US20110117171A1

US20110117171A1 - Implantable bone graft materials

Info

Publication number: US20110117171A1
Application number: US12/949,069
Authority: US
Inventors: Mora Carolynne Melican; Isaac Gilliam Sanford; Jonathan Allen Hodges; Shrikumar Ambujakshan Nair; Martyn Kerry Darby
Original assignee: Affinergy Inc
Current assignee: Affinergy Inc
Priority date: 2009-11-18
Filing date: 2010-11-18
Publication date: 2011-05-19
Also published as: WO2011063152A1; US20110117167A1; US20110117168A1; US20110223142A1; US20110117169A1; WO2011063128A1; WO2011063140A3; US8691946B2; US20110117166A1; US8779089B2; WO2011063165A1; WO2011063140A2; WO2011063165A9; US20110117165A1

Abstract

Compositions and methods are provided for promoting bone growth. An implantable bone graft material is provided comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide. The implantable bone graft materials are useful for promoting bone growth in a subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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.

GRANT STATEMENT

The invention was made with government support under Grant No. 5R44DE020760-03 and Grant No. 2R44DE018071-02 awarded by the National Institute of Dental and Craniofacial Research and under Grant No. 1R43AR054229-01 awarded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases. The government has certain rights in the invention.

FIELD

The presently disclosed subject matter relates to implantable bone graft materials for promoting bone growth.

BACKGROUND

Approximately 500,000 bone-grafting procedures are performed each year in the United States. Autologous iliac crest bone graft continues to be the gold standard, because it provides essential elements for bone formation: progenitor cells, an osteoconductive matrix, and osteoinductive molecules. However, iliac crest harvest is associated with a significant number of complications and often provides an inadequate volume of graft. Bone marrow aspirate (BMA) contains osteoinductive factors and can be harvested at point-of-care without the same 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 a prolonged period of time. In addition, multipotent Mesenchymal Stem Cells (MSCs) have been added to bone graft substitutes and explored for use in tissue engineering applications, such as spinal fusions and bone defect repairs. Bone marrow derived mesenchymal stem cells (BMSCs) are well characterized in their ability to differentiate into cells and tissues of mesodermal origin. Adipose derived stem cells (ASCs) have been proposed as an alternative to BMSCs for cell therapy because these cells are found in the stromal vascular fraction (SVF) of processed lipoaspirate which is often more readily attainable than BMA.
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). TCP-based bone graft substitutes often containing collagen are used commonly in lumbar spinal fusion because TCP is absorbed 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 osteogenic cells throughout the healing process. The presently disclosed subject matter provides such a bone graft substitute.

SUMMARY

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 comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell 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 comprises a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide, and wherein the presence of the graft material promotes bone growth.
In one embodiment, the presently disclosed subject matter provides a method for capturing stem cells onto an implantable bone graft material, the method comprising contacting a sample comprising stem cells with the graft material, wherein the graft material comprises a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide, and wherein the stem cells comprised in the sample are captured onto the graft material through binding to the attached stem cell 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 stem cells 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 stem cell binding peptide, wherein the stem cells comprised in the sample are captured onto the graft material through binding to the attached stem cell binding peptide; and delivering to the subject the graft material comprising the captured stem cells, wherein the graft material having the captured stem cells promotes bone growth in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting one method for covalently attaching a stem cell binding peptide to a substrate comprising amino functional groups.

FIG. 2 is a schematic diagram depicting one method for covalently attaching a stem cell binding peptide to a substrate comprising amino functional groups.

FIG. 3 is a schematic diagram depicting methods for covalently attaching a stem cell binding peptide to a substrate having an amino functional group.

FIG. 4 is a schematic diagram depicting one method for covalently attaching a stem cell binding peptide to a substrate comprising amino functional groups.

FIG. 5 is a schematic diagram depicting one method for covalently attaching a stem cell binding peptide to a substrate comprising amino functional groups.

FIG. 6 is a schematic diagram depicting the chemistry for covalently attaching a stem cell binding peptide to a polyanhydride polymer, polymaleic anhydride (PMA), through the reactive amines on the peptide.

FIG. 7 is a schematic diagram depicting exemplary chemistry for covalently attaching a stem cell binding peptide to chitosan.

FIG. 8 is a schematic diagram depicting exemplary chemistry for covalently attaching a stem cell binding peptide to chitosan.

FIG. 9 is a schematic diagram depicting exemplary chemistry for covalently attaching a stem cell binding peptide to hyaluronic acid.

FIG. 10 is a schematic diagram depicting exemplary chemistry for covalently attaching a stem cell binding peptide to hyaluronic acid.

FIG. 11 is a schematic diagram depicting exemplary chemistry for introducing an amino functional group on cellulose for subsequent covalent attachment of a stem cell binding peptide.

FIG. 12 is a schematic diagram depicting exemplary chemistry for covalently attaching a stem cell binding peptide to oxidized cellulose.

FIG. 13 is a schematic diagram depicting one method for covalently attaching more than one stem cell binding peptide to a substrate comprising amino functional groups.

FIG. 14 is a table showing an alignment of cell binding peptides from a phage display library selection.

FIG. 15 is a bar graph showing the ability of stem cell binding peptide SEQ ID NO: 4 to specifically bind human mesenchymal stem cells (hMSCs) from bone marrow compared to a number of other cells types including human adipose-derived mesenchymal stem cells (hASCs), human dermal fibroblasts, rodent MSCs, red blood cells (RBCS), monocytes, lymphocytes and granulocytes. The y axis shows biotinylated stem cell binding peptide SEQ ID NO: 4 reactivity as percent positivity relative to Neutravidin-PE staining without the addition of biotinylated peptide.

FIG. 16 is a bar graph showing the ability of biotinylated stem cell binding peptide SEQ ID NO: 4 (“test”) to capture human MSCs from a homogeneous cultured cell population relative to a non-binding control peptide (“control”). The capture of MSCs bound to biotinylated peptide was performed with MILTENYI BIOTEC Streptavidin microbeads loaded into LS columns.

FIGS. 17A-17C are flow cytometry histograms of cells showing selective capture of MSCs on biotinylated stem cell binding peptide SEQ ID NO: 16 (“test”; panel C) attached to CELLECTION magnetic beads. The CELLECTION beads have streptavidin coupled to a magnetic particle through a DNA linker. Three different cell types, human MSCs, IM-9, and U937 cells were differentially labeled with CELLTRACKER dye and mixed in a ratio such that the MSCs represented ˜4% of the starting cell mixture (the percentage of each cell type in the starting mixture was 38% IM9, 56% U937, and 4% MSC (panel A). Panel B is a no peptide control.

FIG. 18 is a bar graph showing the ability of biotinylated stem cell binding peptide SEQ ID NO: 4 (“test”) to capture human MSCs directly from bone marrow aspirate in comparison to a negative peptide control and to two separate antibodies against the CD105 stem cell antigen (CD105) and the MSCA-1 stem cell antigen (MSCA-1), respectively. The capture of MSCs bound to biotinylated peptide was performed with MILTENYI BIOTEC Streptavidin microbeads loaded into LS columns. The y axis shows colony forming units (CFUs) counted after 14 days in culture.

FIGS. 19A and 19B are images showing the ability of a collagen sponge having covalently attached stem cell binding peptide SEQ ID NO: 4 (panel A) to capture cultured human MSCs labeled with fluorescent CELLTRACKER Green dye compared to an unmodified collagen sponge (panel B). The stem cell binding peptide SEQ ID NO: 4 was modified at the carboxyl terminus with a PEG-10 spacer and a lysine residue. After contact with MSCs, sponges were transferred and incubated with 2% fetal bovine serum. Sponge images (16 ms, 10×) were taken after a 19 hour incubation.

