METHODS AND COMPOSITIONS FOR CAPTURE OF CELLS

- AFFINERGY, INC.

Compositions and methods are provided for capturing cells. Cell binding peptides are provided that bind to one or more of stem cells, fibroblasts, or endothelial cells. In the methods, a sample containing cells is contacted with a cell binding peptide attached to a substrate, and the cells present in the sample are captured onto the substrate through binding to the cell binding peptide.

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Description
GRANT STATEMENT

The invention was made with government support under Grant No. 1R43AR054229-01 awarded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, under Grant No. R43HL087501-02 awarded by the National Heart, Lung and Blood Institute, and under Grant No. R43HL091590-01A1 awarded by the National Heart, Lung and Blood Institute. The government has certain rights in the invention.

FIELD

The presently disclosed subject matter relates to the capture of cells onto a biocompatible substrate.

BACKGROUND

Multipotent stem cells are known to play a role in healing and repair in response to trauma, disease or disorder. Stem cell mediated repair and healing are achieved by proliferation and differentiation of the stem cells into specialized cell types. For example, mesenchymal stem cells (MSCs) can differentiate into cell types such as bone, cartilage, fat, ligament, muscle, and tendon. In the case of defects in bone, mesenchymal stem cells from the bone marrow, periosteum, and surrounding soft tissue proliferate and differentiate into specialized bone cells. Stem cells can be obtained from embryonic or adult tissues of humans or other animals. As a result of the healing activity of stem cells, much focus has been placed on using stem cells as a treatment to aid in the remodeling of damaged tissue into healthy tissue.

TGF-βhas also shown promise in wound healing in animal models with the application of TGF-β1 and TGF-β2 generating enhanced biomechanical strength of a healed wound (Franz et al., 2001, J. Surgical Res., 97:109-16; Werner & Grose, 2003, Physiol. Rev, 83:835-70). While TGF-βs exert an array of responses, their early up-regulation promotes healing, and exogenous application of TGF-β1 and 2 promotes healing (Franz et al., 2001, J. Surgical Res., 97:109-16; Werner & Grose, 2003, Physiol. Rev, 83:835-70). In addition, JUVISTA (recombinant TGF-(33) is being developed by RENOVO as a treatment to prevent scarring (Wadman, 2005, Nature, 436:1079-80). Further, TGF-β1 has been a target of research in meniscus and cartilage repair, but there are currently no clinical methods to deliver and prolong the retention of the growth factor at the site of injury. Meniscus tears have historically been treated by menisectomy. However, removal of the meniscus dramatically increases the risk of osteoarthritis; therefore, meniscus repair surgeries are increasingly preferred as interventions. Multiple techniques have been developed for arthroscopic surgery, and the most preferred methods rely on “all-inside” meniscal repair devices with sutures. Although surgical repair has been successful for tears in the outer zone of the meniscus, the more common tears occur in the inner meniscus, which is avascular and heals poorly after surgery. Therefore, interventions with biological therapeutics such as stem cells and growth factors are an active area of research to compensate for the poor intrinsic healing capacity of the inner zone of the meniscus. Therefore, a medical device that can deliver TGF-β to the local environment and retain it throughout the healing process would have significant advantages over the current meniscal repair devices.

Thus, there remains a need for systems to deliver and retain cells and/or growth factors to a site of tissue in need of healing or repair. The presently disclosed subject matter provides such systems.

SUMMARY

A method is provided for capturing cells, comprising contacting a sample comprising cells with a cell binding peptide attached to a substrate, wherein the cells comprised in the sample are captured onto the substrate through binding to the cell binding peptide. In one embodiment the cell binding peptide binds to one or more of stem cells, fibroblasts, or endothelial cells and the sample comprising cells comprises one or more of stem cells, fibroblasts, or endothelial cells.

In one embodiment, a method is provided for delivering cells to a subject comprising, contacting a sample comprising cells with a cell binding peptide attached to a substrate, wherein the cells comprised in the sample are captured onto the substrate through binding to the cell binding peptide, releasing the cells from the cell binding peptide, and delivering the released cells to the subject.

In one embodiment, a method is provided for enhancing cell attachment to a synthetic matrix comprising, contacting a sample comprising cells with a collagen having a covalently attached cell binding peptide, wherein the cells comprised in the sample are captured onto the collagen through binding to the attached cell binding peptide; and contacting the collagen having the cells bound to the attached cell binding peptide with the synthetic matrix, wherein the attachment of the cells bound to the attached cell binding peptide to the synthetic matrix is enhanced relative to free cells. In one embodiment, the synthetic matrix is a cell culture plate.

In one embodiment, a method is provided for promoting tissue integration of an implantable device, comprising coating the implantable device with a polymer having a covalently attached cell binding peptide; contacting the coated implantable device with a sample comprising cells, wherein the cells comprised in the sample are captured onto the collagen substrate through binding to the attached cell binding peptide; and delivering the coated implantable device having the bound cells to a subject for promoting tissue integration of the implantable device. In one embodiment, the polymer is collagen and the synthetic matrix is polyether ether ketone (PEEK).

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

FIG. 6 is a schematic diagram depicting the chemistry for covalently attaching a 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 binding peptide to chitosan.

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

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

FIG. 10 is a schematic diagram depicting exemplary chemistry for covalently attaching a 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 binding peptide.

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

FIG. 13 is a schematic diagram depicting one method for covalently attaching more than one 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.

FIGS. 16A-16C are graphs showing the ability of cell binding peptide SEQ ID NO: 4 to bind endothelial cells compared to other cell types. The cell binding peptide was incubated with human umbilical vein endothelial cells (HUVECs), endothelial colony forming cells (ECFCs), umbilical artery smooth muscle cells (UASMCs) or whole blood cells. The data in FIGS. 16A & 16B show that SEQ ID NO: 4 cross-reacted strongly with ECFCs and HUVECs, weakly with UASMCs, and had no cross-reactivity with peripheral blood cells. The cell binding peptide was assayed for its ability to selectively isolate endothelial cells versus smooth muscle cells in the presence of whole blood. The data in FIG. 16C show that the cell binding peptide isolated 8-fold more HUVECs compared to UASMCs.

