.005 wt%, <0.002 wt%, <0.001 wt%, etc.). In some embodiments, the composite material and / or one or more components thereof have been processed (e.g., freeze-dried, lyophilized, etc.) to remove water from the composite and / or components. In some embodiments, a bioactive component (e.g., a bioactive peptide, a bioactive factor, etc.) is active when delivered within the dry composite material.

[0090] In some embodiments, the materials herein allow for orders of magnitude lower therapeutic BMP-2 doses for successful spinal fusion (e.g., 10-fold lower doses of rhBMP-2 than water-based PAs, a 1000-fold reduction compared to clinical doses).

[0091] The compositions described herein are used in medical and veterinary applications, such as bone / tissue repair, bioactive factor delivery, and regenerative medicine. The exemplary composite materials described herein are used for bone regeneration (e.g., for spinal fusion applications). However, the techniques described herein can be used to create composites containing peptide amphiphile nanofibers displaying different bioactive peptides and / or containing different bioactive factors. For example, the bioactive peptides and / or factors included in the composite can be tailored to promote cell adhesion or mimic the function of growth factors. In addition to bone repair, the materials can be used in the regeneration / repair of cartilage or other connective tissue, muscle, skin, etc. In some embodiments, the relative percentages and types of polymers and / or ceramics can be tailored to create composites that are more or less compressible, moldable, viscous, stable, biodegradable, etc.

[0092] In some embodiments, methods are provided for administering the composite materials described herein to a subject. In the exemplary case of a bone defect or injury (e.g., to create a spinal fusion), the materials described herein are molded into the treatment site. In some embodiments, the exemplary materials described herein promote bone formation at the treatment site.

[0093] In some embodiments, the materials described herein are used in posterolateral spinal fusion procedures, where bone graft substitutes are implanted within the spinal muscle space. The treatment site has an irregular shape, requiring a moldable material. Furthermore, once the surgical site is closed, surrounding muscle tissue exerts forces against the implant, and patient movement can also change the position of the implant. The implant must maintain its integrity, remain in place, and resist compression against these forces. Exemplary materials described herein are used to meet the requirements of this procedure.

[0094] experiment [Example]

[0095] [Example 1] Composite synthesis Provided below are procedures for the synthesis of exemplary composite materials within the scope of the present specification. The composite materials described below are of particular use in the repair of bone defects. Alternative components, procedures, materials, and applications are contemplated and are within the scope of the present specification.

[0096] Step 1a: Preparing a peptide amphiphile solution containing rhBMP-2 A bioactive PA supramolecular polymer has been identified that can enhance rhBMP-2 signaling, thereby reducing the therapeutic dose required to achieve bone growth (Lee, SS, Hsu, EL, Mendoza, M., Ghodasra, J., Nickoli, MS, Ashtekar, A., Polavarapu, M., Babu, J., Riaz, RM, Nicolas, J.D. and Nelson, D., 2015. Gel scaffolds of BMP-2 binding peptide amphiphile nanofibers for spinal arthrodesis. Advanced healthcare materials, 4(1), pp. 131-141.; incorporated by reference in its entirety). This bioactive PA has been used in bone repair applications, such as spinal fusion surgery, because existing rhBMP-2 products require supraphysiological doses to achieve sufficient bone growth and can result in dangerous side effects (although effective). This PA-based supramolecular polymer contains two PA molecules: a bioactive PA capable of binding BMP-2 and a dilute PA without any bioactive components (Figure 1). The bioactive PA supramolecular polymer was prepared by dissolving 1.2 wt% (12 mg / mL) dilute PA in water containing 15 mM NaOH and 1.2 wt% (12 mg / mL) bioactive PA in water containing 30 mM NaOH, mixing equal volumes of the PA solutions, resulting in a final PA solution with equal wt% of both PAs (0.6 wt% each). The PA mixture was then heated (thermally annealed) at 80 °C (e.g., in a water bath or oven) for 30 min and then allowed to cool to room temperature. Typically, the PA solution was allowed to cool slowly and passively overnight, although shorter or longer cooling times can be used. To the cooled PA mixture, rhBMP-2 solution was added to achieve a final PA concentration of 1 wt% (10 mg / mL).