FIGS. 20A-20D are images of differentiated MSCs captured from bone marrow aspirate with stem cell binding peptide SEQ ID NO: 4. Following a 21 day incubation in adipocyte differentiation media, captured MSCs were fixed and stained with Oil Red O to determine the extent of adipogenesis. Panel A shows undifferentiated MSCs and panel B shows adipocyte differentiated MSCs. The image of the adipose differentiated cells (panel B) contains a larger magnification inset where the lipid vacuoles are clearly visible. Following a 14 day incubation in osteoblast differentiation media, the captured MSCs were stained with Alizarin Red S to reveal mineralizing osteoblasts. Panel C shows undifferentiated MSCs and panel D shows osteoblast differentiated MSCs.

FIGS. 21A-21D are images of differentiated MSCs captured from bone marrow aspirate with a stem cell binding peptide SEQ ID NO: 4. In FIG. 21 sections from the periphery (panels A and B) or center (panels C and D) were incubated with an antibody against aggrecan (ABCAM) (panels B and D) or with secondary detection reagents only as a control (panels A and C), then counterstained with DAPI to reveal cell nuclei.

DETAILED DESCRIPTION

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.

DEFINITIONS

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 stem cell binding peptide” or reference to “a 1 unit polyethylene glycol (“mini-PEG” or “MP”)” includes a plurality of such stem cell binding peptides or such polyethylene glycol units, and so forth.
The term “adipose tissue” as used herein, for the purposes of the specification and claims, includes the term “liposuction aspirate”. Therefore, the term “stromal vascular fraction of adipose tissue” also means “stromal vascular fraction of liposuction aspirate”.
The term “substrate” is used, for the purposes of the specification and claims, to refer to any material that is biologically compatible with stem cells to which a stem cell binding peptide can be attached for the purpose of capturing stem cells 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 stem cell 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 stem cell binding peptide to a “substrate” it is meant that attachment of the stem cell binding peptide is to a polymer comprised in the substrate. Therefore, the attachment of a stem cell binding peptide to a “substrate” is referring to attachment of the stem cell 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 alpha-tricalcium phosphate, beta-tricalcium phosphate, tetra-tricalcium phosphate, dicalcium phosphate, calcium carbonate, barium carbonate, calcium sulfate, barium sulfate, hydroxyapatite, biphasic calcium phosphate, biphasic 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 stem cell 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 to which a stem cell 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 stem cell 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 stem cell 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 stem cell 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 “stem cell 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 stem cell (i.e., the stem cell is the binding “target” of the stem cell binding peptide). In one embodiment, the stem cell binding peptide is any one of SEQ ID NOs: 1-20 (see Example 1). The stem cell 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 stem cell binding peptide of the presently disclosed subject matter. A stem cell 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.
Stem cell binding peptides of 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 Table 1 (SEQ ID NOs: 1-16), as long as the binding properties of the exemplary stem cell binding peptides to their stem cell targets are substantially retained. Thus, the stem cell 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 stem cell 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 stem cell binding peptide can have an amino acid sequence consisting essentially of a sequence of an exemplary stem cell binding peptide or a stem cell 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 stem cell binding activity of the exemplary stem cell 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, stem cell binding peptides useful in the presently disclosed subject matter include those peptides that are conservatively substituted variants of the stem cell binding peptides set forth in SEQ ID NOs: 1-16, and those peptides that are variants having at least 65% sequence identity or greater to the stem cell binding peptides set forth in SEQ ID NOs: 1-16, wherein all of the variant stem cell binding peptides useful in the presently disclosed subject matter substantially retain the ability to bind to stem cell. In one embodiment of the presently disclosed subject matter, a useful stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-16, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16, wherein the variant stem cell binding peptide substantially retains the ability to bind stem cell.
Stem cell 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 stem cell 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 stem cell binding peptide and the polymer. In some embodiments, the spacer also serves the function of a linker (i.e. to attach the stem cell 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 stem cell binding peptide from the polymer. In some cases the spacer can serve to position the stem cell binding peptide at a distance and in a spatial position suitable for stem cell binding and capture and/or in some cases the spacer can serve to increase the solubility of the stem cell binding peptide. Spacers can increase flexibility and accessibility of the stem cell binding peptide to stem cell, as well as increase the stem cell 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 stem cell to the stem cell binding peptides is prevented or substantially impaired. Thus, the term “stem cell binding peptide” encompasses any of a variety of forms of stem cell 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 stem cell binding peptide derivative that has substantially retained stem cell 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 stem cell binding peptide in the presence of biological fluids where proteases can be present. In addition, as used herein, the term “stem cell 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 stem cell binding peptides are covalently attached to the polymer. The term “attached” in reference to a stem cell binding peptide of the presently disclosed subject matter being attached to a polymer means, for the purposes of the specification and claims, a stem cell binding peptide being immobilized on the polymer by covalent attachment by any means that will enable binding of stem cell onto the peptide-modified polymer such that the bound stem cell retains its biological activity including its ability to differentiate into other cell types. In one embodiment, the linkers/spacers for use in attaching stem cell 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 stem cell binding peptide or in some cases it binds to the “spacer” already attached to the stem cell binding peptide. Preferably, the attachment of the stem cell 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 stem cell peptide binding behavior to such an extent that binding of the stem cell to the stem cell 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 stem cell binding peptide to the polymer and spacing the peptide from the polymer. In many embodiments herein, the linkers used to attach the stem cell 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 stem cell binding peptides of the presently disclosed subject matter can comprise a functional group that is intrinsic to the stem cell binding peptide (e.g., amino groups on lysine), or the functional group can be introduced into the stem cell binding peptide by chemical modification to facilitate covalent attachment of the stem cell 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 stem cell binding peptide. The stem cell 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, stem cell 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,
,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 polymer to which the binding peptide will be attached.
The stem cell binding peptides can be covalently attached to the substrate polymer through one or more anchoring (or linking) groups on the substrate polymer and the stem cell binding peptide. The stem cell binding peptides of the presently disclosed subject matter can comprise a functional group that is intrinsic to the stem cell binding peptide, or the stem cell 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,
,{tilde over (β)}-unsaturated carbonyl, alkyl halide, azide, epoxide, NHS ester, lysine, and cysteine groups on the surface of the substrate. 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 stem cell binding peptide is attached to a substrate polymer in a two step process (see FIG. 1; Mikulec & Puleo, 1996, J. Biomed. Mat. Res., Vol 32, 203-08). In the first step, the anchoring (or linking) groups (i.e., amino groups on a collagen for example) on the surface of a substrate polymer are activated by an acylating reagent (4-nitrophenyl chloroformate). In the second step, a lysine residue which has been introduced along with a PEG10 spacer at the C-terminus of a stem cell binding peptide is reacted with the activated chloroformate intermediate on the substrate polymer surface, resulting in attachment of the stem cell binding peptide to the substrate polymer.
By way of non-limiting example, in one embodiment, a stem cell binding peptide is covalently attached to a substrate polymer comprising an amino functional group (see FIG. 2). FIG. 2 exemplifies attachment of a stem cell binding peptide comprising an aldehyde group at one terminus to a substrate polymer that comprises an amino functional group. The stem cell binding peptide comprising an aldehyde functional group is treated with the substrate polymer amino groups under reductive amination conditions to give attached stem cell binding peptide. In another embodiment not depicted in FIG. 2, a stem cell binding peptide comprising an amine functional group is reacted with the substrate polymer amino groups via a homobifunctional linker such as, for example, glutaraldehyde, to yield a covalently attached stem cell binding peptide (Simionescu et. al., 1991, J. Biomed Mater. Res., 25:1495-505).
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 stem cell binding peptide having an amino group (FIG. 3). The stem cell binding peptide with attached linking group is then reacted through the remaining N-hydroxysuccinimide ester with an amino group on the substrate polymer to form a peptide-substrate conjugate (FIG. 3). The homobifunctional N-hydroxysuccinimide ester depicted in FIG. 3 is BS³crosslinking reagent (THERMO SCIENTIFIC, Rockford, Ill.). As stated herein previously, the length and type of spacer groups between the two reactive end groups on the NHS ester can vary.
By way of non-limiting example, in one embodiment, a stem cell binding peptide is covalently attached to a substrate polymer having amino functional groups in a two-step process using a disulfide linkage (see FIG. 4; Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, 1996; pp. 150-151). First, the substrate polymer containing amino groups is reacted with 2-iminothiolane resulting in the introduction of thiol groups on the substrate polymer. Simultaneous addition of 4,4′-dithiodipyridine or 6,6′-dithiodinicotinic acid results in rapid capping of the newly-introduced thiol as a pyridyl disulfide. Second, the stem cell binding peptide containing a free thiol is attached covalently to the substrate polymer through a thiol-disulfide exchange resulting in a disulfide bond between the substrate and stem cell binding peptide.
By way of non-limiting example, in one embodiment, a stem cell binding peptide is attached covalently to a substrate polymer comprising amino functional groups in a similar process using a disulfide linkage (see FIG. 5; Carlsson et al., 1978, Biochem. J., 173:723-37). The substrate polymer is first functionalized with amine groups using known methods (if the amino groups are not intrinsic to the material of the substrate polymer). Next, a thiol-cleavable, heterobifunctional (amine- and sulfhydryl-reactive) compound (LC-SPDP; THERMO SCIENTIFIC, Rockford, Ill.) is reacted with the amino-functionalized substrate polymer. The stem cell binding peptide is reacted with the LC-SPDP modified substrate polymer.