FIG. 17 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. 18A-18C 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. 19 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. 20A and 20B 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. 21A-21C demonstrate the ability of collagen having covalently attached cell binding peptide SEQ ID NO: 4 to capture and retain endothelial cells. FIG. 21A shows a peptide dependent increase in cell retention on peptide-modified collagen, with a 3-fold increase in cell retention for the peptide-modified collagen over unmodified collagen. The data in FIG. 21B show that the collagen coating was similar for all the samples shown in FIG. 21A. Peptide-modified collagen retained 7-fold more HUVECs than unmodified collagen (FIG. 21C).

FIGS. 22A-22B are graphs showing that the phenotype, viability, and anti-thrombogenic protein expression profiles for endothelial cells are not altered in the presence of collagen modified with cell binding peptide SEQ ID NO: 4. Cells cultured in the presence of peptide-modified collagen, collagen with free peptide, or collagen alone for 24 hours maintained their endothelial cell phenotype and their ability to secrete anti-thrombogenic proteins.

FIG. 23 is a graph showing that HUVECs colonize and proliferate equally on unmodified and SEQ ID NO: 4 cell binding peptide-modified collagen.

FIG. 24 is a graph showing HUVEC retention by SEQ ID NO: 4 cell binding peptide-modified- and unmodified-collagen sponges. After 20 hours of incubation with the HUVECs, the cell binding peptide-modified sponge retained 5-fold more cells than the unmodified collagen sponge.

FIGS. 25A-25B are graphs showing the ability of collagen modified with cell binding peptide SEQ ID NO: 4 to capture cells in flow or under shear stress. Glass cover-slips were coated with peptide modified collagen or unmodified collagen and seeded with ECFCs in flow (Panel A) or HUVECs under shear stress (Panel B).

FIGS. 26A-26D 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 0 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. 27A-27D are images of differentiated MSCs captured from bone marrow aspirate with a stem cell binding peptide SEQ ID NO: 4. In FIG. 27, 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.

FIG. 28 is a graph showing enhanced binding of human dermal fibroblasts to cell binding peptide (SEQ ID NO: 4)-modified collagen compared to unmodified collagen. The collagen was coated on a polystyrene microtiter plate.

 DETAILED DESCRIPTION

The presently disclosed subject matter provides compositions and methods for capturing cells and/or growth factors using stem cell binding peptides and/or growth factor binding peptides attached to a substrate. In one embodiment, a sample comprising cells and/or growth factors is contacted with a cell binding peptide and/or a growth factor binding peptide of the presently disclosed subject matter that has been immobilized onto a substrate, such that the cells and/or the growth factors in the sample are captured onto the substrate. In one embodiment the cell binding peptide binds to one or more of stem cells, fibroblasts, or endothelial cells. Samples particularly useful for the presently disclosed methods include, but are not limited to, bone marrow aspirate, liposuction aspirate (or other fat or adipose tissue), platelet-rich plasma (PRP), plasma, or other blood products, and homogeneous and heterogeneous populations of cultured cells. In one embodiment, the cell binding peptide also captures fibroblasts from the sample. In one embodiment, the captured cells are released from the substrate and provided to a subject to stimulate or enhance tissue repair. In one embodiment, the substrate is an implantable device and the stem cells, fibroblasts, and/or growth factors are captured onto the implantable device substrate which is then provided to a subject to stimulate or enhance tissue repair. The tissue for repair includes any one or more of tendon, muscle, connective tissue, ligament, cardiac tissue, vascular tissue, or dermis.

The methods and compositions 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 cell binding peptide” or reference to “a 1 unit polyethylene glycol (“mini-PEG” or “MP”)” includes a plurality of such 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 cell binding peptides and the growth factor binding peptides of the presently disclosed subject matter are herein collectively referred to as the “binding peptides”. The term “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 cell and is set forth in any one of SEQ ID NOs: 1-20 described in Example 1 (i.e., the cell is the binding “target” of the cell binding peptide). The cell binding peptides of the presently disclosed subject matter bind one or more of stem cells, fibroblasts, or endothelial cells. In addition, the term “stem cell binding peptide” is in some cases herein used interchangeably, for the purposes of the specification and claims, with the terms “cell binding peptide” and “endothelial cell binding peptide” and “fibroblast binding peptide” as certain of the stem cell binding peptides described in Example 1 also bind to fibroblast and endothelial cells (see, e.g., Example 3). The term “growth factor 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 growth factor (i.e., the growth factor is the binding “target” of the growth factor binding peptide). In one embodiment, the growth factor is transforming growth factor beta (TGF-β). In one embodiment, the growth factor is TGF-β and the TGF-βbinding peptide is set forth in SEQ ID NO: 21 (see Examples 13-14). The 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 binding peptide of the presently disclosed subject matter. A 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.

Binding peptides useful in the presently disclosed subject matter also include peptides having one or more substitutions, additions, and/or deletions of residues relative to the sequence of an exemplary cell binding peptide or growth factor binding peptide shown herein at Table 1, as long as the binding properties of the exemplary binding peptides to their targets are substantially retained. Thus, the 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 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 binding peptide can have an amino acid sequence consisting essentially of a sequence of an exemplary binding peptide or a 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 binding activity of the exemplary 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, binding peptides useful in the presently disclosed subject matter include those peptides that are conservatively substituted variants of the binding peptides set forth in SEQ ID NOs: 1-16 (cell binding peptides) and SEQ ID NO: 21 (TGF-β binding peptides), and those peptides that are variants having at least 65% sequence identity or greater to the binding peptides set forth in SEQ ID NOs: 1-16 and SEQ ID NO: 21, wherein all of the variant binding peptides useful in the presently disclosed subject matter substantially retain the ability to bind to their target.

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 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 substrate 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 binding peptide and the substrate. In some embodiments, the spacer also serves the function of a linker (i.e. to attach the binding peptide to the substrate). 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 substrate and spacing the binding peptide from the substrate. In some cases the spacer can serve to position the binding peptide at a distance and in a spatial position suitable for binding and capture and/or in some cases the spacer can serve to increase the solubility of the binding peptide. Spacers can increase flexibility and accessibility of the binding peptide to its target, as well as increase the binding peptide density on the substrate 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 target to the binding peptides is prevented or substantially impaired. Thus, the term “binding peptide” encompasses any of a variety of forms of 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 binding peptide derivative that has substantially retained target binding characteristics can be used in the practice of the presently disclosed subject matter.

