Patent Application
20110117165
The presently disclosed subject matter relates to implantable bone graft materials for promoting bone growth.
Certain growth factors have shown clinical benefit in treatment of bone defects, injuries, disorders, or diseases. In particular, the bone morphogenic proteins (BMP), including BMP-2 and BMP-7, have shown clinical benefit in the treatment of bone fractures and spine fusions. Back pain is one of the leading reasons for physician visits in the United States and in many cases requires surgical intervention. In 2009 in the United States, there were 425,000 spinal fusion surgeries, and the frequency of these surgeries is projected to grow 6% per year. The gold standard for bone graft in spinal fusion is autograft from the iliac crest; however, the use of autograft presents multiple challenges including donor site morbidity, blood loss, limited availability, prolonged operating times, and pseudarthrosis due to a slow rate of fusion. Bone marrow aspirate (BMA) contains osteoinductive factors and can be harvested at point-of-care without the complications of harvesting autogenous bone. However, the current bone graft substitutes are not adequate for the retention and release of osteoinductive factors from BMA over the length of the healing cycle. As a result, there is a large effort to develop bone graft substitutes or extenders that can not only reduce or replace the need for harvest of autogenous bone but also accelerate the rate of fusion (arthrodesis). Tricalcium phosphate (TCP)-based bone graft substitutes often containing collagen are used commonly in lumbar spinal fusion because TCP is resorbed over several months as bone heals. Ceramic bone graft substitutes, such as the MASTERGRAFT and VITOSS line of products, have been used successfully in spinal fusion surgeries (Miyazaki et al., Eur Spine J, 2009, 18:783-99; Khan et al., Am Acad Orthop Surg, 2005, 13:129-37; Neen et al., Spine, 2006, 31:E636-40; Epstein, Spine J, 2009, 9:630-8; Carter, Spine J, 2009, 9:434-8; Epstein, J Spinal Disord Tech, 2006, 19:424-9; Birch, N. and W. L. D'Souza, J Spinal Disord Tech, 2009, 22:434-8; Lerner, T., V., Eur Spine J, 2009, 18:170-9; Knop. et al., Arch Orthop Trauma Surg, 2006, 126:204-10; Epstein, Spine J, 2008, 8:882-7), in particular when used in combination with the recombinant BMP-2-containing product INFUSE (Glassman et al., Spine J, 2007, 7:44-9; Boden et al., Spine, 2002, 27:2662-73; Glassman, Spine, 2005, 30:1694-8). Recombinant BMP-2 is effective but carries a high cost and serious safety risks (Cahill et al., JAMA, 2009, 302:58-66), in part because of leakage away from its carrier and the high dose required to achieve therapeutic levels (Poynton, A. R. and J. M. Lane, Spine, 2002, 27:S40-8).
Therefore, there is an unmet clinical need in bone repair and spinal fusion surgery for a safe, cost-effective bone graft substitute that can maintain a sustained dose of osteoinductive factors throughout the healing process. The presently disclosed subject matter provides such a bone graft substitute.
The presently disclosed subject matter provides compositions and methods for promoting bone growth. In one embodiment, the presently disclosed subject matter provides an implantable bone graft material, wherein the implantable material consists essentially of a resorbable β-TCP and a resorbable polymer, wherein the β-TCP has a total porosity of about 50% or greater and a particle size ranging from about 100 micron to about 300 micron. In one embodiment, the presently disclosed subject matter provides an implantable bone graft material comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide.
In one embodiment, the presently disclosed subject matter provides a method for promoting bone growth in a subject, the method comprising delivering an implantable bone graft material to a subject, wherein the graft material consists essentially of a β-TCP and a resorbable polymer, and wherein the presence of the graft material promotes bone growth. In one embodiment, the presently disclosed subject matter provides a method for promoting bone growth in a subject, the method comprising delivering an implantable bone graft material to a subject, wherein the graft material comprises a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, and wherein the presence of the graft material having attached BMP binding peptide promotes bone growth. In one embodiment, the presently disclosed subject matter provides a method for capturing BMP onto an implantable bone graft material, the method comprising contacting a sample comprising BMP with the graft material, wherein the graft material comprises a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, and wherein the BMP comprised in the sample is captured onto the graft material through binding to the attached BMP binding peptide. In one embodiment, the presently disclosed subject matter provides a method for promoting bone growth in a subject, the method comprising contacting a sample comprising BMP with an implantable bone graft material, wherein the graft material comprises a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, wherein the BMP comprised in the sample is captured onto the graft material through binding to the attached BMP binding peptide; and delivering to the subject the graft material comprising the captured BMP, wherein the presence of the captured BMP promotes bone growth in the subject.
The presently disclosed subject matter provides compositions and methods for promoting bone growth. The compositions and methods of the presently disclosed subject matter are described in greater detail herein below.
While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a BMP binding peptide” or reference to “a 1 unit polyethylene glycol (“mini-PEG” or “MP”)” includes a plurality of such BMP binding peptides or such polyethylene glycol units, and so forth.
The term “substrate” is used, for the purposes of the specification and claims, to refer to any material that is biologically compatible with a BMP and to which a BMP binding peptide can be attached for the purpose of capturing BMP onto the substrate. In one embodiment the substrate is in the form of an implantable device. Therefore, the terms “substrate”, “implantable device”, “implantable bone graft material”, “bone void filler”, and “bone graft substitute” are herein used interchangeably for the purposes of the specification and claims to refer to an implantable medical device for promoting bone formation. In one embodiment, the implantable bone graft material comprises a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide. Accordingly, the term “substrate” is used interchangeably herein with the term “polymer” for the purposes of the specification and claims when referring to the attachment of a BMP binding peptide to a “substrate” it is meant that attachment of the BMP binding peptide is to a polymer comprised in the substrate. Therefore, the attachment of a BMP binding peptide to a “substrate” is referring to attachment of the BMP binding peptide to the polymer comprised in the substrate.
In one embodiment, the implantable bone graft material of the presently disclosed subject matter is a composite of a resorbable ceramic (e.g., TCP) and a resorbable polymer and, therefore, the terms “implantable device”, “implantable bone graft material”, “bone void filler”, “bone graft substitute”, “composite”, and “collagen/TCP composite” are also in some cases used interchangeably for the purposes of the specification and claims. In one embodiment, the implantable bone graft material comprises a composite of a resorbable ceramic and a resorbable polymer. In one embodiment, the ceramic and the polymer are present at a weight ratio ranging from about 10:1 ceramic to polymer to about 2:1 ceramic to polymer. In one embodiment, the weight ratio of the ceramic to the polymer is from about 2:1 (about 66% ceramic to about 33% polymer), from about 3:1 (about 75% ceramic to about 25% polymer), from about 4:1 (about 80% ceramic to about 20% polymer), from about 9:1 (about 90% ceramic to about 10% polymer), from about 10:1 (about 99% ceramic to about 1% polymer).