By way of non-limiting example, in one embodiment, a stem cell 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 stem cell 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 stem cell binding peptide modified with the maleimido group.
By way of non-limiting example, in one embodiment, a stem cell 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 stem cell binding peptide shown in the schematic diagram in FIG. 6 (Pompe, et al., 2003, Biomacromolecules, 4(4):1072-9).
By way of non-limiting example, in one embodiment, a stem cell binding peptide is covalently attached to a chitosan. The chemical scheme is shown in FIG. 7. First, the amino group on chitosan is protected with phthaloyl group. The hydroxyl group on chitosan is then reacted with chloroacetic acid to give an acid handle on chitosan. The stem cell binding peptide amine is coupled to the acid group on the chitosan to give the stem cell binding peptide-chitosan conjugate. The phthaloyl group is then removed using hydrazine.
By way of non-limiting example, in one embodiment a stem cell binding peptide is covalently attached to a chitosan. The chemical scheme is shown in FIG. 8. First, the amino group on chitosan is protected with a phthaloyl group. The hydroxyl group on chitosan is then converted to a bromo group under standard halogenation conditions. The stem cell binding peptide amine is reacted with halogenated chitosan to give the stem cell binding peptide-chitosan conjugate. The phthaloyl group is finally removed by reacting with hydrazine.
By way of non-limiting example, in one embodiment a stem cell 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 FIG. 3, is useful for attaching the stem cell binding peptide through the amino group on chitosan.
By way of non-limiting example, in one embodiment a stem cell binding peptide is covalently attached to a hyaluronan (HA). The chemical scheme is shown in FIG. 9. The hyaluronan is chemically modified at the carboxylic acid group on the glucuronate units. The carboxylic group is activated using carbonyl diimidazole (CDI). The activated HA is then reacted with the amino group of stem cell binding peptide to yield the peptide-HA conjugate.
By way of non-limiting example, in one embodiment, a stem cell binding peptide is covalently attached to a hyaluronan (HA). The chemical scheme is shown in FIG. 10. Hyaluronan is chemically modified at the carboxylic acid group on the glucuronate units. The carboxylic group is activated using water a soluble carbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) along with HOBt. The activated HA is coupled with the amino group of a stem cell binding peptide to yield the peptide-HA conjugate.
By way of non-limiting example, in one embodiment, a stem cell binding peptide is covalently attached to cellulose. The chemical scheme is shown in FIG. 11. Hydroxyl groups on the polysaccharide are first reacted with epichlorohydrin to introduce an epoxide. Ring opening of the epoxide by reaction with aqueous ammonia provides free amino groups that can function as anchors for peptide conjugation using chemistry described in previous embodiments (Matsumoto, et al. (1980) J. Biochem., 87: 535-540).
By way of non-limiting example, in one embodiment, a stem cell binding peptide is covalently attached to oxidized cellulose. The chemical scheme is shown in FIG. 12. Sulfhydryl groups are introduced by reaction of carboxylates on the oxidized cellulose with cystamine and EDC followed by reduction with dithiothreitol (DTT). Activation of sulfhydryls with 6,6′-dithiodinicotinic acid (DTNA) followed by a sulfhydryl-containing stem cell binding peptide results in covalent attachment of the peptide to the oxidized cellulose through a disulfide bond. In another embodiment not depicted in FIG. 12, the sulfhydryl modified oxidized cellulose is reacted with a maleimide or other Michael acceptor on the stem cell binding peptide resulting in covalent attachment through a thioether bond. In another embodiment not depicted in FIG. 12, carboxyl groups on oxidized cellulose are activated with EDC and 1-hydroxybenzotriazole (HOBt) followed by reaction with cell binding peptide containing a free amine group. This results in conjugation of peptide to the oxidized cellulose through an amide bond (this chemistry is exemplified in FIG. 10). In another embodiment not depicted in FIG. 12, a stem cell binding peptide can be covalently attached to oxidized cellulose through the aldehyde groups on the oxidized cellulose. In this example, a stem cell 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 FIG. 2 (the chemistry is the same as that in FIG. 2 except that the functional groups on the polymer substrate and cell binding peptide are reversed).
By way of non-limiting example, in one embodiment, a cell binding peptide can be covalently attached to an oxidized dextran polymer substrate by reductive amination as described above for oxidized cellulose. More specifically, a stem cell 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 FIG. 2 (the chemistry is the same as that in FIG. 2 except that the functional groups on the polymer substrate and cell binding peptide are reversed).
By way of non-limiting example, in one embodiment, more than one stem cell binding peptide is attached to a substrate polymer. Attaching multiple stem cell 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 stem cell binding peptides are covalently attached to a substrate polymer using any of the chemical schemes shown in FIGS. 1-12. In each of the chemical schemes depicted in FIGS. 1-12, the substrate polymer having a functional group is reacted with two or more different stem cell binding peptides that each comprise a functional group to covalently attach the two or more stem cell binding peptides to the substrate polymer based on simple competition between the stem cell binding peptides. In particular, for example, in the case of the chemical schemes depicted in FIGS. 1 and 2, the modified substrate is reacted with two or more different stem cell binding peptides that each comprise an amino group or an aldehyde group (i.e., the two different stem cell binding peptides replace the single peptide depicted in FIGS. 1 and 2), to covalently attach the two or more stem cell binding peptides to the substrate polymer through the amino or aldehyde group, respectively. In the case of the chemical schemes depicted in FIGS. 4 and 5, the modified substrate polymer is reacted with two or more different stem cell binding peptides that each comprise a thiol group, to covalently attach the two or more stem cell binding peptides to the substrate through the thiol group (i.e., the “HS-Peptide” in FIGS. 4 and 5 in this embodiment represents two or more different stem cell binding peptides).
By way of non-limiting example, in one embodiment, two different stem cell binding peptides are covalently attached to a substrate polymer comprising amino groups using the chemical scheme shown in FIG. 13. In this embodiment, the amino groups on the substrate polymer are modified with maleimido groups. The modified substrate polymer is then reacted with a stem cell binding peptide comprising both a thiol group and an aldehyde group to covalently attach the stem cell binding peptide to the substrate polymer through the thiol group. Next, the substrate-stem cell binding peptide conjugate is reacted with another stem cell binding peptide having a hydrazine group, to give a second covalent bond through the aldehyde-hydrazine (see FIG. 13). Alternatively, in one embodiment, the same chemical scheme is utilized but with the substrate modified with a thiol group and the stem cell binding peptide modified with the maleimido group. In addition to using this scheme to covalently attach different stem cell binding peptides, the scheme is also useful for attaching the same stem cell binding peptide.
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 stem cell 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.
A wide range of stem cell binding peptides are useful in the compositions and methods of the presently disclosed subject matter. By way of non-limiting example, the stem cell binding peptides described herein at Example 1 (SEQ ID NOs: 1-20) are useful in the presently disclosed subject matter. In addition, stem cell binding peptides useful in the presently disclosed subject matter include those peptides that are conservatively substituted variants of the stem cell binding peptides set forth in SEQ ID NOs: 1-16, and those peptides that are variants having at least 65% sequence identity or greater to the stem cell binding peptides set forth in SEQ ID NOs: 1-16, wherein all of the variant stem cell binding peptides useful in the presently disclosed subject matter substantially retain the ability to bind to stem cells.
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 stem cell 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, dextran, polysaccharides, 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 stem cell binding peptide, and wherein the stem cell 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 stem cell 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”), an 11 unit polyethylene glycol (“PEG10”), and a 1 unit polyethylene glycol (“mini-PEG” or “MP”), and combinations thereof. In one embodiment, the stem cell binding peptide is attached to the polymer with or without a spacer.
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 stem cell 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%. In one embodiment, the ceramic is β-TCP having a particle size ranging from about 100 micron to about 300 micron and a pore diameter of 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 stem cell binding peptide, wherein the covalently attached stem cell binding peptide is present at a range of about 1-200 μmol peptide/gram polymer.
In one embodiment of the presently disclosed subject matter, a method is provided for promoting bone growth in a subject by delivering to the subject the implantable bone graft material comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide, wherein the presence of the graft material 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 stem cells onto an implantable bone graft material by contacting a sample comprising stem cells with the graft material comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide, wherein the stem cells comprised in the sample are captured onto the graft material through binding to the attached stem cell 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), allogeneic stem cells, a homogeneous or heterogeneous population of cultured cells, adipose tissue, stromal vascular fraction of adipose tissue, blood, blood products, platelets, platelet-rich plasma (PRP), umbilical cord blood, embryonic tissues, placenta, amniotic epithelial cells, or combinations or derivatives thereof.
In one embodiment, a method is provided for promoting bone growth in a subject by contacting a sample comprising stem cells with the implantable bone graft material comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide, wherein the stem cells comprised in the sample are captured onto the graft material through binding to the attached stem cell binding peptide, and delivering to the subject the graft material comprising the captured stem cells, wherein the presence of the graft material having the captured stem cells 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, the sample comprises autologous bone, allograft bone, xenograft bone, bone marrow aspirate (BMA), allogeneic stem cells, a homogeneous or heterogeneous population of cultured cells, adipose tissue, stromal vascular fraction of adipose tissue, blood, blood products, platelets, platelet-rich plasma (PRP), umbilical cord blood, embryonic tissues, placenta, amniotic epithelial cells, or combinations or derivatives thereof.
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.