The term “substrate” is used, for the purposes of the specification and claims, to refer to any material that is biologically compatible with cells and/or growth factors and to which a binding peptide can be attached for the purpose of capturing the cells and/or growth factors onto the substrate. The binding peptide attached to the substrate can be one or more of a cell binding peptide or a growth factor binding peptide, or combinations thereof. In one embodiment the substrate is in the form of an implantable device. In one embodiment, the substrate can comprise any material and can be present in any form that is desirable and conducive to capturing stem cells and/or growth factors onto the substrate such that the stem cells retain their native biological activity including their ability to differentiate into one or more cells of mesenchymal tissue lineage and/or the growth factors retain their biological growth factor activity. For example, a substrate of the presently disclosed subject comprises one or more materials including, but not limited to, one or more materials selected from: metal, glass, plastic, gel, hydrogel, silica gel, synthetic matrix, polymer, collagen, ceramic, agarose, biopolymer, polysaccharide, dextran, keratin, silk, cellulose derivative, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitosan, chitin, hyaluronic acid, decellularized tissue, or tissue, or derivatives or combinations thereof. In one embodiment, the substrate is in the form of beads, coated beads, gel, hydrogel, mesh, fibrous form, hollow fibers, or sheets, or any of these forms having a biocompatible coating. In one embodiment, the substrate is coated, for example, with a polymer such as collagen.

The term “implantable device” generally refers to a structure that is introduced into a human or animal body to restore a function of a damaged tissue or to provide a new function. An implantable device can be created using any biocompatible material. Representative implantable devices include but are not limited to: hip endoprostheses, artificial joints, jaw or facial implants, tendon and ligament replacements, tendon wraps, surgical meshes, meshes for hernia repair, skin replacements, bone replacements and artificial bone screws, bone graft devices, bone void fillers, cardiac patches, vascular prostheses, heart pacemakers, artificial heart valves, breast implants, penile implants, stents, catheters, shunts, nerve growth guides, intraocular lenses, wound dressings, and tissue sealants. The implantable device substrate can comprise, for example, any one or more of materials including a metal, a glass, a plastic, a gel, a polymer, a synthetic matrix, a biopolymer, a polysaccharide, a collagen, an injectable collagen, a hydrogel, a ceramic, a decellularized tissue, a tissue, a demineralized bone matrix, an extracellular matrix, a dermal matrix, or combinations or derivatives thereof.

The term “attached” in reference to a binding peptide of the presently disclosed subject matter being “attached” to a substrate means, for the purposes of the specification and claims, a binding peptide being immobilized on the substrate by any means that will enable capture of the binding peptide target onto the substrate that allows for the retention of biological activity of the binding peptide target. A binding peptide can be attached to a substrate by any one of covalent bonding, non-covalent bonding including, one or more of hydrophobic interactions, Van der Waals forces, hydrogen bonds, ionic bonds, magnetic force, or avidin-, streptavidin-, and Neutravidin-biotin bonding.

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 binding peptide in the presence of biological fluids where proteases can be present. In addition, as used herein, the term “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).

In one embodiment, the binding peptides are covalently attached to a substrate. In one embodiment the binding peptides are covalently attached to a polymer comprised in the substrate. In one embodiment, the linkers/spacers for use in attaching binding peptides to substrates have at least two chemically active groups (functional groups), of which one group binds to the substrate, and a second functional group binds to the binding peptide or in some cases it binds to the “spacer” already attached to the binding peptide. Preferably, the attachment of the binding peptides to the substrate 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 peptide binding behavior to such an extent that binding of the target to the 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 binding peptide to the substrate and spacing the peptide from the substrate. In many embodiments herein, the linkers used to attach the binding peptide to the substrate 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 binding peptides of the presently disclosed subject matter can comprise a functional group that is intrinsic to the binding peptide (e.g., amino groups on lysine), or the functional group can be introduced into the binding peptide by chemical modification to facilitate covalent attachment of the binding peptide to the substrate. Similarly, the substrate can comprise a functional group that is intrinsic to the substrate (e.g., amino groups on collagen), or the substrate can be modified with a functional group to facilitate covalent attachment to the binding peptide. The binding peptide can be covalently attached to the substrate 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, substrate), and an opposite end having a second reactive functionality to specifically link to a second molecule (e.g, 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 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 substrate to which the binding peptide will be attached.

The binding peptides can be covalently attached to the substrate through one or more anchoring (or linking) groups on the substrate and the binding peptide. The binding peptides of the presently disclosed subject matter can comprise a functional group that is intrinsic to the binding peptide, or the binding peptide can be modified with a functional group to facilitate covalent attachment to the substrate 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, ,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 (e.g., amino groups on a collagen or on a polyamine-containing substrate) or the anchoring groups can be introduced into the substrate by chemical modification.

By way of non-limiting example, in one embodiment, a binding peptide is attached to a substrate 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 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 binding peptide is reacted with the activated chloroformate intermediate on the substrate surface, resulting in attachment of the binding peptide to the substrate.

By way of non-limiting example, in one embodiment, a binding peptide is covalently attached to a substrate comprising an amino functional group (see FIG. 2). FIG. 2 exemplifies attachment of a binding peptide comprising an aldehyde group at one terminus to a substrate that comprises an amino functional group. The binding peptide comprising an aldehyde functional group is treated with the substrate amino groups under reductive amination conditions to give attached binding peptide. In another embodiment not depicted in FIG. 2, a binding peptide comprising an amine functional group is reacted with the substrate amino groups via a homobifunctional linker such as, for example, glutaraldehyde, to yield a covalently attached 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 binding peptide having an amino group (FIG. 3). The binding peptide with attached linking group is then reacted through the remaining N-hydroxysuccinimide ester with an amino group on the substrate to form a peptide-substrate conjugate (FIG. 3). The homobifunctional N-hydroxysuccinimide ester depicted in FIG. 3 is BS3 crosslinking 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 binding peptide is covalently attached to a substrate 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 containing amino groups is reacted with 2-iminothiolane resulting in the introduction of thiol groups on the substrate. 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 binding peptide containing a free thiol is attached covalently to the substrate through a thiol-disulfide exchange resulting in a disulfide bond between the substrate and binding peptide.