The term “resorbable ceramic” is herein used interchangeably for the purposes of the specification and claims with the term “ceramic”. The term “resorbable ceramic” is used herein, for the purposes of the specification and claims, to refer to particulate ceramic mineral or inorganic filler useful for promoting bone formation. The term “resorbable ceramic” is herein used interchangeably, for the purposes of the specification and claims, with the terms “ceramic” and “inorganic fillers”. The ceramics of the presently disclosed subject matter include, by non-limiting example, synthetic and naturally occurring inorganic fillers such as alphatricalcium phosphate, beta-tricalcium phosphate, tetra-tricalcium phosphate, dicalcium phosphate, calcium carbonate, barium carbonate, calcium sulfate, barium sulfate, hydroxyapatite, biphasic calcium phosphate (e.g., composite between HA and β-TCP), bioglass, bone particles, and combinations and mixtures thereof. In certain embodiments the ceramic comprises a polymorph of calcium phosphate. Preferably, the ceramic is beta-tricalcium phosphate.
The term “resorbable polymer” is herein used interchangeably for the purposes of the specification and claims with the term “polymer”. The term “resorbable polymer” is used herein, for the purposes of the specification and claims, to refer to a natural resorbable polymer or a synthetic resorbable polymer suitable for use in the implantable medical device of the presently disclosed subject matter. Natural resorbable polymers of the of the presently disclosed subject matter include, by non-limiting example, collagen, fibrillar collagen, Type I collagen, bovine collagen, porcine collagen, human recombinant collagen, keratin, silk, polysaccharides, dextran, cellulose derivatives, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), chitosan, chitin, and hyaluronic acid. In some embodiments, a BMP binding peptide is covalently attached to the natural resorbable polymer. In some cases, the term “resorbable polymer” is used herein, for the purposes of the specification and claims, to refer to a synthetic resorbable polymer. Synthetic resorbable polymers of the presently disclosed subject matter include, by non-limiting example, aliphatic polyesters, polyanhydrides and poly(orthoester)s, and homopolymers, such as, for example, poly(glycolide) (PGA), poly(lactide) (PLLA), poly(ε-caprolactone), poly(trimethylene carbonate) and poly(p-dioxanone), and copolymers, such as for example poly(lactide-co-glycolide) (PLGA), poly(ε-caprolactone-co-glycolide), poly(glycolide-co-trimethylene carbonate), tyrosine-based polycarbonates, and tyrosine-based polyarylates. The synthetic resorbable polymers of the presently disclosed subject matter can be derivatives of the foregoing polymers, and/or statistically random copolymers, segmented copolymers, block copolymers, or graft copolymers of the foregoing polymers. In some embodiments, a BMP binding peptide is attached to the synthetic resorbable polymer of the presently disclosed subject matter. Synthetic resorbable polymers of the presently disclosed subject matter to which a BMP binding peptide can be attached include, by non-limiting example, aliphatic polyesters, polyanhydrides, and poly(orthoester)s. The synthetic resorbable polymers of the presently disclosed subject matter to which a BMP binding peptide can be attached can be derivatives of the foregoing polymers, and/or statistically random copolymers, segmented copolymers, block copolymers, or graft copolymers of the foregoing polymers. In one example, a synthetic “resorbable polymer” to which a BMP binding peptide can be attached, for the purposes of the specification and claims, means a polyanhydride polymer where the anhydride groups are not present in the backbone of the polymer and the portion of the polyanhydride polymer chain that will not be hydrolyzed in vivo is small enough to allow efficient clearance through the renal system. Polymaleic anhydride (PMA) having molecular weight of about 5,000 Dalton or less is one example of a resorbable polyanhydride polymer for the purposes of the specification and claims. In another example, the synthetic resorbable polymer to which the BMP binding peptide is attached is a block co-polymer of polymaleic anhydride having molecular weight of about 5,000 Dalton or less and a co-polymer comprising a biodegradable functionality, wherein the co-polymer is selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polycaprolactone, poly-3-hydroxybutyrate, poly(p-dioxanone) and copolymers thereof, polyhydroxyalkanoate, poly(propylene fumarate), poly(ortho esters), and polyanhydrides, and combinations thereof.
The term “BMP binding peptide” is used herein, for the purposes of the specification and claims, to refer to an amino acid chain comprising a peptide that can bind to a bone morphogenic protein (BMP) (i.e., the BMP is the binding “target” of the BMP binding peptide). The BMPs are members of the transforming growth factor beta (TGF-β) superfamily that share a set of conserved cysteine residues and a high level of sequence identity overall. Over 15 different BMPs have been identified, and most BMPs stimulate the cascade of events that lead to new bone formation and are considered to be osteoinductive factors (see, e.g., U.S. Pat. Nos. 5,013,649; 5,635,373; 5,652,118; and 5,714,589; also reviewed by Reddi and Cunningham (1993) J. Bone Miner. Res. 8 Supp. 2: S499-S502; Issack and DiCesare (2003) Am. J. Orthop. 32: 429-436; and Sykaras & Opperman (2003) J. Oral Sci. 45: 57-73). The BMP's, including BMP-2 and BMP-7, have shown clinical benefit in the treatment of bone fractures and spine fusions. Preferably, the BMP binding peptides of the presently disclosed subject matter bind to one or more of BMP-2, BMP-4, BMP-6, or BMP-7. In one embodiment, the BMP binding peptide is set forth in US Patent Application Publication No. US20060051395A1. In one embodiment, the BMP binding peptide is set forth in US Patent Application Publication No. US20060051395A1 and is identified therein as one of SEQ ID No's: 11-28, 44-74, or 77-94 (i.e., these are the SEQ ID NO identifiers for the previously published patent application rather than this current one). In one embodiment, the BMP binding peptide is set forth in US Patent Application Publication No. US20090098175A1. In one embodiment, the BMP binding peptide is set forth in US Patent Application Publication No: US20090098175A1 and is identified therein as one of SEQ ID No's: 1-12 (i.e., these are the SEQ ID NO identifiers for the previously published patent application rather than this current one). In one embodiment, the BMP binding peptide is set forth in US Patent Application Publication No. US 2006/0051395A1 or US 2009/0098175A1, and is any one of SEQ ID NOs: 1-10 (see Table 1 herein below). The BMP binding peptides of the presently disclosed subject matter can include naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof; however, an antibody is specifically excluded from the scope and definition of a BMP binding peptide of the presently disclosed subject matter. A BMP binding peptide used in accordance with the presently disclosed subject matter can be produced by chemical synthesis, recombinant expression, biochemical, or enzymatic fragmentation of a larger molecule, chemical cleavage of larger molecule, a combination of the foregoing or, in general, made by any other method in the art, and preferably isolated.