EXAMPLES

Example 1

Identification of Cell Binding Peptides by Phage Display

Peptides that bind human mesenchymal stem cells (MSCs) were identified by phage display biopanning. MSCs were culture amplified from plated bone marrow aspirate (LONZA, <4 passages). After biopanning, individual plaques were picked, grown overnight, and tested for MSC binding activity using flow cytometry according to the following procedure. Phage supernatant was incubated with MSCs for 30 min on ice. Cells were washed twice with Dulbecco's Phosphate Buffered Saline (DPBS) containing 2% FBS, then incubated with 50 μl of anti-M13 antibody labeled with the fluorophore phycoerythrin (PE). After 30 minutes on ice, cells were washed twice with DPBS containing 2% FBS and binding data acquired on a BD FACSARRAY flow cytometer. For the phage displaying MSC binding activity, DNA sequences were analyzed and translated into peptide sequences using Vector NTI DNA Analysis software (see FIG. 14 and Table 1; SEQ ID NOs: 1-15).

TABLE 1

Stem Cell Binding Peptides

SEQ ID	Amino acid sequence
NO:	(single letter code)

1	SSMYFSPLHTWQSAPSTSGAE

2	SSFRFQRLEDWNYPSNTDNAE

3	SSGYMQFGHLLDWTGSPSGSR

4	SSFWDVCQGDGTCYGGGSR

5	VANPFTYLSAWSNPL

6	ETLIFSKLGQWGNSLS

7	GYMQFGHLLDWTGSP

8	SVYRFDSLTTWSSNQ

9	GSWSFGTLGPWSSSQ

10	WLGNFNALTDWPTDS

11	TSGFFGSLDTWPPTL

12	NYWNFGPLEDYS

13	SVLHFHPMKSYD

14	NSIYFSPLRDYQ

15	GHFEYGRLQSIL

In addition to the cell binding sequences in Table 1 above, a consensus stem cell binding sequence was designed based on the sequences for the stem cell binders shown in FIG. 14. Specifically, the following sequence: SSFRFGPLGTWNYPSTDNAE (SEQ ID NO: 16) was designed based on sequences in FIG. 14 (SEQ ID NOs: 5-15) which showed a high level of stem cell binding activity, and the observation that non-binding sequences contain a larger number of negatively charged residues in the amino and carboxyl terminal regions, and a larger number of positively charged residues in the central region, than the sequences showing stem cell binding activity.
In addition to consensus cell binding sequence (SEQ ID NO: 16), the following sequence motifs SEQ ID NOs: 17-18 were generated based on the stem cell binding activity observed for the peptide sequences in Table 1 and FIG. 14:
SEQ ID NO: 17:

X₁X₂FX₄X₅LX₇X₈WX₁₀X₁₁X₁₂X₁₃X₁₄,

wherein “X₁” is F, M, L, Y, W, or N; wherein “X₂” is R, Q, P, I, Y or S; wherein “X₄” is G, S, Q, T, or D; wherein “X₅” is P, R, Y, K, H, or S; wherein “X₇” is G, H, E, S, L, or T; wherein “X₈” is T, D, A, Q, or P; wherein “X₁₀” is N, Q, S, G, or T; wherein “X₁₁” is Y, S, N, or G; wherein “X₁₂” is P, A, S, or N; wherein “X₁₃” is S, P, L, or Q; and wherein “X₁₄” is T, S, or N.
SEQ ID NO: 18:

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄,

wherein “X₁” is F, W, L, Y, M, or I; wherein “X₂” is N, Y, R, P, Q, I, F, or E; wherein “X₃” is F or Y; wherein “X₄” is G, S, T, Q, N, H, or D; wherein “X₅” P, R, Y, T, S, K, H, or A; wherein “X₆” is L or M; wherein “X₇” is T, G, E, S, R, Q, L, K, H, or D; wherein “X₈” is D, T, S, Q, P, or A; wherein “X₉” is W, Y, or I; wherein “X₁₀” is P, N, Q, S, G, L, D, or T; wherein “X₁₁” is Y, S, N, T, P, or G; wherein “X₁₂” is P, A, S, T, D, or N; wherein “X₁₃” is S, P, L, or Q; and wherein “X₁₄” is S or N.
Mutagenesis of Cell Binding Peptide Sequence SEQ ID NO: 4. A focused phage display library was generated around the SEQ ID NO: 4 sequence with each nucleotide position varying in identity at a ratio of 91:3:3:3, with the original nucleotide being the dominant form. This is considered a form of “light” mutagenesis, retaining the majority of residue identities with a few amino acid identity changes. The construction of this “degenerate” phage library was performed according to the methods described in Kay et al., 1996. Individual phage were picked from the degenerate phage library and binding to MSCs was assessed by flow cytometry as described herein above. Forty eight phage “binders” (binding comparable to wild type SEQ ID NO: 4) and 48 phage “non-binders” (phage binding reduced to level of a control without a polypeptide insert) were re-amplified, retested, and submitted for DNA sequencing to determine the insert amino acid sequences.
Based on these results, the following first cell binding sequence motif was generated:
SEQ ID NO: 19:

X₁-Z₂-Z₃-X₄-X₅-C-X₇-X₈-X₉-G-T-C-X₁₃-G-G-G,

wherein “X₁” is S, N, T, I, V, or G; wherein “Z₂” and “Z₃” are F, W, or Y; wherein “X₄” is D, E, W, N, Q, or G; wherein “X₅” is V, M, or A; wherein “X₇” is Q, P, E, L, H, R, or A; wherein “X₈” is G, A, V, or R; wherein “X₉” is D, N, or E; and wherein “X₁₃” is Y, W, or H.
Further, based on these results, the following second cell binding sequence motif was generated:
SEQ ID NO: 20:

X₁-X₂-W-X₄-X₅-C-X₇-X₈-X₉-G-T-C-X₁₃-G-G-G,

wherein “X₁” is S, N, T, I, V, or G; wherein “X₂” is F or Y; wherein “X₄” is D, E, W, N, Q, or G; wherein “X₅” is V, M, or A; wherein “X₇” is Q, P, E, L, H, R, or A; wherein “X₈” is G, A, V, or R; wherein “X₉” is D, N, or E; and wherein “X₁₃” is Y, W, or H.

Example 2

Generation of Synthetic Binding Peptides

Peptide Synthesis. Binding peptide sequences were synthesized using standard solid-phase peptide synthesis techniques on a SYMPHONY Peptide Synthesizer (PROTEIN TECHNOLOGIES, Tucson, Ariz.) using standard Fmoc chemistry (HBTU/HOBT activation, 20% piperidine in DMF for Fmoc removal). N-α-Fmoc-amino acids (with orthogonal side chain protecting groups; NOVABIOCHEM). After all residues were coupled, simultaneous cleavage and side chain deprotection was achieved by treatment with a trifluoroacetic acid (TFA) cocktail. Crude peptide was precipitated with cold diethyl ether and purified by high-performance liquid chromatography on a WATERS Analytical/Semi-preparative HPLC unit on VYDAC C18 silica column (preparative 10 μm, 250 mm×22 mm) using a linear gradient of water/acetonitrile containing 0.1% TFA. Homogeneity of the synthetic peptides was evaluated by analytical RP-HPLC (VYDAC C18 silica column, 10 μm, 250 mm×4.6 mm) and the identity of the peptides confirmed with MALDI-TOF-MS. Biotinylated peptides were generated similarly, with a GSSGK(biotin) sequence or other spacer group added to the C-terminus of the peptide.

Example 3

Cell Binding Peptide Specificity

The synthetic biotinylated cell binding peptide SEQ ID NO: 4 was examined for its ability to specifically bind MSCs compared to a number of other cells types including adipose-derived mesenchymal stem cells (ASCs), dermal fibroblasts, rodent MSCs, red blood cells, monocytes, lymphocytes and granulocytes. The cell binding peptide was biotinylated as described herein at Example 2. Cultured cells of each type were either purchased (ASCs and dermal fibroblasts) or isolated from rodent bone marrow (rodent MSCs) or human blood. Cells were first harvested and resuspended at 10⁶/mL in PBS+2% fetal bovine serum (FBS). An aliquot of cells (50 μL) was incubated in 50 μL of peptide solution (25 μM in PBS+FBS) for 30 min at 4° C. Cells were then washed twice in PBS+FBS with 300×g centrifugation for 5 min between washes. Fluorescently-tagged neutravidin (Neutravidin-PE from INVITROGEN) was then added to the cells to label biotinylated peptide bound to cells. Neutravidin-PE was diluted 1:250 from stock and applied at 50 μL to cells. Cells were then washed in PBS+FBS, and acquired on a BD FACSARRAY. Peptide reactivity was then measured as percent positivity relative to Neutravidin-PE staining without the addition of biotinylated peptide. Cell binding peptide SEQ ID NO: 4 was observed to have high binding to human MSCs, human ASCs and dermal fibroblasts (see FIG. 15).

Example 4

Capture of Cultured MSCs with Cell Binding Peptide Attached to a Substrate

In this experiment the ability of the biotinylated cell binding peptide SEQ ID NO: 4 to capture human MSCs from a homogeneous cultured cell population was examined. The cell binding peptide SEQ ID NO: 4 was biotinylated as described herein at Example 2. The cell binding peptide SEQ ID NO: 4, and a non-binding control peptide, were added to separate 300 μl volumes of MILTENYI BIOTEC Streptavidin microbeads at a concentration of 20 μM. These beads are made from iron filings, coated with a dextran coating which is functionalized with streptavidin moieties. Peptide was incubated with beads for 45 min on ice. The 300 μl of peptide coated microbeads were added to pre-equilibrated LS columns outside of the magnetic field to evenly distribute the magnetic beads throughout the columns. Columns were then placed into a magnetic field, and excess peptide was washed away with buffer while retaining the peptide coated microbeads. Cultured and expanded human MSCs from bone marrow aspirate (160,000 cells; LONZA) were added to each LS column and allowed to pass through by gravity flow. Flowthrough was collected and cycled through the columns 5 times, after which the columns were washed with 5 ml buffer. Bound cells were eluted by removing columns from the magnetic field and flushing with 5 ml buffer into 15 ml conical tubes. The collected flowthrough with 5 ml wash and eluted cells were spun down, resuspended in a smaller volume, and counted by hemacytometer (see FIG. 16). The data in FIG. 16 show capture of approximately 70% of the MSCs by the cell binding peptide SEQ ID NO: 4 compared to only about 10% capture by the control peptide.

Example 5

Capture of MSCs from a Mixed Cell Population with Cell Binding Peptide Attached to a Substrate

In this experiment, biotinylated cell binding peptide, SEQ ID NO: 16, was attached to CELLECTION magnetic beads. The CELLECTION beads have streptavidin coupled to a magnetic particle through a DNA linker. Three different cell types, human MSCs, IM-9, and U937 cells were differentially labeled with CELLTRACKER dye and mixed in a ratio such that the MSCs represented ˜4% of the starting cell mixture (the percentage of each cell type in the starting mixture was 38% IM9, 56% U937, and 4% MSC). The cell binding peptide, SEQ ID NO: 16, was biotinylated as described herein at Example 2, as was a general cell binding peptide for use as a control peptide in the experiment. Magnetic particles with no peptide attached, as well as magnetic beads having attached either the cell binding peptide, SEQ ID NO: 16, or the control peptide were tested for their ability to capture human MSCs. The starting cell mixture was incubated with magnetic beads having either attached peptide or no peptide, the beads were washed, and the captured cells were released from the beads by DNase treatment. The cells were measured by flow cytometry histograms shown in 17A-17C. The starting cell mixture is shown in FIG. 17A. The magnetic beads without peptide captured little or no cells (FIG. 17B). The magnetic beads with attached cell binding peptide SEQ ID NO: 16 captured a cell population that was 96% MSCs (FIG. 17C).