By way of non-limiting example, in one embodiment, a binding peptide is attached covalently to a substrate 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 is first functionalized with amine groups using known methods (if the amino groups are not intrinsic to the material of the substrate). Next, a thiol-cleavable, heterobifunctional (amine- and sulfhydryl-reactive) compound (LC-SPDP; THERMO SCIENTIFIC, Rockford, Ill.) is reacted with the amino-functionalized substrate. The binding peptide is reacted with the LC-SPDP modified substrate.

By way of non-limiting example, in one embodiment, a binding peptide is attached covalently to a substrate 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 comprising amino functional groups and then the modified substrate is reacted with a binding peptide having a free thiol group. Alternatively, in one embodiment, the same chemical scheme is utilized but with the substrate modified with a thiol group and the binding peptide modified with the maleimido group.

By way of non-limiting example, in one embodiment, a 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 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 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 binding peptide amine is coupled to the acid group on the chitosan to give the binding peptide-chitosan conjugate. The phthaloyl group is then removed using hydrazine.

By way of non-limiting example, in one embodiment a 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 binding peptide amine is reacted with halogenated chitosan to give the binding peptide-chitosan conjugate. The phthaloyl group is finally removed by reacting with hydrazine.

By way of non-limiting example, in one embodiment a 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 binding peptide through the amino group on chitosan.

By way of non-limiting example, in one embodiment a 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 binding peptide to yield the peptide-HA conjugate.

By way of non-limiting example, in one embodiment, a 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 soluble carbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl) carbodimide (EDC) along with HOBt. The activated HA is coupled with the amino group of a binding peptide to yield the peptide-HA conjugate.

By way of non-limiting example, in one embodiment, a 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 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 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 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 cell binding peptide can be covalently attached to oxidized cellulose through the aldehyde groups on the oxidized cellulose. In this example, a cell binding peptide having a free amine undergoes reductive amination with the aldehyde group on the 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 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 substrate by reductive amination as described above for oxidized cellulose. More specifically, a cell binding peptide having a free amine undergoes reductive amination with the aldehyde group on the 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 substrate and cell binding peptide are reversed).

By way of non-limiting example, in one embodiment, more than one binding peptide is attached to a substrate. Attaching multiple binding peptides to a single substrate is only limited by practical considerations related to the method of attachment. For example, in one embodiment, two different binding peptides are covalently attached to a substrate using any of the chemical schemes shown in FIGS. 1-12. In each of the chemical schemes depicted in FIGS. 1-12, the substrate having a functional group is reacted with two or more different binding peptides that each comprise a functional group to covalently attach the two or more binding peptides to the substrate based on simple competition between the 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 binding peptides that each comprise an amino group or an aldehyde group (i.e., the two different binding peptides replace the single peptide depicted in FIGS. 1 and 2), to covalently attach the two or more binding peptides to the substrate through the amino or aldehyde group, respectively. In the case of the chemical schemes depicted in FIGS. 4 and 5, the modified substrate is reacted with two or more different binding peptides that each comprise a thiol group, to covalently attach the two or more 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 binding peptides).

By way of non-limiting example, in one embodiment, two different binding peptides are covalently attached to a substrate comprising amino groups using the chemical scheme shown in FIG. 13. In this embodiment, the amino groups on the substrate are modified with maleimido groups. The modified substrate is then reacted with a binding peptide comprising both a thiol group and an aldehyde group to covalently attach the binding peptide to the substrate through the thiol group. Next, the substrate-binding peptide conjugate is reacted with another 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 binding peptide modified with the maleimido group. In addition to using this scheme to covalently attach different binding peptides, the scheme is also useful for attaching the same binding peptide.

Cell and/or growth factor capture onto a substrate having an attached cell binding peptide and/or growth factor binding peptide of the presently disclosed subject matter can be performed using any of a wide array of separation methods known to those of skill in the art. In particular, for example, useful separation methods include, but are not limited to, adsorption chromatography, such as with an adsorption column or an adsorption membrane, or by using density centrifugation. In one embodiment, the substrate is also an implantable device substrate and the cells and/or growth factors are captured directly onto the implantable device using the forgoing methods. In one embodiment, the forgoing separation methods are not used and the cells and/or growth factors are captured onto the implantable device endogenously in vivo.

The presently disclosed subject matter provides compositions and methods for capturing cells and/or growth factors onto a substrate to which the binding peptides of the presently disclosed subject matter are attached. In one embodiment, a sample comprising one or more binding peptide targets (i.e., cells and/or growth factors) is contacted with a substrate to which one or more binding peptides of the presently disclosed subject matter is attached, such that the binding peptide target(s) in the sample are captured onto the substrate. In one embodiment, the sample is a sample comprising stem cells, fibroblasts, and/or growth factors and the sample is contacted with a substrate comprising an attached stem cell binding peptide and/or an attached growth factor binding peptide, such that the stem cells and/or growth factors in the sample are captured onto the substrate. In one embodiment, the stem cell binding peptide also binds to fibroblasts. In one embodiment, the attached growth factor binding peptide is a TGF-β binding peptide.

The sample comprising stem cells, fibroblasts, and/or growth factors is not limited to a particular source. Samples particularly useful for the presently disclosed methods include, by non-limiting example, bone marrow, allogeneic stem 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, tissue punch, omentum, or a homogeneous or heterogeneous population of cultured cells, or combinations or derivatives thereof. The captured stem cells of the presently disclosed subject matter are capable of differentiating into one or more cells of mesenchymal tissue lineage including, for example, bone, fat, tendon, cartilage, ligament, muscle, or marrow stroma.

In one embodiment, the captured cells and/or growth factors are released from the substrate and provided to a subject to stimulate or enhance tissue repair. In one embodiment, the substrate is an implantable device and the cells and/or growth factors are captured directly onto the implantable device which is then provided to a subject to stimulate or enhance tissue repair. In one embodiment, the implantable device having one or more attached cell binding peptides and/or growth factor binding peptides is provided directly to a subject to stimulate or enhance tissue repair through endogenous cell and/or growth factor capture. The tissue for repair includes by non-limiting example any one or more of tendon, muscle, connective tissue, ligament, cardiac tissue, vascular tissue, or dermis.