[0097] The rhBMP-2 concentration can be adjusted to the desired amount. As an example, when this supramolecular polymer was tested in a rat spinal fusion model, the desired rhBMP-2 dose was 10–100 ng / animal (5–50 ng / scaffold, two scaffolds per animal). At the size scale of a rat spine, each scaffold requires approximately 130 μL of PA solution, delivered via a structural component such as a polymer / ceramic putty. Therefore, a solution containing 1 wt% PA and 50 ng rhBMP-2 in 130 μL is desired and is prepared as follows: starting with a 2.307 μg / mL (2307 ng / mL) rhBMP-2 solution, add 50 μL of rhBMP-2 solution to 250 μL of 1.2 wt% annealed PA solution. This results in a solution with 1 wt% PA and 384.5 ng / mL rhBMP-2, or 0.3845 ng / μL rhBMP-2. This solution contains 0.3845 ng / μL of rhBMP-2, so 130 μL of this solution contains 50 ng of rhBMP-2.

[0098] For the specific purpose described in step 1a (preparing PA implants for a rat spinal fusion model), an exemplary solution containing 1% by weight PA and 0.3845 ng / μL rhBMP-2 was identified as preferred. For other embodiments of the present invention, the supramolecular polymer may be at a different concentration in the solution, or the solution may contain different concentrations or types of bioactive growth factors or other therapeutic agents. For example, the PA solution may be mixed with recombinant transforming growth factor beta-1 (TGFβ-1). In addition to growth factors and their peptidomimetics, the PA solution may also be mixed with small molecule drugs such as resveratrol. Furthermore, for example, when the supramolecular polymer is used to bind endogenous growth factors when implanted in the body, the PA solution may contain no exogenous growth factors or therapeutic agents at all.

[0099] Step 1b: Prepare the polymer / ceramic putty The bioactive components (supramolecular polymer and rhBMP-2) prepared in step 1a are a liquid solution that cannot be directly implanted into the defect. The implant must be able to maintain its shape and remain in place, ideally allowing the surgeon to mold the implant to fit irregular shapes. A composite putty of bioactive components is made by weighing out a ratio of 46.7% PEG 1450, 23.3% PEG 600, and 30% ceramic (PEG:polyethylene glycol polymer). In this exemplary embodiment of the present invention for spinal fusion surgery, the ceramic is tricalcium phosphate (TCP) particles. As an example, if a final putty of 700 mg is desired, the following materials are weighed: 326.9 mg PEG 1450, 163.1 mg PEG 600, and 210 mg TCP. This particular combination of polymers has been found to result in a putty-like material with favorable surgical handling properties at room temperature. However, any water-soluble material with these handling properties may be used in embodiments within the scope of this specification. For example, other polymers that may be used include polyethylene glycol, polyacrylic acid copolymers, and polyvinyl alcohol.

[0100] The two PEG components were placed in the same container and heated at 50°C until a clear liquid was observed. For the exemplary amounts listed above, approximately 15 minutes of heating was sufficient when 50°C was used for heating. After a clear liquid was observed, the PEG solution was removed from the heat. The PEG polymer almost immediately began to form a white waxy solid. TCP particles were added to this waxy solid.

[0101] Step 2: Combine the PA solution (containing any added growth factors or pharmaceuticals) with the putty The PA solution was added to the PEG and TCP mixture. For each 700 mg putty, 520 μL of PA solution was added. The water from the PA solution immediately dissolved the PEG, creating a "slush-like" material. The material was mixed well to evenly distribute the TCP particles. The PEG, TCP, PA, and rhBMP-2 mixture was lyophilized to remove the water. The resulting material was compressed into the form of a putty material. PEG provides moldability, and TCP provides compression resistance. In an exemplary embodiment of the present invention for spinal fusion surgery, PA and rhBMP-2 are the bioactive components contained within the putty. Other drying methods, such as evaporation, can be used to remove water from the material.

[0102] [Example 2] Effect of TCP on handling properties and biological function TCP comes in many different forms. In an exemplary embodiment of the present invention for spinal fusion surgery, described in Example 1, "small" and "large" TCP particles were investigated. The small TCP particles ranged in size from 53 to 250 μm, while the large TCP particles ranged in size from 250 to 1000 μm. While the small TCP particles handled like a powder, the large TCP particles were visually distinct particulate matter. Consequently, the "small TCP putty" was a smooth putty with no detectable individual particles during handling, whereas the "large TCP putty" felt rougher during handling (particles detectable by touch). The addition of small or large TCP resulted in putties with improved handling properties. The putty material held together, had sufficient compression resistance for implantation into defects, and exhibited an increased storage modulus (over 200-fold) compared to materials lacking TCP. Furthermore, surgeons experienced in spinal fusion surgery tested the putties and determined that, although both had acceptable handling properties for direct implantation into irregularly shaped defects, the large TCP particle version had greater compression resistance. This is because the large TCP particles have better resistance to compression compared to the small TCP particles that are treated like a powder, which is reflected in the slightly higher storage modulus of the large TCP putty.