Example 6

Capture of MSCs from Bone Marrow Aspirate with Cell Binding Peptide Attached to a Substrate

This experiment was performed to examine the ability of cell binding peptide SEQ ID NO: 4 to capture MSCs directly from bone marrow aspirate in comparison to two separate antibodies against the CD105 I antigen (CD105) and the MSCA-1 stem cell antigen (MSCA-1). This experiment employed biotinylated peptides with streptavidin-coated MILTENYI magnetic beads. The peptides were biotinylated as described herein at Example 2. In addition to cell binding peptide SEQ ID NO: 4, a negative control peptide that does not bind to MSCs was included in the experiment. For the antibody capture experiment, magnetic beads having covalently attached CD105 or MSCA-1 antibody were employed (MILTENYI). Prior to incubation with the peptides and antibodies, bone marrow aspirate (BMA) was mixed with 5 volumes of 10 mM ammonium chloride for 1-2 min at room temperature to lyse red blood cells. The lysate was centrifuged for 5 min at 300×g and the supernatant discarded. The cell pellet was washed with wash buffer (PBS+0.5% bovine serum albumin+0.5 mM EDTA) and cells were resuspended in 25 mM peptide at a concentration of 10⁸per mL. For peptide binding studies, the cell suspension was incubated with the biotinylated peptides for 30 min at 4° C. to allow for binding. After incubation, cells were spun down at 300×g for 5 min, and peptide solution was aspirated. Cells were then rinsed twice with wash buffer, with centrifugation between washes. Cells were resuspended in 80 μL of wash buffer per 10⁷cells. Streptavidin-coated beads were then added at 20 μL per 80 μL of cells. The following procedure was performed for the cell binding experiment with the CD105 and MSCA-1 antibodies. First, 20 μL of magnetic beads having attached CD105 or MSCA-1 antibody were added to 80 μL of the resuspended cells. Bead and cell mixtures were then incubated for 20 min at 4° C. The mixtures were centrifuged to pellet the cells, and the cells were washed once to remove unbound beads. The cell pellet was then resuspended in 1 mL of wash buffer, and loaded into an equilibrated LS purification column attached to a MIDIMACS separator (MILTENYI). The separator contains a magnet, which causes the magnetic beads to adhere to the column, while unbound materials flow through the column. Column was then washed three times with 3 mL of wash buffer. Column was then removed from the magnet, and cell-bound beads were eluted by flushing the column with 5 mL of wash buffer in a clean 15 mL conical tube. The eluants were then plated and cultured. Colony forming units (CFUs) were counted after 14 days in culture. The results are shown in FIG. 18. MSC capture by stem cell binding peptide SEQ ID NO: 4 is as efficient as capture by either of the CD105 or MSCA-1 antibodies (FIG. 18). In contrast, no MSC capture was observed with the negative peptide control. Cells captured with this method were also examined for immunoreactivity for a number of antigens. When comparing stem cell binding peptide SEQ ID NO: 4 and CD105 isolated cells, no changes were observed for immunoreactivity for a number of antibodies including: CD29(+), CD44(+), CD73(+), CD105(+), CD166(+), CD90(+), CD45(−), and CD34 (−). These data suggest that stem cell binding peptide SEQ ID NO: 4 is capable of isolating an MSC cell population that is phenotypically similar to cells isolated by CD105 isolation.

Example 7

Covalent Attachment of Cell Binding Peptide to Collagen Substrate

Cell binding peptide SEQ ID NO: 4 was covalently attached to a collagen substrate using p-nitrophenyl chloroformate chemistry (see FIG. 1). HELISTAT collagen sponge (INTEGRA LIFE SCIENCES, Plainsboro, N.J.) was used as the collagen substrate. The cell binding peptide SEQ ID NO: 4 was modified at the carboxyl terminus with a PEG-10 spacer and a lysine residue.
Collagen sponge substrate modification. HELISTAT collagen (15 sponges, 21.6 mg) was placed in a peptide reactor vessel flushed with nitrogen. The amount of surface amines on the collagen was estimated at ˜35 μmol/g based on quantitative ninhydrin assay. The vessel was charged with 10 mL anhydrous acetonitrile and DIEA (50 μL). Excess (100-fold) p-nitrophenylchloroformate (35 mg, 173 μmol) was added to the vessel and flushed with nitrogen. The reaction vessel was shaken for 4 h on a vortexer (low setting). The reaction mixture was filtered and the sponges were washed thoroughly with 10 mL of DCM, anhydrous (3×) with shaking and then dried under nitrogen. Based on quantitative ninhydrin assay, the majority of surface amines were consumed during this step. This was also confirmed by hydrolyzing a collagen sponge sample with 0.1N NaOH at 22° C. for 15 min to release the nitrophenylate ions, which were quantified spectrophotometrically at 405 nm (ε=1.7×10⁴M⁻¹cm⁻¹). The collagen-pNP sponges were then reacted directly with peptide.
Peptide coupling. Cell binding peptide SEQ ID NO: 4 (with PEG-10-Lys modification; 4 mg) was taken in 4 mL anhydrous acetonitrile and DMF mixture (1:1) in a polypropylene tube flushed with nitrogen. DIEA (50 μL) was added to bring the pH to ˜9. The collagen-pNP (11 sponges; 15.8 mg) was added to the peptide solution. The reactor was flushed with nitrogen and vortexed overnight. The yellow reaction solution was carefully collected and the sponges were thoroughly washed with anhydrous acetonitrile. The washes were carefully pooled. The sponges were flushed and dried under nitrogen. The extent of peptide loading was determined spectrophotometrically at 405 nm by quantifying the p-nitrophenylate ion displaced by the peptide. The peptide loading was determined to be 28.48 μmol peptide/g of collagen.
Soluble collagen substrate modification. In another experiment, cell binding peptide SEQ ID NO: 4 was covalently attached to a soluble collagen substrate using homobifunctional N-hydroxysuccinimide ester, BS³crosslinking reagent (THERMO SCIENTIFIC, Rockford, Ill.) (the chemistry is depicted in FIG. 3). First the peptide having a spacer and lysine residue at the carboxyl terminus was reacted with the BS³crosslinking reagent and the complex purified by HPLC. An excess of BS³was used to minimize peptide dimerization. The activated peptide was then added to soluble bovine, type I collagen (BD BIOSCIENCES, San Jose, Calif.) in a phosphate buffer (pH=8) resulting in conjugation to collagen via lysine amino groups on collagen. The reaction resulted in 80-90% of the peptide-BS³complexes coupled to the collagen.
Fibrillar collagen substrate modification. In another experiment, cell binding peptide SEQ ID NO: 4 is covalently attached to a fibrillar collagen substrate using homobifunctional N-hydroxysuccinimide ester, BS³crosslinking reagent (THERMO SCIENTIFIC, Rockford, Ill.) (the chemistry is depicted in FIG. 3). First, the peptide having a spacer and a lysine residue at the C-terminus is reacted with BS³, and the resulting complex is purified by HPLC. An excess of BS³is used to minimize peptide dimerization. The BS³-activated peptide is conjugated to fibrillar collagen in PBS buffer by addition of approximately 20 μmol peptide per gram of matrix. After washing and freeze-drying the material the peptide loading efficiency is evaluated, for example, by trypsin assay. Briefly, the tissues are placed in trypsin digestion buffer (50 mM Tris-HCl, 0.15 mM NaCl, 10 mM CaCl₂, pH 7.5) containing 10 μg/mL trypsin for 18 h at 37° C. resulting in cleavage of the peptide and release of a peptide fragment into the supernatant. An HPLC assay is used to measure the amount of peptide fragment released using a standard curve generated from trypsin digestion of unconjugated peptide.