In one embodiment, a method is provided for capturing stem cells, fibroblasts, and/or growth factors onto a substrate, comprising contacting a sample comprising stem cells, fibroblasts, and/or growth factors with a cell binding peptide and/or a growth factor binding peptide attached to a substrate, wherein the cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-20, and wherein the stem cells, fibroblasts, and/or growth factors comprised in the sample are captured onto the substrate. In one embodiment, the cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-16, and peptides that are variants having at least 65% sequence identity or greater to the SEQ ID NOs: 1-16, wherein all of the variant binding peptides useful in the presently disclosed subject matter substantially retain the ability to bind to stem cells. In one embodiment, the substrate is an implantable device. In one embodiment, the sample comprises bone marrow, adipose tissue, platelet-rich plasma (PRP), platelets, or other blood products, omentum, tissue punch, or a homogeneous or heterogeneous population of cultured stem cells, or combinations or derivatives thereof. In one embodiment, the sample comprises stem cells and fibroblasts (e.g., in the case of the sample being liposuction aspirate) and the cell binding peptide attached to the substrate captures fibroblasts in addition to capturing the stem cells comprised in the sample. In one embodiment, the implantable device comprises a metal, a glass, a plastic, a ceramic, a gel, a hydrogel, silica gel, a polymer, a synthetic matrix, a biopolymer, dextran, agarose, keratin, silk, cellulose derivative, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitosan, chitin, hyaluronic acid a polysaccharide, a collagen, an injectable collagen, a decellularized tissue, a tissue, a demineralized bone matrix, an extracellular matrix, a dermal matrix, a surgical mesh, a mesh for hernia repair, a cardiac patch, a tendon wrap, a bone void filler, or an orthopedic joint replacement, or derivatives or combinations thereof. In one embodiment, the substrate is an implantable device comprising an attached cell binding peptide. In one embodiment, the implantable device further comprises an attached growth factor binding peptide. In one embodiment, the substrate is an implantable device comprising one or more growth factor binding peptides. In one embodiment, the implantable device is a cardiac patch comprising an attached cell binding peptide.

The cell and/or growth factor capture onto the substrate can be performed using any of an array of methods known to those of skill in the art. Accordingly, in one embodiment, the substrate can be in a form including, but not limited to, beads, coated beads, gel, hydrogel, mesh, fibrous form, hollow fibers, or sheets. In one embodiment, the cell and/or growth factor capture method comprises contacting a sample comprising stem cell cells, fibroblasts, and/or growth factors with a cell binding peptide and/or a growth factor binding peptide attached to the substrate and performing the capture according to methods well known in the art, such as by performing the capture by an adsorption column, an adsorption membrane, or a density centrifugation. In one embodiment of the presently disclosed subject matter, a device is provided for chromatography for capturing stem cells, fibroblasts, and/or growth factors from a sample, the device comprising a stem cell binding peptide and/or a growth factor binding peptide of the presently disclosed subject matter attached to a substrate. In one embodiment of the presently disclosed subject matter, a filter apparatus is provided for capturing stem cells, fibroblasts, and/or growth factors from a sample, the filter apparatus comprising a substrate to which a stem cell binding peptide and/or a growth factor binding peptide of the presently disclosed subject matter is attached. In one embodiment, the stem cells, fibroblasts, and/or growth factors are captured onto the binding peptide attached to the substrate. In one embodiment, the stem cell binding peptide also binds fibroblasts, and the fibroblasts are captured along with the stem cells onto the substrate.

In one embodiment of the presently disclosed subject matter, the captured cells and/or growth factors are released from the substrate. In one embodiment, the captured cells are released from the stem cell binding peptide bound to the substrate by physical or chemical means. In one embodiment, the stem cell binding peptides to which the stem cells and/or fibroblasts are bound are released from the substrate through chemical means or by enzymatic cleavage. For example, in one embodiment, a method is provided for capturing stem cells, fibroblasts, and/or growth factors comprising contacting a sample comprising stem cells, fibroblasts, and/or growth factors with a stem cell binding peptide and/or a growth factor binding peptide attached to a substrate, wherein the stem cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-20, and wherein the cells and/or growth factors comprised in the sample are captured onto the substrate, and further comprising a step of releasing the captured cells and/or growth factors from the substrate. In one embodiment, the step of releasing the captured cells and/or growth factors from the substrate is by a physical means comprising shaking or centrifugation. In one embodiment, the step of releasing the captured cells and/or growth factors from the substrate is by a change in pH, a change in salt concentration, or a competitive inhibition binding with molecules that compete with the binding of the captured cells and/or growth factors to the binding peptide(s). In one embodiment, the step of releasing the captured cells and/or growth factors from the substrate is by cleaving the binding peptide, to which the captured cells and/or growth factors are bound, from the substrate. Accordingly, in this embodiment the binding peptide can comprise one or more modifications to the peptide N-terminus, peptide C-terminus, or within the peptide amino acid sequence, to allow for its cleavage from the substrate. In one embodiment, the binding peptide comprises a disulfide bond and the peptide is cleaved from the substrate by addition of a reducing agent to cleave the disulfide bond such as, for example, dithiothreitol (DTT) or tris[2-carboxyethyl] phosphine (TCEP). In one embodiment, the binding peptide comprises an enzyme cleavage sequence and the peptide is cleaved from the substrate by addition of an enzyme that can cleave the sequence in the peptide such as, for example, the enzyme trypsin. In one embodiment, the modification to the binding peptide to facilitate release from the substrate comprises a disulfide group cleavable by addition of a reducing agent or comprises an amino acid sequence cleavable by addition of an enzyme.