[0103] The size of the TCP particles (small: 53-250 μm, large: 250-1000 μm) affects the bioactivity of the putty. In an embodiment of the present invention for spinal fusion surgery, when the putty is made with large TCP particles, the material is able to achieve 100% fusion in a rat spinal fusion model using 100 ng of rhBMP-2 per rat (Figure 3). In this animal model, this is considered a low dose of rhBMP-2, which is advantageous because supraphysiological rhBMP-2 doses can result in undesirable and dangerous side effects. When the large TCP particles are replaced with an equal weight of small TCP particles and all other putty components are the same, the fusion rate is significantly lower (Figure 3). Some fusion was observed even when the putty did not contain BMP-2 (Figure 3). The large TCP putty achieved a fusion rate of approximately 42% without delivering BMP-2 (Figure 3), indicating some mobilization of endogenous growth factors. Small TCP putties that do not deliver BMP-2 achieved less fixation compared to large TCP putties (17% vs. 42%), indicating that the TCP particles provide some bioactivity.

[0104] In PEG / TCP carriers for bioactive supramolecular polymers and rhBMP-2, the ratio of PEG to TCP can affect mechanical and handling properties. While PEG is water-soluble, TCP is not, which may affect the implant degradation rate and bioactivity in vivo. An exemplary embodiment containing 30% large TCP particles by weight was able to achieve 100% in a rat spinal fusion model using a low dose of rhBMP-2 (Figure 3). In addition to favorable biological function, this embodiment also had favorable handling properties, as verified by surgeons and demonstrated in rheological experiments. Different ratios of TCP particles were investigated by varying the weight percentage of TCP to 30%, 15%, and 10%. When these putties were examined by surgeons experienced in spinal fusion surgery, it was found that the putties with 15% and 10% TCP by weight had lower compression resistance than the putty with 30% TCP by weight. However, all were deemed usable for spinal fusion implantation. The lower TCP percentage correlated with a slightly lower storage modulus of the putty (Figure 4).

[0105] The weight percent of TCP in the putty can also affect bioactivity. When TCP constitutes 30% of the putty by weight, the putty is more effective at promoting spinal fusion at low BMP-2 doses in a rat model, even achieving 100% fusion with 10 ng of rhBMP-2 (Figure 5). For the purpose of inducing bone growth to promote spinal fusion, 30% TCP by weight appears highly advantageous, resulting in an unprecedented 1000-fold dose reduction compared to clinically used therapeutic doses. For other therapeutic applications, other ratios of TCP to other components may be desirable.

[0106] [Example 3] Further characterization and testing PA structure in putty A putty containing 30% large TCP particles was further characterized. In some embodiments, in aqueous solution, PA nanofibers contain PA molecules held together by non-covalent β-sheet structures. In the putty, PA is not present in aqueous solution but instead resides, for example, between PEG polymer chains. FTIR (Fourier Transform Infrared Spectroscopy) experiments on an exemplary putty showed the presence of β-sheet structure ( FIG. 6A ), indicating that PA remains assembled into nanofiber-like structures within the non-aqueous putty.

[0107] Protein distribution within the putty The BMP-2-conjugated PA contains an epitope designed to bind the BMP-2 growth factor, whereas the negatively charged nanofibers can nonspecifically bind charged proteins. To visualize protein distribution within the putty, fluorescently tagged bovine serum albumin (BSA, considered a "model" protein) was loaded into a putty containing fluorescently tagged PA and fluorescently tagged PEG (Figure 6B). These images show colocalization of the PA with BSA (Figure 6B).

[0108] Release of BMP-2 from PA-containing putties One limitation of existing clinical products that use collagen carriers to deliver BMP-2 is that collagen has no affinity for BMP-2. Therefore, upon implantation, a burst release of BMP-2 occurs, and BMP-2 does not remain at the site where new bone is needed. PA nanofibers are designed to bind BMP-2 growth factors and retain them at the implant site. When BMP-2 was loaded into both collagen and putty materials, the putty exhibited a slower release rate (Figure 6C).