Example 8

Capture of Cultured MSCs with a Cell Binding Peptide Covalently Attached to Collagen

This experiment measured the ability of collagen sponge having covalently attached stem cell binding peptide SEQ ID NO: 4 to capture cultured human MSCs compared to unmodified collagen sponge. The cell binding peptide SEQ ID NO: 4 was modified at the carboxyl terminus with a PEG-10 spacer and a lysine residue. The modified cell binding peptide SEQ ID NO: 4 was covalently attached to the collagen sponge as described in Example 7. For the MSC capture experiment, human MSCs were labeled with fluorescent CELLTRACKER Green dye. Unmodified and peptide modified HELISTAT sponge coupons (d=5 mm, thickness=2.5 mm) were used in the experiment. The sponge coupons were pre-wetted in PBS+2% FBS. Sponge coupons were transferred to a suspension of human MSCs (˜25,000 cells in 1 ml PBS+2% FBS). The sponge coupons were incubated with the cells for ˜3 hr at RT rotating. Images were taken of the sponge coupons immediately after the incubation with cells and again after a transfer to and 19 hr incubation in 1 ml PBS+2% FBS (see FIGS. 19A-19B). The images of the peptide modified (right panel) and unmodified (left panel) sponge coupons shown in FIGS. 19A and 19B demonstrate the significantly improved ability of the peptide modified sponges to capture and retain MSCs. In addition, release of MSCs from the sponges was quantified by measuring both fluorescence and cell count following centrifugation of the sponges to release unbound MSCs (data not shown). Further, after the 19 hr incubation the sponge coupons were digested with collagenase to liberate the remaining bound cells, and the cells were similarly quantified (data not shown). For the peptide modified sponges, approximately 10% of the MSCs were detected in the sponge effluent following incubation with the cells, while approximately 40% of the MSCs were detected in the sponge effluent for the unmodified sponge coupons. Very few cells were detected in the sponge effluent after the 19 hr incubation (1 ml PBS+2% FBS) for either the peptide modified or unmodified sponges. However, after collagenase digestion of the collagen sponges to liberate bound cells, approximately 90% of the total cell count was detected for the peptide modified sponge and approximately 60% of the total cell count was detected for the unmodified sponge. Accordingly, the collagen sponges covalently modified with stem cell binding peptide SEQ ID NO: 4 captured significantly more MSCs than unmodified sponges.

Example 9

Differentiation of MSCs Captured with Stem Cell Binding Peptide into Adipocytes, Osteoblasts, or Chondrocytes

This experiment was performed to determine whether MSCs captured with a stem cell binding peptide retained the ability to differentiate into cells of mesenchymal origin.
Adipocyte differentiation. After capture of MSCs using cell binding peptide SEQ ID NO: 4 with the MILTENYI magnetic system according to Example 6, the ability of the captured cells to differentiate into an adipocyte lineage was examined. The MSCs present in the MILTENYI column eluants (see Example 6) were cultured for 21 days in complete medium (DMEM, 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine) supplemented with 0.5 mM isobutyl methylxanthine, 1 μM dexamethasone, 10 μM insulin, 200 μM indomethacin, and 1% antibiotic/antimycotic. Following the 21 day incubation, the cells were fixed in 4% paraformaldehyde in PBS for 15 minutes, washed in 60% isopropanol for 5 minutes, and stained with Oil Red O (SIGMA) for 10 minutes to determine the extent of adipogenesis (see FIG. 20, panels A (undifferentiated MSCs) and panel B (adipocyte differentiated MSCs)). The image of the adipose differentiated cells (FIG. 20, panel B) contains a larger magnification inset where the lipid vacuoles are clearly visible. The results shown in panels A and B of FIG. 20 show that the adipogenesis pathway remains intact for the MSCs after capture from bone marrow using stem cell binding peptide SEQ ID NO: 4.
Osteoblast differentiation. For osteogenic differentiation, the MSCs present in the MILTENYI column eluants (see Example 6) were cultured for 14 days in complete medium supplemented with 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate, 10 mM β-glycerophosphate, and 1% antibiotic/antimycotic. Calcium mineralization was measured by Alizarin Red S (SIGMA) staining to reveal mineralizing osteoblasts (see FIG. 20, panel C (undifferentiated MSCs) and panel D (osteoblast differentiated MSCs)). The results shown in panels C and D of FIG. 20 show that the osteogenic pathway remains intact for the MSCs after capture from bone marrow using stem cell binding peptide SEQ ID NO: 4.
Chondrocyte differentiation. To examine chondrocyte differentiation, the MSCs present in the MILTENYI column eluants (see Example 6) were pelleted by centrifugation at 150×g for 5 minutes. Cells were washed once with 1 ml MACS NH CHONDRODIFF Medium without disturbing the pellet. Cells were spun again and 1 mL CHONDRODIFF Medium was added to each pellet. Every third day, medium was aspirated and replaced with fresh pre-warmed medium. After 24 days in culture, cells exposed to CHONDRODIFF medium formed cartilage plugs or nodules, whereas control cells formed only loose or small nodules. Cells were washed once with PBS and fixed in neutral buffered formalin overnight. Sections were embedded in paraffin and sectioned at 5 microns. Sections were examined by hemotoxylin and eosin staining (data not shown). Sections were further examined by immunostaining for aggrecan, a major structural component of cartilage. In FIG. 21, sections from the periphery (panels A and B) or center (panels C and D) were incubated with an antibody against aggrecan (ABCAM) (panels B and D) or with secondary detection reagents only as a control (panels A and C), then counterstained with DAPI to reveal cell nuclei. After 24 days in the culture medium, aggrecan could be detected in the nodules formed from the MSCs captured by cell binding peptide SEQ ID NO: 4. Control images of captured cells grown in the absence of differentiation medium could not be taken due to the small and loose nature of nodules.

Example 10

Collagen/TCP Composite Bone Void Filler

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. 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, a collagen/TCP composite was generated for use as a bone graft substitute. In addition, the collagen/TCP composite having a stem cell binding peptide covalently attached to the collagen portion of the composite is generated according to the following procedure.
Unmodified collagen/TCP composite. A collagen/TCP composite was generated using unmodified (i.e. collagen without peptide attachment) bovine type I fibrillar collagen (KENSEY NASH, Exton, Pa.). 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). A slurry of the collagen (ranging from about 2-4% collagen) was homogenized and mixed 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. After lyophilization (VIRTIS ADVANTAGE PLUS), the distribution of the β-TCP in the collagen matrix was determined to be substantially uniform by X-ray analysis (FAXITRON, Lincolnshire, Ill.; data not shown).
Stem cell peptide-modified collagen/TCP composite. A stem cell binding peptide such as for example, SEQ ID NO: 4, is covalently attached to fibrillar Type I collagen at a peptide load density ranging from about 1 to 100 μmol peptide/g collagen according to the procedure described herein above at Example 7 using BS³reagent). A collagen/TCP composite comprising the stem cell peptide-modified collagen is generated as follows. The TCP component of the composite is β-TCP. The peptide modified collagen in slurry form (about 2-4% collagen) is homogenized and mixed by hand 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 is modified with stem cell binding peptide both before and after mixing with the β-TCP.
After lyophilization the unmodified collagen/TCP composite was in the form of a sponge that is formable into a putty upon hydration. There was no discernable loss of β-TCP filler from the composite after hydration in saline (1.5 μl saline/mg composite) and puttying, and the composite retained its form after being shaped. In addition, the hydrated and puttied composite is able to be pushed through a 4.5 mm tube such as used in a non-invasive spinal fusion type surgery.

Example 11

Collagen/TCP Composite in Strip Formulation

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 collagen/TCP composite described in Example 10 was 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 collagen/TCP composite described in Example 10 was formulated into a strip by dehydrothermal treatment.
In the first example, a composite from Example 10 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, liquid retention, and compression resistance under a 50 g weight. In another example, a composite generated as described in Example 10 was placed in an vacuum oven (SHELDON, Cornelius, Oreg.) at a temperature ranging from 100-110° C., at 29.5 inches Hg, and for 48-162 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.

Example 12

Covalent Attachment of Cell Binding Peptide to Polyanhydride Polymer

A stem cell 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 FIG. 6. PMA ˜5,000 MW is dissolved in anhydrous dimethylformamide (DMF) and peptide is dissolved in DMF with excess diisopropylethylamine (DIEA). The peptide solution is heated with the PMA solution at 40° C. overnight, for example, and the reaction mixture quenched with water. The crude PMA-peptide conjugate is filtered and analyzed. For example, the extent of substitution on the polyanhydride polymer can be estimated by integration of ¹H-NMR peaks from the peptide together with the integrals of key reference peaks on the polymer to provide an estimate of the level of peptide substitution. In another example, size exclusion chromatography is used by monitoring the UV absorption of the peptide along with a known amount of unconjugated PMA. The degree of peptide substitution is estimated from the mass of the lyophilized product and the UV absorbance of the peptide component.