In one embodiment, a method is provided for stimulating and/or enhancing tissue repair in a subject, comprising capturing stem cells and/or fibroblasts onto a substrate by contacting a sample comprising stem cells and/or fibroblasts with one or more stem cell binding peptides SEQ ID NOs: 1-20 attached to the substrate, wherein the stem cells and/or fibroblasts are captured onto the substrate; releasing the captured stem cells and/or fibroblasts from the substrate; and providing the released stem cells and/or fibroblasts to the subject in need of the tissue repair. In one embodiment, the cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-16, and peptides that are variants having at least 65% sequence identity or greater to the SEQ ID NOs: 1-16, wherein all of the variant binding peptides useful in the presently disclosed subject matter substantially retain the ability to bind to stem cells. In one embodiment, the tissue for repair comprises any one or more of bone, cartilage, tendon, muscle, connective tissue, or dermis. In one embodiment, the stem cell binding peptide also binds fibroblasts and the fibroblasts are captured and released along with the stem cells. In one embodiment, the stem cells and/or fibroblasts are delivered to the subject by injection at the site in need of tissue repair. In one embodiment, the stem cells and/or fibroblasts are delivered to the subject by injection at the site in need of tissue repair in combination with an implantable device such as, for example, an injectable collagen. In one embodiment, the stem cells and/or fibroblasts are delivered to the subject in need of tissue repair in combination with an implantable device, for example, by incubating the released cells with the implantable device prior to implantation of the device into the subject. In one embodiment, the tissue for repair is cardiac, adipose tissue, or bone tissue. In one embodiment, the released stem cells are incubated with an implantable device comprising a bone void filler prior to delivery of the implantable device to the subject. In one embodiment, the released stem cells are incubated with an implantable device comprising a cardiac patch prior to delivery of the implantable device to the subject. In one embodiment, the released stem cells and/or fibroblasts are incubated with an implantable device for soft tissue repair prior to delivery of the implantable device to the subject. In one embodiment, the released stem cells and/or fibroblasts are incubated with an implantable device comprising a mesh for hernia repair prior to delivery of the implantable device to the subject. In one embodiment, the released stem cells are incubated with an implantable device comprising an orthopedic joint replacement prior to delivery of the implantable device to the subject.

In one embodiment, a method is provided for capturing stem cells and/or fibroblasts onto an implantable device, comprising contacting a sample comprising stem cells and/or fibroblasts with a stem cell binding peptide attached to the implantable device, wherein the stem cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-20, and wherein the stem cells and/or fibroblasts comprised in the sample are captured onto the implantable device through binding to the stem cell binding peptide. In one embodiment, the cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-16, and peptides that are variants having at least 65% sequence identity or greater to the SEQ ID NOs: 1-16, wherein the variant binding peptides substantially retain the ability to bind to stem cells. In one embodiment, the implantable device comprises one or more of a metal, a glass, a plastic, a ceramic, a polymer, a synthetic matrix, a gel, a hydrogel, a biopolymer, a polysaccharide, a collagen, an injectable collagen, a tissue, a demineralized bone matrix, an extracellular matrix, a dermal matrix, a decellularized tissue, a surgical mesh, a mesh for hernia repair, a cardiac patch, a tendon wrap, a bone void filler, or an orthopedic joint replacement, or combinations or derivatives thereof. In one embodiment, the sample comprising stem cells and/or fibroblasts comprises bone marrow aspirate, liposuction aspirate (or other adipose tissue), platelets, platelet rich plasma (PRP), or other blood products, omentum, tissue punch, or a homogeneous or heterogeneous population of cultured stem cells, or derivatives thereof. In one embodiment, the captured stem cells are capable of further differentiating into one or more cells of mesenchymal tissue lineage selected from the group consisting of bone, adipose tissue, tendon, cartilage, ligament, muscle, or marrow stroma. In one embodiment, the stem cell binding peptide captures fibroblasts comprised in the sample along with the stem cells onto the implantable device. In one embodiment, the stem cell binding peptide is attached to the implantable device covalently. In one embodiment, the implantable device further comprises an attached growth factor binding peptide, the sample comprising stem cells and/or fibroblasts further comprises one or more growth factors, and the growth factor(s) comprised in the sample is captured onto the implantable device through binding to the growth factor binding peptide. In one embodiment, the growth factor is TGF-β. In one embodiment, the growth factor binding peptide is a TGF-β binding peptide comprising SEQ ID NO: 21. In one embodiment, the growth factor binding peptide is selected from the group consisting of SEQ ID NO: 21 and conservatively substituted variants thereof, and peptides that are variants having at least 65% sequence identity or greater to SEQ ID NO: 21, wherein the variant binding peptides substantially retain the ability to bind to TGF-β.

In one embodiment, a method is provided for capturing one or more growth factors onto an implantable device, comprising contacting a sample comprising the growth factor with a growth factor binding peptide attached to the implantable device, wherein the growth factor is TGF-β, and wherein the growth factor comprised in the sample is captured onto the implantable device through binding to the growth factor binding peptide. In one embodiment, the growth factor is TGF-β and the growth factor binding peptide is a TGF-β binding peptide comprising SEQ ID NO: 21. In one embodiment, the growth factor binding peptide is selected from the group consisting of SEQ ID NO: 21 and conservatively substituted variants thereof, and peptides that are variants having at least 65% sequence identity or greater to SEQ ID NO: 21, wherein the variant binding peptides substantially retain the ability to bind to TGF-β. In one embodiment, the growth factor binding peptide is attached to the implantable device covalently. In one embodiment, the implantable device comprises one or more of a metal, a glass, a plastic, a ceramic, a polymer, a synthetic matrix, a gel, a hydrogel, a biopolymer, a polysaccharide, a collagen, an injectable collagen, a tissue, a demineralized bone matrix, an extracellular matrix, a dermal matrix, a decellularized tissue, a surgical mesh, a mesh for hernia repair, a cardiac patch, a tendon wrap, a bone void filler, or an orthopedic joint replacement, or combinations or derivatives thereof. In one embodiment, the sample comprising one or more growth factors comprises bone marrow aspirate, liposuction aspirate (or other adipose tissue), platelets, platelet rich plasma (PRP), or other blood products, omentum, tissue punch, or a homogeneous or heterogeneous population of cultured stem cells, or derivatives thereof.