BMP binding peptides useful in the presently disclosed subject matter also include peptides having one or more substitutions, additions, and/or deletions of residues relative to the sequence of an exemplary binding peptide shown in US Patent Application Publication No. US 2006/0051395A1 or US 2009/0098175A1, as long as the binding properties of the exemplary BMP binding peptides to their BMP targets are substantially retained. Thus, the BMP binding peptides include those that differ from the exemplary sequences by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and include BMP binding peptides that share sequence identity with the exemplary peptide of at least 65%, 66%, 67%, 68%, 69%, 70%, 71%; 72%, 73%, 74%, 75%, 80%, 81%; 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%; 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Sequence identity can be calculated manually or it can be calculated using a computer implementation of a mathematical algorithm, for example, GAP, BESTFIT, BLAST, FASTA, and TFASTA, or other programs or methods known in the art. Alignments using these programs can be performed using the default parameters. A BMP binding peptide can have an amino acid sequence consisting essentially of a sequence of an exemplary BMP binding peptide or a BMP binding peptide can have one or more different amino acid residues as a result of substituting an amino acid residue in the sequence of the exemplary binding peptide with a functionally similar amino acid residue (a “conservative substitution”); provided that the peptide containing the conservative substitution will substantially retain the BMP binding activity of the exemplary BMP binding peptide not containing the conservative substitution. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine, or methionine for another; the substitution between asparagine and glutamine, the substitution of one large aromatic residue such as tryptophan, tyrosine, or phenylalanine for another; the substitution of one small polar (hydrophilic) residue for another such as between glycine, threonine, serine, and proline; the substitution of one basic residue such as lysine, arginine, or histidine for another; or the substitution of one acidic residue such as aspartic acid or glutamic acid for another. Accordingly, BMP binding peptides useful in the presently disclosed subject matter include those peptides that are conservatively substituted variants of the BMP binding peptides in US Patent Application Publication Nos. US 2006/0051395A1 and US 2009/0098175A1, and those peptides that are variants having at least 65% sequence identity or greater to the BMP binding peptides in US Patent Application Publication Nos. US 2006/0051395A1 and US 2009/0098175A1, wherein all of the variant BMP binding peptides useful in the presently disclosed subject matter substantially retain the ability to bind to BMP. In one embodiment of the presently disclosed subject matter, a useful BMP binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-7 (see Table 1 herein below), conservatively substituted variants of SEQ ID NOs: 1-7, and variants having at least 65% sequence identity to SEQ ID NOs: 1-7, wherein the variant BMP binding peptide substantially retains the ability to bind BMP.
BMP binding peptides can include L-form amino acids, D-form amino acids, or a combination thereof. Representative non-genetically encoded amino acids include but are not limited to 2-aminoadipic acid; 3-aminoadipic acid; β-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2′-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine; N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline; norvaline; norleucine; ornithine; and 3-(3,4-dihydroxyphenyl)-L-alanine (“DOPA”). Representative derivatized amino acids include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.
Further, a BMP binding peptide according to the presently disclosed subject matter can include one or more modifications, such as by addition of chemical moieties, or substitutions, insertions, and deletions of amino acids, where such modifications provide for certain advantages in its use, such as to facilitate attachment to the polymer with or without a spacer or to improve peptide stability. The term “spacer” is used herein, for the purposes of the specification and claims, to refer to a compound or a chemical moiety that is optionally inserted between a BMP binding peptide and the polymer. In some embodiments, the spacer also serves the function of a linker (i.e. to attach the BMP binding peptide to the polymer). Therefore, the terms “linker” and “spacer” can be used interchangeably herein, for the purposes of the specification and claims, when performing the dual functions of linking (attaching) the peptide to the polymer and spacing the BMP binding peptide from the polymer. In some cases the spacer can serve to position the BMP binding peptide at a distance and in a spatial position suitable for BMP binding and capture and/or in some cases the spacer can serve to increase the solubility of the BMP binding peptide. Spacers can increase flexibility and accessibility of the BMP binding peptide to BMP, as well as increase the BMP binding peptide density on the polymer surface. Virtually all chemical compounds, moieties, or groups suitable for such a function can be used as a spacer unless adversely affecting the binding behavior to such an extent that binding of the BMP to the BMP binding peptides is prevented or substantially impaired. Thus, the term “BMP binding peptide” encompasses any of a variety of forms of BMP binding peptide derivatives including, for example, amides, conjugates with proteins, conjugates with polyethylene glycol or other polymers, cyclic peptides, polymerized peptides, peptides having one or more amino acid side chain group protected with a protecting group, and peptides having a lysine side chain group protected with a protecting group. Any BMP binding peptide derivative that has substantially retained BMP binding characteristics can be used in the practice of the presently disclosed subject matter.
Further, a chemical group can be added to the N-terminal amino acid of a binding peptide to block chemical reactivity of the amino terminus of the peptide. Such N-terminal groups for protecting the amino terminus of a peptide are well known in the art, and include, but are not limited to, lower alkanoyl groups, acyl groups, sulfonyl groups, and carbamate forming groups. Preferred N-terminal groups can include acetyl, 9-fluorenylmethoxycarbonyl (Fmoc), and t-butoxy carbonyl (Boc). A chemical group can be added to the C-terminal amino acid of a synthetic binding peptide to block chemical reactivity of the carboxy terminus of the peptide. Such C-terminal groups for protecting the carboxy terminus of a peptide are well known in the art, and include, but are not limited to, an ester or amide group. Terminal modifications of a peptide are often useful to reduce susceptibility by protease digestion, and to therefore prolong a half-life of a BMP binding peptide in the presence of biological fluids where proteases can be present. In addition, as used herein, the term “BMP binding peptide” also encompasses a peptide wherein one or more of the peptide bonds are replaced by pseudopeptide bonds including but not limited to a carba bond (CH2—CH2), a depsi bond (CO—O), a hydroxyethylene bond (CHOH—CH2), a ketomethylene bond (CO—CH2), a methylene-oxy bond (CH2—O), a reduced bond (CH2—NH), a thiomethylene bond (CH2—S), an N-modified bond (—NRCO), and a thiopeptide bond (CS—NH).
The BMP binding peptides are covalently attached to the polymer. The term “attached” in reference to a BMP binding peptide of the presently disclosed subject matter being attached to a polymer means, for the purposes of the specification and claims, a BMP binding peptide being immobilized on the polymer by covalent attachment by any means that will enable binding of BMP onto the peptide-modified polymer such that the bound BMP retains biological growth factor activity. In one embodiment, the linkers/spacers for use in attaching BMP binding peptides to polymers have at least two chemically active groups (functional groups), of which one group binds to the polymer, and a second functional group binds to the BMP binding peptide or in some cases it binds to the “spacer” already attached to the BMP binding peptide. Preferably, the attachment of the BMP binding peptides to the polymer is effected through a spacer. Virtually all chemical compounds, moieties, or groups suitable for such a function can be used as a spacer unless adversely affecting the BMP peptide binding behavior to such an extent that binding of the BMP to the BMP binding peptides is prevented or substantially impaired.
Again, the terms “linker” and “spacer” can be used interchangeably herein, for the purposes of the specification and claims, when performing the dual functions of linking (attaching) the BMP binding peptide to the polymer and spacing the peptide from the polymer. In many embodiments herein, the linkers used to attach the BMP binding peptide to the polymer function as both a linker and a spacer. For example, a linker molecule can have a linking functional group on either end while the central portion of the molecule functions as a spacer. The BMP binding peptides of the presently disclosed subject matter can comprise a functional group that is intrinsic to the BMP binding peptide (e.g., amino groups on lysine), or the functional group can be introduced into the BMP binding peptide by chemical modification to facilitate covalent attachment of the BMP binding peptide to the polymer. Similarly, the polymer can comprise a functional group that is intrinsic to the polymer (e.g., amino groups on collagen), or the polymer can be modified with a functional group to facilitate covalent attachment to the BMP binding peptide. The BMP binding peptide can be covalently attached to the polymer with or without one or more spacer molecules.