Example 13

Covalent Attachment of Cell Binding Peptide to Dextran

Cell binding peptide SEQ ID NO: 4 having a C-terminal spacer and lysine residue was covalently attached to dextran (POLYSCIENCES, Inc, PA). Dextran (3-7M; 27.7 mg) was first oxidized by dissolving the dextran in 6 mL of PBS buffer pH 7.5, adding NaIO₄(90 mg), and the vortexing in the dark for 4 hours at room temperature. The reaction mixture was dialyzed against distilled water and lyophilized to give aldehyde activated dextran as a white spongy mass. The peptide was reacted with the aldehyde activated dextran in 0.1 M sodium acetate buffer at pH 5.5 for 3 hours in the dark. Approximately 10 mg of NaCNBH₃was added to the reaction and incubated overnight at room temperature in the dark. Unreacted peptide and other reagents were removed by extensive dialysis against water.
To show that peptide SEQ ID NO: 4 conjugated to soluble dextran described above retained cell binding activity, a stem cell binding competition assay was performed with free peptide SEQ ID NO: 4 by flow cytometry. The SEQ ID NO: 4 peptide-modified dextran was mixed with human MSCs and then incubated with biotinylated stem cell binding peptide SEQ ID NO: 4, the binding of which could be measured by flow cytometry using neutravidin-phycoerythrin. The SEQ ID NO: 4 peptide-modified dextran was a strong competitor of free peptide SEQ ID NO: 4 with a 50% inhibition value below 1 μM, suggesting that the covalently attached stem cell peptide retains its ability to bind MSCs and that the SEQ ID NO: 4 peptide-modified dextran has a higher affinity for MSCs (data not shown).
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.

Claims

1. An implantable bone graft material comprising a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide.

2. The implantable bone graft material of claim 1, wherein the resorbable ceramic and the resorbable polymer are in the form of a composite.

3. The implantable bone graft material of claim 2, wherein the composite is in the form of a sponge, a granulized sponge, a putty, or a strip.

4. The implantable bone graft material of claim 1, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-20.

5. The implantable bone graft material of claim 1, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-16, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16, wherein the variant stem cell binding peptide substantially retains the ability to bind stem cells.

6. The implantable bone graft material of claim 1, wherein the ceramic is selected from the group consisting of calcium 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, biphasic composite between HA and β-TCP, bioglass, bone particles, and combinations and mixtures thereof.

7. The implantable bone graft material of claim 1, wherein the polymer is selected from the group consisting of collagen, fibrillar collagen, Type I collagen, bovine collagen, keratin, silk, dextran, polysaccharides, cellulose derivatives, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitosan, chitin, and hyaluronic acid.

8. The implantable bone graft material of claim 1, wherein 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.

9. The implantable bone graft material of claim 1, wherein the stem cell 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 stem cell 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”), an 11 unit polyethylene glycol (“PEG10”), and a 1 unit polyethylene glycol (“mini-PEG” or “MP”), and combinations thereof.

10. The implantable bone graft material of claim 1, 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.

11. The implantable bone graft material of claim 1, wherein the ceramic is β-TCP and the polymer is bovine Type 1 fibrillar collagen.

12. The implantable bone graft material of claim 12, wherein the weight ratio of β-TCP to bovine Type 1 fibrillar collagen is about 4:1 (about 80% β-TCP to about 20% collagen).

13. The implantable bone graft material of claim 1, wherein the ceramic is β-TCP, 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.

14. The implantable bone graft material of claim 1, wherein the polymer is collagen and wherein the ceramic is β-TCP, wherein the β-TCP has a total porosity of about 70%, wherein the β-TCP has a particle size ranging from about 100 micron to about 300 micron and a pore diameter of less than 100 micron.

15. 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 stem cell binding peptide, and wherein the presence of the graft material promotes bone growth.

16. The method of claim 15, wherein the resorbable ceramic and the resorbable polymer are in the form of a composite.

17. The method of claim 16, wherein the composite is in the form of a sponge, a granulized sponge, a putty, or a strip.

18. The method of claim 15, wherein the site for promoting bone growth is the spine.

19. The method of claim 15, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-20.

20. The method of claim 15, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-16, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16, wherein the variant stem cell binding peptide substantially retains the ability to bind stem cells.

21. The method of claim 15, wherein the ceramic is β-TCP and wherein the polymer is collagen, wherein the β-TCP has a total porosity of about 70%, wherein the β-TCP has a particle size ranging from about 100 micron to about 300 micron and a pore diameter of less than 100 micron, and wherein the weight ratio of the β-TCP to collagen is about 4:1 (about 80% (β-TCP to about 20% collagen).

22. A method for capturing stem cells onto an implantable bone graft material, the method comprising contacting a sample comprising stem cells with the graft material, wherein the graft material comprises a composite of a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide, and wherein the stem cells comprised in the sample are captured onto the graft material through binding to the attached stem cell binding peptide.

23. The method of claim 22, wherein the sample comprising stem cells comprises autologous bone, allograft bone, xenograft bone, bone marrow aspirate (BMA), allogeneic stem cells, a homogeneous or heterogeneous population of cultured cells, adipose tissue, stromal vascular fraction of adipose tissue, blood, blood products, platelets, platelet-rich plasma (PRP), umbilical cord blood, embryonic tissues, placenta, amniotic epithelial cells, or combinations or derivatives thereof.

24. The method of claim 22, wherein the resorbable ceramic and the resorbable polymer are in the form of a composite.

25. The method of claim 24, wherein the composite is in the form of a sponge, a granulized sponge, a putty, or a strip.

26. The method of claim 23, wherein the site for promoting bone growth is the spine.

27. The method of claim 22, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-20.

28. The method of claim 22, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-16, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16, wherein the variant stem cell binding peptide substantially retains the ability to bind stem cells.

29. The method of claim 22, wherein the ceramic is β-TCP, wherein the polymer is collagen, wherein the β-TCP has a total porosity of about 70%, wherein the β-TCP has a particle size ranging from about 100 micron to about 300 micron and a pore diameter of less than 100 micron, and wherein the weight ratio of the β-TCP to collagen is about 4:1 (about 80% (β-TCP to about 20% collagen).

30. A method for promoting bone growth in a subject, the method comprising:

a. contacting a sample comprising stem cells with an implantable bone graft material, wherein the graft material comprises a composite of a resorbable ceramic and a resorbable polymer, wherein the polymer comprises a covalently attached stem cell binding peptide, wherein the stem cells comprised in the sample are captured onto the graft material through binding to the attached stem cell binding peptide; and

b. delivering to the subject the graft material comprising the captured stem cells, wherein the presence of the graft material having the captured stem cells promotes bone growth in the subject.

31. The method of claim 30, wherein the sample comprising stem cell comprises autologous bone, allograft bone, xenograft bone, bone marrow aspirate (BMA), allogeneic stem cells, a homogeneous or heterogeneous population of cultured cells, adipose tissue, stromal vascular fraction of adipose tissue, blood, blood products, platelets, platelet-rich plasma (PRP), umbilical cord blood, embryonic tissues, placenta, amniotic epithelial cells, or combinations or derivatives thereof.

32. The method of claim 30, wherein the resorbable ceramic and the resorbable polymer are in the form of a composite.

33. The method of claim 32, wherein the composite is in the form of a sponge, a granulized sponge, a putty, or a strip.

34. The method of claim 30, wherein the site for promoting bone growth is the spine.

35. The method of claim 30, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-20.

36. The method of claim 30, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-16, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16, wherein the variant stem cell binding peptide substantially retains the ability to bind stem cell.

37. The method of claim 30, wherein the ceramic is β-TCP and wherein the polymer is collagen, wherein the β-TCP has a total porosity of about 70%, a particle size ranging from about 100 micron to about 300 micron, and a pore diameter of less than 100 micron, and wherein the weight ratio of the β-TCP to bovine Type 1 fibrillar collagen is about 4:1 (about 80% β-TCP to about 20% collagen).