In one embodiment, an implantable device is provided comprising an attached stem cell binding peptide and/or one or more attached growth factor binding peptides, wherein the stem cell binding peptide comprises a sequence selected from the group consisting of: SEQ ID NOs: 1-20. In one embodiment, the cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-16 and conservatively substituted variants thereof, and peptides that are variants having at least 65% sequence identity or greater to SEQ ID NOs: 1-16, wherein the variant binding peptides substantially retain the ability to bind to cells. In one embodiment, the implantable device comprises a metal, a glass, a plastic, a ceramic, a gel, a hydrogel, a polymer, a synthetic matrix, a biopolymer, a polysaccharide, a collagen, an injectable collagen, a tissue, a decellularized tissue, a demineralized bone matrix (DBM), an extracellular matrix, a dermal matrix, a surgical mesh, a mesh for hernia repair, a cardiac patch, a tendon wrap, a bone void filler, or an orthopedic joint replacement, or derivatives or combinations thereof. In one embodiment, the binding peptide is attached covalently to the implantable device. In one embodiment, the growth factor is TGF-β. In one embodiment, the implantable device comprises the TGF-β binding peptide comprising SEQ ID NO: 21. In one embodiment, the growth factor binding peptide is SEQ ID NO: 21 and conservatively substituted variants thereof, and peptides that are variants having at least 65% sequence identity or greater to SEQ ID NO: 21, wherein the variant binding peptides substantially retain the ability to bind to TGF-β.

In one embodiment, the implantable device comprises the TGF-β binding peptide comprising SEQ ID NO: 21 and the material comprised in the implantable device is collagen. In one embodiment, the TGF-β binding peptide is attached covalently to the collagen. In one embodiment, the implantable device is a surgical mesh or a mesh for hernia repair.

In one embodiment, a method is provided for stimulating and/or enhancing tissue repair in a subject, comprising delivering to the subject an implantable device comprising stem cells, fibroblasts, and/or growth factors captured by contacting a sample comprising stem cells, fibroblasts, and/or growth factors with the implantable device comprising an attached stem cell binding peptide and/or growth factor binding peptide, wherein the stem cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-20, and wherein the stem cells, fibroblasts, and/or growth factors comprised in the sample are captured onto the implantable device through binding to the corresponding binding peptide. In one embodiment, the cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-16, and peptides that are variants having at least 65% sequence identity or greater to the SEQ ID NOs: 1-16, wherein the variant binding peptides substantially retain the ability to bind to stem cells. In one embodiment, the cell binding peptide also binds fibroblasts and the fibroblasts are captured onto the implantable device along with the stem cells. In one embodiment, the cell binding peptide and/or the growth factor binding peptide is covalently attached to the implantable device. In one embodiment, the growth factor is TGF-β, and the attached growth factor binding peptide is a TGF-β binding peptide comprising SEQ ID NO: 21.

In one embodiment, the tissue for repair comprises any one or more of bone, cartilage, tendon, adipose tissue, muscle, connective tissue, or dermis. In one embodiment, the sample comprising stem cells, fibroblasts, and/or growth factors comprises bone marrow aspirate, liposuction aspirate (or other adipose tissue), platelets, platelet rich plasma (PRP), or other blood products, omentum, tissue punch, or a homogeneous or heterogeneous population of cultured stem cells, or derivatives thereof. In one embodiment, the implantable device comprises an injectable polymer and the implantable device comprising the stem cells, fibroblasts, and/or growth factors is delivered to the subject by injection at the site in need of tissue repair. In one embodiment, the injectable polymer is collagen. In one embodiment, the injectable polymer is polysaccharide, the tissue for repair is cardiac tissue, and the cell binding peptide selected from the group consisting of SEQ ID NOs: 1-20 is covalently attached to the polysaccharide. In one embodiment, the implantable device comprises a cardiac patch. In one embodiment, the implantable device comprises a surgical mesh. In one embodiment, the implantable device comprises a mesh for hernia repair comprising collagen, and the cell binding peptide selected from the group consisting of SEQ ID NOs: 1-20 is covalently attached to the collagen mesh. In one embodiment, the cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-16, and peptides that are variants having at least 65% sequence identity or greater to the SEQ ID NOs: 1-16, wherein the variant binding peptides substantially retain the ability to bind to stem cells.

In one embodiment, a method is provided for stimulating and/or enhancing tissue repair in a subject, comprising delivering to the subject an implantable device comprising an attached stem cell binding peptide and/or growth factor binding peptide, wherein the cell binding peptide is selected from the group consisting of SEQ ID NOs: 1-20, wherein the stem cell binding peptide and/or growth factor binding peptide is available for capture of endogenous stem cells and/or growth factors. In one embodiment, the stem cell binding peptide also binds fibroblasts, wherein the stem cell binding peptide is available for capture of endogenous fibroblasts. In one embodiment, stem cell binding peptide and/or growth factor binding peptide is attached covalently to the implantable device. In one embodiment, the attached growth factor binding peptide is a TGF-β binding peptide comprising a SEQ ID NO: 21. In one embodiment, the tissue for repair comprises any one or more of bone, cartilage, tendon, adipose tissue, muscle, connective tissue, or dermis. In one embodiment, the implantable device comprises a cardiac patch, wherein the cardiac patch comprises an attached stem cell binding peptide selected from the group consisting of SEQ ID NOs: 1-20.

In one embodiment, a method is provided for capturing cells, comprising contacting a sample comprising cells with a cell binding peptide attached to a substrate, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16, and wherein the cells comprised in the sample are captured onto the substrate through binding to the cell binding peptide. In one embodiment, the cell binding peptide binds to one or more of stem cells, fibroblasts, or endothelial cells and the sample comprising cells comprises one or more of stem cells, fibroblasts, or endothelial cells. In one embodiment, the sample comprising cells comprises bone marrow, allogeneic stem 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, tissue punch, omentum, or a homogeneous or heterogeneous population of cultured cells, or combinations or derivatives thereof. In one embodiment, the method further comprises a step of releasing the captured cells from the substrate. In one embodiment, the captured cells are culture expanded on the substrate prior to release from the substrate. In one embodiment, the step of releasing the captured stem cells is a physical means comprising shaking or centrifugation. In one embodiment, the step of releasing the captured stem cells is a chemical means comprising a change in pH, a change in salt concentration, or a competitive inhibition binding with molecules that compete with the captured stem cells. In one embodiment, the cell binding peptide is attached to the substrate through a spacer. In one embodiment, the spacer is selected from the group consisting of amino acids, 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 cell binding peptide comprises one or more modifications to the peptide N-terminus, peptide C-terminus, or within the peptide amino acid sequence, to allow for attachment of the cell binding peptide to the substrate or release of the cell binding peptide from the substrate. In one embodiment, the modification is selected from the group consisting of amino acids, cysteine, lysine, hydroxyl group, thiol group, aldehyde group, 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”), a group cleavable by addition of a reducing agent, an amino acid sequence cleavable by addition of an enzyme, and combinations thereof. In one embodiment, the substrate comprises metal, glass, plastic, synthetic matrix, silica gel, polymer, polysaccharide, dextran, agarose, biopolymer, collagen, hydrogel, gel, keratin, silk, cellulose derivative, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitosan, chitin, hyaluronic acid or derivatives or combinations thereof. In one embodiment, the substrate is in the form of beads, coated beads, gel, hydrogel, mesh, fibrous form, hollow fibers, or sheets. In one embodiment, the cell capture is performed by an adsorption column, an adsorption membrane, or a density centrifugation.