For example, linkers/spacers are known to those skilled in the art to include, but are not limited to, chemical compounds (e.g., chemical chains, compounds, reagents, and the like). The linkers/spacers may include, but are not limited to, homobifunctional linkers/spacers and heterobifunctional linkers/spacers. Heterobifunctional linkers/spacers, well known to those skilled in the art, contain one end having a first reactive functionality (or chemical moiety) to specifically link a first molecule (e.g, polymer), and an opposite end having a second reactive functionality to specifically link to a second molecule (e.g, BMP binding peptide). It is evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional can be employed as a linker/spacer with respect to the presently disclosed subject matter such as, for example, those described in the catalog of the PIERCE CHEMICAL CO., Rockford, Ill.; amino acid linkers/spacers that are typically a short peptide of between 3 and 15 amino acids and often containing amino acids such as glycine, and/or serine; and wide variety of polymers including, for example, polyethylene glycol. In one embodiment, representative linkers/spacers comprise multiple reactive sites (e.g., polylysines, polyornithines, polycysteines, polyglutamic acid and polyaspartic acid) or comprise substantially inert peptide spacers (e.g., polyglycine, polyserine, polyproline, polyalanine, and other oligopeptides comprising alanyl, serinyl, prolinyl, or glycinyl amino acid residues). In one embodiment, representative spacers between the reactive end groups in the linkers include, by non-limiting example, the following functional groups: aliphatic, alkene, alkyne, ether, thioether, amine, amide, ester, disulfide, sulfone, and carbamate, and combinations thereof. The length of the spacer can range from about 1 atom to 200 atoms or more. In one embodiment, linkers/spacers comprise a combination of one or more amino acids and another type of spacer or linker such as, for example, a polymeric spacer.
Suitable polymeric spacers/linkers are known in the art, and can comprise a synthetic polymer or a natural polymer. Representative synthetic polymer linkers/spacers include but are not limited to polyethers (e.g., poly(ethylene glycol) (“PEG”), 11 unit polyethylene glycol (“PEG10”), or 1 unit polyethylene glycol (“mini-PEG” or “MP”), poly(propylene glycol), poly(butylene glycol), polyesters (e.g., polylactic acid (PLA) and polyglycolic acid (PGA)), polyamines, polyamides (e.g., nylon), polyurethanes, polymethacrylates (e.g., polymethylmethacrylate; PMMA), polyacrylic acids, polystyrenes, and polyhexanoic acid, and combinations thereof. Polymeric spacers/linkers can comprise a diblock polymer, a multi-block copolymer, a comb polymer, a star polymer, a dendritic or branched polymer, a hybrid linear-dendritic polymer, a branched chain comprised of lysine, or a random copolymer. A spacer/linker can also comprise a mercapto(amido)carboxylic acid, an acrylamidocarboxylic acid, an acrlyamido-amidotriethylene glycolic acid, 7-aminobenzoic acid, and derivatives thereof.
In one embodiment, the binding peptide comprises one or more modifications to the peptide N-terminus, peptide C-terminus, or within the peptide amino acid sequence, to facilitate covalent attachment of the binding peptide to a substrate polymer with or without a spacer. The binding peptides can comprise one or more modifications including, but not limited to, addition of one or more groups such as hydroxyl, thiol, carbonyl, carboxyl, ester, carbamate, hydrazide, hydrazine, isocyanate, isothiocyanate, amino, alkene, dienes, maleimide, α,β-unsaturated carbonyl, alkyl halide, azide, epoxide, N-hydroxysuccinimide (NHS) ester, lysine, or cysteine. In addition, a binding peptide can comprise one or more amino acids that have been modified to contain one or more chemical groups (e.g., reactive functionalities such as fluorine, bromine, or iodine) to facilitate linking the binding peptide to a spacer molecule or to the substrate polymer to which the binding peptide will be attached.
The BMP binding peptides can be covalently attached to the substrate polymer through one or more anchoring (or linking) groups on the substrate polymer and the BMP binding peptide. The BMP binding peptides of the presently disclosed subject matter can comprise a functional group that is intrinsic to the BMP binding peptide, or the BMP binding peptide can be modified with a functional group to facilitate covalent attachment to the substrate polymer with or without a spacer. Representative anchoring (or linking) groups include by non-limiting example hydroxyl, thiol, carbonyl, carboxyl, ester, carbamate, hydrazide, hydrazine, isocyanate, isothiocyanate, amino, alkene, dienes, maleimide, α,β-unsaturated carbonyl, alkyl halide, azide, epoxide, NHS ester, lysine, and cysteine groups on the surface of the substrate polymer. The anchoring (or linking) groups can be intrinsic to the material of the substrate polymer (e.g., amino groups on a collagen or on a polyamine-containing polymer) or the anchoring groups can be introduced into the substrate polymer by chemical modification.
By way of non-limiting example, in one embodiment, a BMP binding peptide is attached to a substrate polymer in a two step process (see
By way of non-limiting example, in one embodiment, a BMP binding peptide is covalently attached to a substrate polymer comprising an amino functional group (see
By way of non-limiting example, in one embodiment, a homobifunctional linker possessing N-hydroxysuccinimide esters at both ends is reacted at one end with the BMP binding peptide having an amino group (
By way of non-limiting example, in one embodiment, a BMP binding peptide is covalently attached to a substrate polymer having amino functional groups in a two-step process using a disulfide linkage (see
By way of non-limiting example, in one embodiment, a BMP binding peptide is attached covalently to a substrate polymer comprising amino functional groups in a similar process using a disulfide linkage (see
By way of non-limiting example, in one embodiment, a BMP binding peptide is attached covalently to a substrate polymer via a thioether bond formed by reaction of a thiol and maleimide (O'Sullivan et al., 1979, Anal. Biochem., 100:100-8). In one embodiment, the maleimide is added to a substrate polymer comprising amino functional groups and then the modified substrate polymer is reacted with a BMP binding peptide having a free thiol group. Alternatively, in one embodiment, the same chemical scheme is utilized but with the substrate polymer modified with a thiol group and the BMP binding peptide modified with the maleimido group.