[0109] Large Animal Models Following its success in a small-animal model (rat spinal fusion), a putty containing 30% large TCP particles was tested in a widely accepted, rigorous large-animal model (rabbit posterolateral spinal fusion). For comparison, an ACS (absorbable collagen sponge, used in the current clinical product, INFUSE™) implant delivering BMP-2 was also tested. ACS implants delivering 60 μg of BMP-2 per rabbit achieved a 50% fusion rate, whereas lowering the BMP-2 dose to 30 μg resulted in a 0% fusion rate (Figure 7). However, when the putty was used to deliver an ultra-low dose of 1 μg of BMP-2 per rabbit, approximately 83% of the animals successfully fused (Figure 7). A putty without BMP-2 achieved approximately 92% fusion (Figure 7), demonstrating the mobilization of endogenous BMP-2 and the bioactivity of the TCP particles. In a rat model, putty without BMP-2 achieved a fixation rate of 42%, lower than the 92% achieved in rabbits, a larger animal with a larger defect size. This is likely due to variations in the bone decortication procedure during surgery, as the amount of decortication to expose osteoprogenitor cells can affect the ability of bone regeneration. The data demonstrate that putty without BMP-2 has the ability to induce bone growth without osteogenic growth factors.

[0110] array SEQ ID NO: 1 TSPHVPYGGGS SEQ ID NO:2 AAVV SEQ ID NO: 3 AAAVVV SEQ ID NO: 4 VVAA SEQ ID NO: 5 VVVAAA SEQ ID NO: 6 TSPHVPYGGSEEEAAVVVK SEQ ID NO: 7 EEEAAAVVV

Claims

1. (a) peptide amphiphilic nanofibers; (b) a polymer component; and (c) ceramic component A composition comprising a composite material comprising:

2. The peptide amphiphile nanofiber comprises a supramolecular assembly of a peptide amphiphile, the peptide amphiphile comprising: (i) a hydrophobic non-peptidic segment; (ii) a structural peptide segment; and (iii) charged segments The composition of claim 1 comprising:

3. The composition of claim 1 , wherein the polymer component comprises polyethylene glycol (PEG).

4. 4. The composition of claim 3, wherein the polymer component comprises a PEG selected from PEG200, PEG300, PEG400, PEG500, PEG550, PEG600, PEG700, PEG800, PEG900, PEG1000, PEG1450, PEG3350, PEG4500, PEG8000, or combinations thereof.

5. The composition of claim 4 , wherein the polymer component comprises PEG 600 and PEG 1450.

6. The composition of claim 1 , wherein the ceramic component comprises hydroxyapatite (HA), tricalcium phosphate (TCP), bioglass, or calcium sulfate.

7. The composition of claim 6 , wherein the ceramic component comprises a TCP.

8. The composition of claim 1 , wherein the composite material further comprises a bioactive factor.

9. 10. The composition of claim 1, wherein the composite material comprises less than 5 wt. % peptide amphiphile nanofibers, 60-80 wt. % polymer component, and 20-40 wt. % ceramic component.

10. 10. The composition of claim 9, wherein the composite material comprises less than 1 wt. % peptide amphiphile nanofibers, 65-75 wt. % polymer component, and 25-35 wt. % ceramic component.

11. 11. The composition of claim 10, wherein the composite material comprises less than 1% by weight of peptide amphiphile nanofibers, about 69% by weight of a polymer component, and about 30% by weight of a ceramic component.

12. The composition of claim 2 , wherein all or a portion of the peptide amphiphile further comprises a biologically active peptide segment.

13. The composition of claim 12, wherein the bioactive peptide segment mimics the biological function of a bioactive factor and / or cellular component or is capable of binding to a bioactive factor and / or cellular component.

14. 14. The composition of claim 13, wherein the bioactive factors and / or cellular components are selected from bone morphogenic proteins, transforming growth factors, epidermal growth factors, growth differentiation factors, human endothelial growth factors, granulocyte-macrophage colony-stimulating factors, nerve growth factors, vascular endothelial growth factors, fibroblast growth factors, insulin-like growth factors, cartilage-derived morphogenetic proteins, platelet-rich plasma, platelet-derived growth factors, insulin growth factor 1, and platelet-derived growth factors.

15. Peptide amphiphilic nanofibers (i) a bioactive peptide amphiphile comprising (A) a hydrophobic nonpeptidic segment, (B) a structural peptide segment, (C) a charged segment, and (D) a bioactive peptide; and (ii) a diluent peptide amphiphile comprising (A) a hydrophobic non-peptidic segment, (B) a structural peptide segment, and (C) a charged segment; The composition of claim 12, comprising a supramolecular assembly of

16. 16. The composition of claim 15, wherein (i) and (ii) are present in a ratio of 1:10 to 10:

1.