By way of non-limiting example, in one embodiment, a BMP binding peptide is covalently attached through a non-backbone anhydride group of a polyanhydride polymer, polymaleic acid (PMA), through a reactive lysine group on the BMP binding peptide shown in the schematic diagram in
By way of non-limiting example, in one embodiment, a BMP binding peptide is covalently attached to a chitosan. The chemical scheme is shown in
By way of non-limiting example, in one embodiment a BMP binding peptide is covalently attached to a chitosan. The chemical scheme is shown in
By way of non-limiting example, in one embodiment a BMP binding peptide is covalently attached to chitosan through the amino group on chitosan. For example, a chemical scheme using a homobifunctional N-hydroxysuccinimide ester, such as that described for
By way of non-limiting example, in one embodiment a BMP binding peptide is covalently attached to a hyaluronan (HA). The chemical scheme is shown in
By way of non-limiting example, in one embodiment, a BMP binding peptide is covalently attached to a hyaluronan (HA). The chemical scheme is shown in
By way of non-limiting example, in one embodiment, a BMP binding peptide is covalently attached to cellulose. The chemical scheme is shown in
By way of non-limiting example, in one embodiment, a BMP binding peptide is covalently attached to oxidized cellulose. The chemical scheme is shown in
By way of non-limiting example, in one embodiment, a BMP-2 binding peptide can be covalently attached to an oxidized dextran polymer substrate by reductive amination as described above for oxidized cellulose. More specifically, a BMP-2 binding peptide having a free amine undergoes reductive amination with the aldehyde group on the polymer substrate to yield an amine bond as shown in
By way of non-limiting example, in one embodiment, more than one BMP binding peptide is attached to a substrate polymer. Attaching multiple BMP binding peptides to a single substrate polymer is only limited by practical considerations related to the method of attachment. For example, in one embodiment, two different BMP binding peptides are covalently attached to a substrate polymer using any of the chemical schemes shown in
By way of non-limiting example, in one embodiment, two different BMP binding peptides are covalently attached to a substrate polymer comprising amino groups using the chemical scheme shown in
The presently disclosed subject matter provides compositions and methods for promoting bone growth. In one embodiment, an implantable bone graft material is provided consisting essentially of a resorbable β-TCP and a resorbable polymer, wherein the β-TCP has a total porosity of about 50% or greater and a particle size ranging from about 100 micron to about 300 micron. In one embodiment, the resorbable β-TCP and the resorbable polymer are in the form of a composite. In one embodiment, the composite is in the form of a sponge, a granulized sponge, a putty, or a strip. In one embodiment, the total porosity of the β-TCP is about 70%. In one embodiment, the diameter of the pores in the β-TCP is less than 100 micron. In one embodiment, the polymer is selected from the group consisting of collagen, fibrillar collagen, Type I collagen, bovine collagen, keratin, silk, polysaccharides, dextran, cellulose derivatives, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitosan, chitin, hyaluronic acid, aliphatic polyesters, polyanhydrides, poly(orthoester)s, poly(glycolide), poly(lactide), poly(ε-caprolactone), poly(trimethylene carbonate), poly(p-dioxanone), poly(lactide-co-glycolide), poly(ε-caprolactone-co-glycolide), poly(glycolide-co-trimethylene carbonate), poly(lactide-co-ε-caprolactone), tyrosine-based polycarbonates, tyrosine-based polyarylates, and copolymers and derivatives thereof. In one embodiment, the β-TCP and the polymer are present at a weight ratio ranging from about 10:1 β-TCP to polymer to about 2:1 β-TCP to polymer. In one embodiment, the polymer is collagen and the weight ratio of β-TCP to collagen is about 4:1 (80% β-TCP to about 20% collagen).
In one embodiment, a method is provided for promoting bone growth in a subject by delivering the implantable bone graft material consisting essentially of a β-TCP and a resorbable polymer to a subject, wherein the presence of the graft material promotes bone growth. In one embodiment, a method is provided for promoting spinal fusion in a subject by delivering the implantable bone graft material consisting essentially of a β-TCP and a resorbable polymer to a subject, wherein the presence of the graft material promotes spinal fusion. In one embodiment, the implantable bone graft material consisting essentially of a β-TCP and a resorbable polymer is in the form of a composite. In one embodiment, the composite is in the form of a sponge, a granulized sponge, a putty, or a strip. In one embodiment, the implantable bone graft material is mixed or contacted with saline, bone marrow aspirate (BMA), blood, platelet rich plasma (PRP), or recombinant BMP, or combinations or derivatives thereof prior to or during delivery to the subject.
In one embodiment of the presently disclosed subject matter, an implantable bone graft material is provided comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of a composite. In one embodiment, the composite is in the form of a sponge, a granulized sponge, a putty, or a strip. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of an injectable bone graft material. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of a moldable cement. In one embodiment, the BMP binding peptide binds one or more of BMP-2, BMP-4, BMP-6, or BMP-7.
In one embodiment of the presently disclosed subject matter, an implantable bone graft material is provided comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, wherein the ceramic is selected from the group consisting of calcium phosphate, tetra-tricalcium phosphate, dicalcium phosphate, calcium carbonate, calcium sulfate, barium carbonate, barium sulfate, alphatricalcium phosphate (α-TCP), tricalcium phosphate (TCP), betatricalcium phosphate (β-TCP), hydroxyapatite (HA), biphasic calcium phosphate (e.g., composite between HA and β-TCP), bioglass, bone particles, and combinations and mixtures thereof. In one embodiment, the polymer is selected from the group consisting of collagen, fibrillar collagen, Type I collagen, porcine collagen, human recombinant collagen, bovine collagen, keratin, silk, polysaccharides, dextran, cellulose derivatives, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitosan, chitin, and hyaluronic acid. In one embodiment, the polymer is a block co-polymer of polymaleic anhydride having molecular weight of about 5,000 Dalton or less and a co-polymer comprising a biodegradable functionality, wherein the co-polymer is selected from the group consisting of polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polycaprolactone, poly-3-hydroxybutyrate, poly(p-dioxanone) and copolymers thereof, polyhydroxyalkanoate, poly(propylene fumarate), poly(ortho esters), and polyanhydrides, and combinations thereof.
In one embodiment of the presently disclosed subject matter, an implantable bone graft material is provided comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, and wherein the BMP binding peptide comprises one or more modifications to the peptide N-terminus, peptide C-terminus, or within the peptide amino acid sequence to facilitate linkage of the BMP binding peptide to the polymer with or without a spacer, wherein the modification is selected from the group consisting of aldehyde group, hydroxyl group, thiol group, amino group, amino acids, lysine, cysteine, acetyl group, polymers, synthetic polymers, polyethers, poly(ethylene glycol) (“PEG”), a 11 unit polyethylene glycol (“PEG10”), and a 1 unit polyethylene glycol (“mini-PEG” or “MP”), and combinations thereof. In one embodiment, the BMP binding peptide is attached to the polymer with or without a spacer. In one embodiment, the BMP binding peptide comprises the mini-PEG modification and the thiol group modification.
In one embodiment of the presently disclosed subject matter, an implantable bone graft material is provided comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, and wherein the ceramic and the polymer are present at a weight ratio ranging from about 10:1 ceramic to polymer to about 2:1 ceramic to polymer. In one embodiment, the ceramic is β-TCP and the polymer is bovine Type I fibrillar collagen. In one embodiment, the weight ratio of β-TCP to bovine Type I fibrillar collagen is about 4:1 (about 80% β-TCP to about 20% collagen). In one embodiment, the ceramic is β-TCP having a total porosity of about 50% or greater and a particle size ranging from about 100 micron to about 300 micron. In one embodiment, the polymer is collagen, and the ceramic is β-TCP having total porosity of about 70%, a particle size ranging from about 100 micron to about 300 micron, and a pore diameter less than 100 micron.
In one embodiment of the presently disclosed subject matter, an implantable bone graft material is provided comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, wherein the covalently attached BMP binding peptide is present at a range of about 1-200 μmol peptide/gram polymer, at range of about 5-90 μmol peptide/gram polymer, or at a range of about 5-15 μmol peptide/gram polymer. In one embodiment, the polymer is collagen and the covalently attached BMP binding peptide is present at a range of about 1-200 μmol peptide/gram collagen, at range of about 5-90 μmol peptide/gram collagen, or at a range of about 5-15 μmol peptide/gram collagen.
In one embodiment of the presently disclosed subject matter, a method is provided for promoting bone growth in a subject by delivering the implantable bone graft material comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide to a subject, wherein the presence of the graft material having attached BMP binding peptide promotes bone growth. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of a composite. In one embodiment, the composite is in the form of a sponge, a granulized sponge, a putty, or a strip. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of an injectable bone graft material. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of a moldable cement.
In one embodiment, a method is provided for capturing BMP onto an implantable bone graft material by contacting a sample comprising BMP with the graft material comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, wherein the BMP comprised in the sample is captured onto the graft material through binding to the attached BMP binding peptide. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of a composite. In one embodiment, the composite is in the form of a sponge, a granulized sponge, a putty, or a strip. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of an injectable bone graft material. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of a moldable cement. In one embodiment, the sample comprises autologous bone, allograft bone, xenograft bone, bone marrow aspirate (BMA), blood, platelet rich plasma (PRP), or recombinant BMP, or combinations or derivatives thereof.
In one embodiment, a method is provided for promoting bone growth in a subject by contacting a sample comprising BMP with the implantable bone graft material comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached BMP binding peptide, wherein the BMP comprised in the sample is captured onto the graft material through binding to the attached BMP binding peptide, and delivering to the subject the graft material comprising the captured BMP, wherein the presence of the captured BMP promotes bone growth in the subject. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of a composite. In one embodiment, the composite is in the form of a sponge, a granulized sponge, a putty, or a strip. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of an injectable bone graft material. In one embodiment, the resorbable ceramic and the resorbable polymer are in the form of a moldable cement. In one embodiment, the sample comprises autologous bone, allograft bone, xenograft bone, bone marrow aspirate (BMA), blood, platelet rich plasma (PRP), or recombinant BMP, or combinations or derivatives thereof.
A wide range of BMP binding peptides are useful in the compositions and methods of the presently disclosed subject matter. By way of non-limiting example, the BMP binding peptides described in US Patent Application Publication No's. US 2006/0051395A1 (the BMP binding peptides identified therein as one of SEQ ID No's: 11-28, 44-74, or 77-94) and US 200910098175A1 (the BMP binding peptides identified therein as one of SEQ ID No's: 1-12) are useful in the presently disclosed subject matter. In particular, the BMP binding peptides described in US Patent Application Publication No's. US 2006/0051395 A1 and US 2009/0098175A1 shown herein below at Table 1 (SEQ ID NOs: 1-10) are useful in the presently disclosed subject matter. In addition, BMP binding peptides useful in the presently disclosed subject matter include those peptides that are conservatively substituted variants of the BMP binding peptides in US Patent Application Publication Nos. US 2006/0051395A1 and US 2009/0098175A1, and those peptides that are variants having at least 65% sequence identity or greater to the BMP binding peptides in US Patent Application Publication Nos. US 2006/0051395A1 and US 2009/0098175A1, wherein all of the variant BMP binding peptides useful in the presently disclosed subject matter substantially retain the ability to bind to BMP. In one embodiment of the presently disclosed subject matter, a useful BMP binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-7 (see Table 1 herein below), conservatively substituted variants of SEQ ID NOs: 1-7, and variants having at least 65% sequence identity to SEQ ID NOs: 1-7, wherein the variant BMP binding peptide substantially retains the ability to bind BMP.
The following examples are provided to further describe certain aspects of the presently disclosed subject matter and are not intended to limit the scope of the presently disclosed subject matter.
BMP binding peptides SEQ ID NOs:1-4 & 8-10 were identified as described in US Patent Application Publication No. US20060051395A1. Briefly, the peptides were identified by phage display using immobilized BMP-2 as a substrate for the phage library selections. Synthetic peptides SEQ ID NOs: 1-2 were determined to bind to BMP-2 with a relative EC50 value of 0.8 nM and 0.9 nM, respectively (data not shown). Separately, the relative binding affinity of peptides SEQ ID NOs: 1-2 for BMP-2 is shown in
BMP binding peptides SEQ ID NOs: 5-7 were identified as described in US Patent Application Publication No. US20090098175A1. Briefly, the peptides were identified by phage display using immobilized GDF-7 (GDF-7 is BMP-12) as a substrate for the phage library selections. In addition to binding GDF-7 on which the phage selection was based, a relative EC50 value of 2.0 nM, 1.8 nM, and 1.1 nM, respectively, for BMP-2 was measured for peptides SEQ ID NOs: 5-7 (data not shown). In addition to binding BMP-2, the data in
In this experiment, a bone morphogenic protein (BMP) binding peptide SEQ ID NO: 2 was covalently attached to a collagen substrate using a disulfide linkage (see
Peptide synthesis. BMP binding peptides (including SEQ ID NO: 2) were synthesized by solid-phase peptide synthesis techniques either manually in glass reaction vessels or on a RAININ SYMPHONY PEPTIDE SYNTHESIZER multiplex automated peptide synthesizer (PROTEIN TECHNOLOGIES INC., Tucson Ariz.). Homogeneity of each synthetic peptide was evaluated by analytical RP-HPLC (WATERS ANALYTICAL/SEMI-PREPARATIVE HPLC), and the identity of the peptide confirmed with electrospray ionization mass spectrometry.
Collagen substrate modification. A slurry of fibrillar type I bovine collagen (1-3% collagen; KENSEY NASH, Exton, Pa.) was reacted with 2-iminothiolane (0.09-2.0 mg/ml) and either 4,4′-dithiodipyridine or 6,6′-dithiodinicotinic acid (DTNA) ranging from 0.4-9 mg/ml with gentle agitation at room temperature for about 24 h. The reaction mixture was isolated from the collagen using vacuum filtration. The collagen material was washed 4 times with phosphate buffer pH 8 and 2 times with PBS pH 7.4. For each wash, the mixture was agitated and then the wash buffer removed by vacuum.
Peptide coupling. BMP binding peptide SEQ ID NO: 2 (with N-terminal spacer and thiol group) at a concentration ranging from about 0.25-1.0 mg/mL in PBS was added to the activated collagen substrate described above. The reaction mixture was incubated with agitation at room temperature for about 24 hours. The resulting degree of peptide covalent attachment to the collagen ranged from about 5-90 μmol peptide/g collagen.
In another example, BMP binding peptides SEQ ID NO: 1 and SEQ ID NO: 2 were each covalently attached to a collagen matrix (HELISTAT sponge, INTEGRA LIFE SCIENCES, Plainsboro, N.J.). Amine groups (principally lysine ε-amino) on HELISTAT collagen were modified with sulfosuccinimidyl 6-(3′-[2-pyridyldithio]-propionamido)hexanoate (sulfo-LC-SPDP; THERMO FISHER SCIENTIFIC, Rockford, Ill.) to introduce a thiol-reactive pyridyl disulfide. After washing, sponges were reacted directly with the peptides containing a free thiol group (2.4 mg/mL for both SEQ ID NO: 1 & 2 peptides) resulting in covalent attachment of the peptide via a disulfide. For quantification, the peptides were released by reduction and analyzed by HPLC. The resulting degree of peptide covalent attachment to the HELISTAT collagen was 102 μmol peptide/g collagen (SEQ ID NO: 1) and 65 μmol peptide/g collagen (SEQ ID NO: 2).