17. 17. The composition of claim 16, wherein (i) and (ii) are present in a ratio of 1:2 to 2:

1.

18. The composition of claim 15 , wherein the hydrophobic, non-peptidic segment comprises an acyl chain.

19. Acyl chain is C ６ -C ２０ 20. The composition of claim 18, comprising:

20. 20. The composition of claim 19, wherein the hydrophobic non-peptidic segment of the diluent peptide amphiphile and the hydrophobic non-peptidic segment of the bioactive peptide amphiphile are the same length.

21. 20. The composition of claim 19, wherein the hydrophobic non-peptidic segment of the diluent peptide amphiphile and the hydrophobic non-peptidic segment of the bioactive peptide amphiphile are of different lengths.

22. The hydrophobic non-peptidic segment of the diluent peptide amphiphile is C １６ and the hydrophobic non-peptidic segment of the bioactive peptide amphiphile is C １２ 22. The composition of claim 21, wherein:

23. 16. The composition of claim 15, wherein the structural peptide segment is an alanine and valine rich peptide segment.

24. 24. The composition of claim 23, wherein the alanine and valine-rich peptide segment comprises AAVV (SEQ ID NO: 2), AAAVVV (SEQ ID NO: 3), VVAA (SEQ ID NO: 4), or VVVAAA (SEQ ID NO: 5).

25. 16. The composition of claim 15, wherein the charged peptide segment is a glutamic acid and / or aspartic acid rich segment.

26. 26. The composition of claim 25, wherein the glutamic acid and / or aspartic acid rich segment comprises 2 to 7 amino acids in length, with 50% or more of the amino acids being selected from Glu (E) and / or Asp (D) residues.

27. 27. The composition of claim 26, wherein the glutamic acid and / or aspartic acid rich segment comprises EE or EEE.

28. The composition of claim 15 , wherein the bioactive peptide is capable of binding to a bioactive factor.

29. 29. The composition of claim 28, wherein the bioactive factor is BMP-2.

30. 30. The composition of claim 29, wherein the bioactive peptide is a BMP-2 binding peptide.

31. 31. The composition of claim 30, wherein the bioactive peptide comprises at least 50% sequence identity with TSPHVPYGGGS (SEQ ID NO: 1).

32. 31. The composition of claim 30, wherein the binding sequence comprises TSPHVPYGGGS (SEQ ID NO: 1).

33. 30. The composition of claim 28, wherein the composition further comprises a bioactive factor.

34. 29. The composition of claim 28, wherein the composition does not include a bioactive factor.

35. A method comprising administering to a subject a composition according to any one of claims 1 to 34.

36. 36. The method of claim 35, wherein the composition is administered to repair bone or tissue damage or defects.

37. (a)(i) a bioactive peptide amphiphile comprising (A) a hydrophobic nonpeptidic segment, (B) a structural peptide segment, (C) a charged segment, and (D) a BMP-2 binding peptide; and (ii) a diluent peptide amphiphile comprising (A) a hydrophobic non-peptidic segment, (B) a structural peptide segment, and (C) a charged segment; Peptide amphiphile nanofibers containing supramolecular assemblies of (b) a PEG-containing polymer component; (c) a ceramic component comprising TCP or HA; and (d) BMP-2 The composition of claim 1 comprising:

38. (a)(i) a bioactive peptide amphiphile comprising (A) a hydrophobic nonpeptidic segment, (B) a structural peptide segment, (C) a charged segment, and (D) a BMP-2 binding peptide; and (ii) a diluent peptide amphiphile comprising (A) a hydrophobic non-peptidic segment, (B) a structural peptide segment, and (C) a charged segment; Peptide amphiphile nanofibers containing supramolecular assemblies of (b) a PEG-containing polymer component; and (c) a ceramic component comprising TCP or HA; Including, The composition of claim 1, wherein the composition does not include BMP-2.

39. 39. A method for promoting bone formation, comprising administering to a subject the composition of claim 37 or 38.

40. 39. A method for repairing bone damage or defects in a subject, comprising administering to the subject a composition according to claim 37 or 38.

41. 40. A method of promoting arthrodesis, comprising administering to a subject a composition according to claim 37 or 38.

42. 40. A method for promoting spinal fusion, comprising administering to a subject the composition of claim 37 or 38.

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