The BMP binding peptide-modified HELISTAT collagen sponges generated in Example 2 were evaluated for ability to bind and retain BMP-2. BMP-2 was added to sponges (n=3) with or without the covalently attached BMP binding peptide (SEQ ID NO: 1 and SEQ ID NO: 2) and then the sponges were challenged with repeated changes in plasma (1, 3, 7, and 24 h). The amount of BMP-2 released into the plasma at each time-point was measured by ELISA (QUANTIKINE, R&D SYSTEMS), and the amount retained on the sponge after 24 h was estimated by Western blot analysis. After 4 h, a large amount of the initial load of BMP-2 was released from the unmodified collagen sponge (
In an experiment to test the biological activity of BMP-2 delivered by BMP binding peptide, alkaline phosphatase secreted by C2C12 cells in response to BMP-2 was measured in the presence and absence of BMP binding peptide in solution. C2C12 cells were incubated with BMP-2 (35 nM) and BMP binding peptide at a concentration ranging from 0-3500 nM. The C2C12 cells were incubated with the BMP-2 and BMP binding peptide for 3 days and the alkaline phosphatase assay was performed as described herein above. The data show that the BMP binding peptide does not interfere with the biological activity of BMP-2 and the BMP binding peptide does not have BMP-2 activity on its own (
Tri-calcium phosphate (TCP)-based bone void fillers or otherwise referred to as bone graft substitutes are used commonly in long bone applications and lumbar spinal fusion, because the TCP is absorbed over several months as bone heals. These products often contain collagen and are often combined with bone marrow aspirate (BMA) to provide osteoinductive factors. BMA can be harvested at point-of-care without the same complications of harvesting autogenous bone. In this Example, collagen/TCP composites were generated for use as a bone graft substitute. Both an unmodified collagen/TCP composite and a collagen/TCP composite having BMP Binding Peptide (SEQ ID NO: 2) covalently attached to the collagen portion of the composite were generated according to the following procedure.
BMP binding peptide SEQ ID NO: 2 was covalently attached to fibrillar Type I bovine collagen at both a low peptide load density (ranging from 5-15 μmol peptide/g collagen) and a high peptide load density (70-100 μmol peptide/g collagen) according to the procedure described herein above at Example 2 (using 6,6′-dithiodinicotinic acid). A collagen/TCP composite was generated using both unmodified collagen (i.e. without peptide attachment) and collagen modified with BMP binding peptide. The TCP used to make the collagen/TCP composite was β-TCP having about 70% porosity and having a particle size distribution of about 100 μm to about 300 μm (CAP BIOMATERIALS, East Troy, Wis.). The pore size of the β-TCP ranged from about 0.5 μm to less than about 70 μm (data not shown).
For each of the BMP binding peptide-modified and unmodified collagen/TCP composites, the collagen slurry was homogenized by hand or in a mixer with the β-TCP at a ratio of about 80% TCP to about 20% collagen while keeping the homogenate chilled and keeping the pH in the range of about pH 3-4. In separate experiments, the collagen was modified with BMP binding peptide both before and after mixing with the β-TCP.
After lyophilization, the collagen/TCP composites were in the form of sponges that are formable into a putty upon hydration. There was no discernable loss of β-TCP filler from the composites after hydration in saline (approximately 1.5 μl saline/mg composite) and puttying, and the composites retained their form after being shaped. In addition, an experiment was performed to evaluate pushing the hydrated and puttied composite through a 4.5 mm tube such as used in a non-invasive spinal fusion type surgery. Specifically, 117 mg of the BMP peptide-modified collagen/TCP was hydrated with 175 μl saline to produce a homogeneous putty. To simulate a cannula, the tip of a 1 ml plastic syringe was cut off to expose the opening of the barrel, which had a diameter of 4.5 mm. The putty was loaded into the top of the syringe and slowly pushed down the barrel with the syringe piston. When it reached the open bottom of the syringe, the material exited the syringe as a cylindrical plug of approximately 0.1 cc and 4.5 mm in diameter.
The ability of the BMP binding peptide-modified and unmodified collagen/TCP composites described in Example 5 to retain BMP-2 after long-term incubation in plasma was tested. Coupons of the unmodified composite and composite modified with low or high densities of BMP binding peptide were sterilized by e-beam sterilization. A 40 μM solution of BMP-2 was added to each of the sterilized composite coupons until the entire BMP-2 solution was absorbed into the coupon. The coupons were then transferred to human plasma and incubated at 37° C. with 100 rpm shaking. The plasma was changed at regular time-points for 6 weeks, and the amount of BMP-2 released into the plasma supernatant was assessed at each time-point by ELISA (
The ability of the BMP binding peptide-modified and unmodified composite sponges from Example 5 to capture BMP-2 from plasma was also tested. The BMP binding peptide-modified composite sponges with the low and the high peptide-loading density were incubated in plasma spiked with 2,600 pg/mL BMP-2 at 37° C. with shaking for 1 week. This concentration of BMP-2 was chosen because it is in the range of physiological levels of BMP-2 in bone marrow aspirate (BMA). At various time-points, 60 μL of plasma supernatant was removed for analysis of BMP-2 levels by ELISA. The levels of BMP-2 in the plasma were the same for the unmodified composite and the no-composite control throughout the course of the experiment (
The ability of each of the bone void filler collagen/TCP composites (unmodified and BMP binding peptide modified at 5-15 μmol peptide/g collagen described in Example 5) to speed bone healing was assessed in a rat calvarial defect model. A defect was introduced into male Sprague Dawley rats (n=8) as described previously (Poehling, et. al, J Periodontol, 2006, 77:1582-90). A full thickness circular defect, 6.8 mm in diameter, was made in the parietal bone, centered across the midline. The disc of bone was removed, and the void was filled with bone graft substitute material hydrated with or without BMA harvested from the tibia (saline was used for hydration without BMA). Tissue was harvested at 4, 8, and 12 weeks. The calvaria were removed and analyzed by micro-computed tomography (μCT) to provide measurements of bone density (SCANCO MEDICAL; Wayne, Pa.). Following μCT, the explants were processed for histology, and the slides were stained with haematoxylin and eosin (H&E). For each sample, 6 sections evenly distributed across the defect were scored by two independent observers, blind to the treatment groups. The slides were scored for osteogenic cellular activity, new bone area, and new bone maturity according to the following procedure. For osteogenic cellular activity, fibroblasts/loose connective tissue, immature cartilage progenitors, immature bone progenitors, giant cells, osteoclasts, osteoblasts, and osteocytes were counted and scored according to the following scale: 1=Rare; 2=Few; 3=Moderate; and 4=Dense. New bone cross-sectional area was scored utilizing the micrometer eye piece to determine the percent of the defect with new bone formed according to the following scale: 1.1=1-10%; 1.2=11-25%; 2.1=26-35%; 2.2=36-50%; 3.0=51-75%; and 4.0=76-100%. New bone maturity was scored according to the following scale: 1=Immature/Unorganized; 2=Immature; 3=Mature; and 4=Mature/Well organized. Data were analyzed by one-way Analysis of Variance (ANOVA). When the Main effect was significant (p<0.05), individual groups were compared by post-hoc analyses with Fisher's PLSD.
The bone graft substitute with covalently attached BMP binding peptide was compared to unmodified collagen/TCP composite (Example 5; no peptide) and two commercially available bone graft substitutes containing TCP: MASTERGRAFT PUTTY (MEDTRONIC SOFAMOR DANEK) and CHRONOS granules (SYNTHES). Animals with empty defects were included as a negative control. Histological analysis revealed increased bone formation at 4, 8, and 12 weeks. At 4 weeks, histology showed greater osteogenic cellular activity and new bone area in the BMP binding peptide-modified group than the unmodified and comparison product groups (data not shown). At 8 weeks, the BMP binding peptide-modified group scored higher for bone maturity and new bone area than any other group. By 12 weeks, none of the empty defects had healed. Among the treatment groups at 12 weeks, there were no significant differences in bone volume assessed by μCT; however, histological analysis revealed marked differences in bone maturity. The peptide-modified group hydrated with BMA had significantly more new bone area, mature new bone, and osteogenic cellular activity than all the other groups (
These data demonstrate that the BMP binding peptide-modified collagen/TCP composite not only performs as well as the comparative commercial products, but it accelerates bone formation and produces significantly more mature new bone. Furthermore, when combined with BMA, the presence of the BMP binding peptide resulted in better bone maturity than the collagen/TCP composite without peptide. This result indicates that the BMP binding peptide-modified composite's ability to deliver bioactive growth factors has significant advantages for bone healing.
As stated above for the results of the animal study for bone healing, the unmodified collagen/TCP composite of the presently described subject matter (no attached BMP binding peptide; Example 5), demonstrated significantly higher osteogenic cellular activity and bone maturity at 12 weeks when hydrated with saline than the commercially available products. This was an unexpected result. Furthermore, in addition to the higher osteogenic cellular activity and bone maturity observed at 12 weeks, the collagen/TCP composite without peptide also demonstrated an unexpected acceleration in bone healing at 4 weeks relative to commercially available MASTERGRAFT (MG) which is also a collagen/TCP composite. At 4 weeks, the unmodified composite when hydrated with either saline or BMA showed greater bone healing than MG as measured by each of the μCT and histological measurements of bone healing examined (see
Bone void fillers are used in some cases for promoting bone growth in spinal fusion applications and numerous animal models of spine fusion are available (e.g., Kraiwattanapong, C., et al., Spine, 2005, 30:1001-7; Magit, D. P., et al., Spine, 2006, 31:2180-8; Choi, Y., et al., Spine, 2007, 32: 36-41; Martin, G. J., et al., Spine, 1999, 24:637-45). Bone void fillers are often formulated into a strip having shape memory when used for spinal fusion applications. In this example, the BMP binding peptide-modified and unmodified collagen/TCP composites described in Example 5 were formulated into a strip having shape memory.
Various methods are available for cross-linking collagen in a collagen/ceramic composite to make a strip that meets the criteria for physical properties in spine fusion applications, such as with chemical agents (e.g., glutaraldehyde), dehydrothermal treatment, ultra-violet irradiation, or a combination of these techniques (see, e.g., Ruijgrok, J. M., et al., Journal of Materials Science: Materials in Medicine, 1994, 5:80-87; Gorham, S. D., et al., Int J Biol Macromol, 1992, 14:129-38; Weadock, K. S., et al., J Biomed Mater Res, 1995. 29: 1373-9. Lew, D. H., et al., J Biomed Mater Res B Appl Biomater, 2007, 82:51-6). In this example, the BMP binding peptide-modified and unmodified collagen/TCP composites described in Example 5 were formulated into a strip by dehydrothermal treatment.
In the first example, a composite from Example 5 (both peptide-modified and unmodified) was compressed between two titanium plates (TIMET, Ofallon, Mo.) held in place with two C-clamps (BESSEY, Leroy, N.Y.). The C-clamps were turned until the composite was compressed to at least half the original height. The clamped composite was placed in an ISOTEMP OVEN OV600G (FISHER SCIENTIFIC, Waltham, Mass.) at 88° C. for 74-112.5 hours. The sample was removed, cooled to room temperature, and hydrated with saline. The hydrated composite exhibited strip-like properties such as shape memory, flexibility, as well as liquid retention and compression resistance under a 50 g weight. In another example, a composite generated as described in Example 5 (peptide-modified and unmodified) was placed in an vacuum oven (SHELDON, Cornelius, Oreg.) at a temperature ranging from 100-110° C., at vacuum 29.5 inches Hg, and for 48-162 hours. The sample was removed, cooled to room temperature under vacuum, and hydrated with saline. The hydrated composite exhibited strip-like properties such as shape memory, flexibility, as well as liquid retention and compression resistance under a 50 g weight.
The ability of the BMP binding peptide-modified and unmodified collagen/TCP composites formulated into strips as described in Example 8 to bind and retain BMP-2 were tested according to the following procedure. Coupons of each of the BMP binding peptide-modified and unmodified composites were loaded with 27 μl of a 40 μM solution of BMP-2 (28 μg). The coupons were then transferred to human plasma and incubated at 37° C. with 100 rpm shaking. The plasma was changed 1 h, 6 h, and 24 h, and the amount of BMP-2 released into the plasma supernatant was assessed at each time-point by ELISA. Both the BMP binding peptide-modified composites (110° C. for 162 h under vacuum) and (88° C. for 112 h+compression) bound and retained BMP-2 as compared to unmodified composite controls (data not shown). Each peptide-modified composite released less than 5% of the BMP-2 originally loaded onto the coupons after 24 h of incubation in plasma (data not shown).
BMP binding peptide is covalently attached to polymaleic anhydride (PMA) using established methods (Pompe, et al., 2003, Biomacromolecules, 4(4):1072-9). First, a spacer, such as for example, GSSGK, is added to a terminus of the peptide and the peptide is attached to the PMA anhydride groups through the reactive terminal lysine amine group on the peptide-spacer. A schematic diagram of one example of this chemistry is shown in
The foregoing description of the specific embodiments of the presently disclosed subject matter has been described in detail for purposes of illustration. In view of the descriptions and illustrations, others skilled in the art can, by applying current knowledge, readily modify and/or adapt the presently disclosed subject matter for various applications without departing from the basic concept of the presently disclosed subject matter; and thus, such modifications and/or adaptations are intended to be within the meaning and scope of the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/262,353, filed Nov. 18, 2009; U.S. Provisional Application No. 61/368,849, filed Jul. 29, 2010; and U.S. Provisional Application No. 61/370,723, filed Aug. 4, 2010, each of which is hereby incorporated in its entirety by reference herein.
The invention was made with government support under Grant No. 5R44GM077753-03 awarded by the National Institute of General Medical Sciences and under Grant No. 5R44DE020760-03 and Grant No. 2R44DE018071-02 awarded by the National Institute of Dental and Craniofacial Research. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61262353 | Nov 2009 | US | |
| 61368849 | Jul 2010 | US | |
| 61370723 | Aug 2010 | US |