In one embodiment, a device is provided for chromatography comprising the a cell binding peptide attached to a substrate, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16.

In one embodiment, a filter apparatus is provided for capturing cells from a sample, the filter apparatus comprising a substrate to which a cell binding peptide is attached, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16.

In one embodiment, a method is provided for delivering cells to a subject, comprising contacting a sample comprising cells with a cell binding peptide attached to a substrate, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, 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 cells comprised in the sample are captured onto the substrate through binding to the cell binding peptide; releasing the cells from the cell binding peptide; and delivering the released cells to the subject.

In one embodiment, a method is provided for delivering cells to a subject, comprising contacting a sample comprising cells with a cell binding peptide attached to a substrate in the form of a bead, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, 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 cells comprised in the sample are captured onto the bead substrate through binding to the cell binding peptide; and delivering the beads having the captured cells to the subject.

In one embodiment a method is provided for enhancing cell attachment to a synthetic matrix comprising, contacting a sample comprising cells with a collagen having a covalently attached cell binding peptide, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, 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 cells comprised in the sample are captured onto the collagen through binding to the attached cell binding peptide; and contacting the collagen having the cells bound to the attached cell binding peptide with the synthetic matrix, wherein the attachment of the cells bound to the attached cell binding peptide to the synthetic matrix is enhanced relative to free cells. In one embodiment, the synthetic matrix is selected from the group consisting of plastic, polypropylene, polystyrene, polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), and polyether ether ketone (PEEK). In one embodiment, the synthetic matrix is a cell culture plate. In one embodiment, the cells are one or more of stem cells, fibroblasts, or endothelial cells.

In one embodiment, a method is provided for promoting tissue integration of an implantable device, comprising coating the implantable device with a polymer having a covalently attached cell binding peptide; contacting the coated implantable device with a sample comprising cells, wherein the cells comprised in the sample are captured onto the collagen substrate through binding to the attached cell binding peptide; and delivering the coated implantable device having the bound cells to a subject for promoting tissue integration of the implantable device. In one embodiment, the polymer is selected from the group consisting of a collagen, a keratin, a silk, a polysaccharide, an agarose, a cellulose derivative, an oxidized cellulose, an oxidized regenerated cellulose, a carboxymethylcellulose, a hydroxypropylmethylcellulose, a chitosan, a chitin, a hyaluronic acid, and derivatives and combinations thereof. In one embodiment, the implantable device comprises one or a combination of plastic, polypropylene, polystyrene, polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), polyether ether ketone (PEEK), or polyether ether ketone (PEEK). In one embodiment, the polymer is collagen and the implantable device comprises polyether ether ketone (PEEK).

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 ul 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 NO: Amino acid sequence (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 5, 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 5, 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).

The synthetic biotinylated binding peptide SEQ ID NO: 4 was further examined for its ability to bind endothelial cells compared to other cell types (see FIG. 16). Biotinylated cell binding peptide SEQ ID NO: 4 (10 μM) was incubated with 25,000 cultured cells (human umbilical vein endothelial cells (HUVECs), endothelial colony forming cells (ECFCs; LONZA), umbilical artery smooth muscle cells (UASMCs) or whole blood cells from freshly collected human blood for 30 min at 4° C. Cells were washed twice and cell-bound peptide was detected with Neutravidin-PE (INVITROGEN) for 30 min at 4° C. Cells were then washed, resuspended and fixed in 2% paraformaldehyde, and acquired on a BD FACSARRAY. The data in FIGS. 16A & 16B show that SEQ ID NO: 4 cross-reacted strongly with ECFCs and HUVECs, weakly with UASMCs, and had no cross-reactivity with peripheral blood cells. ECFCs are clonally expanded cells isolated from human umbilical cord blood, and are considered highly proliferative endothelial progenitor cells (EPCs) based on their clonogenic and proliferative potential. In another experiment, binding peptide SEQ ID NO: 4 was assayed for its ability to selectively isolate endothelial cells versus smooth muscle cells in the presence of whole blood. HUVECs or UASMCs were incubated in the presence of medium containing 20% whole blood and binding peptide SEQ ID NO: 4-conjugated magnetic beads (via biotin-SA) for 30 min. The solutions containing the bead-peptide-cell complexes were then passed through a MILTENYI LS column in a magnetic field. The column was washed, and the remaining peptide-bound cells were then released from the magnetic field and counted. The data in FIG. 16C show that binding peptide SEQ ID NO: 4 isolated 8-fold more HUVECs compared to UASMCs.

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 μM 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. 17). The data in FIG. 17 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 18A-18C. The starting cell mixture is shown in FIG. 18A. The magnetic beads without peptide captured little or no cells (FIG. 18B). The magnetic beads with attached cell binding peptide SEQ ID NO: 16 captured a cell population that was 96% MSCs (FIG. 18C).

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 108 per 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 107 cells. 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. 19. 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. 19). 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 (504). 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=1.7×104 M−1 cm−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, BS3 crosslinking 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 BS3 crosslinking reagent and the complex purified by HPLC. An excess of BS3 was 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-BS3 complexes 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, BS3 crosslinking 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 BS3 is 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. 20A-20B). The images of the peptide modified (right panel) and unmodified (left panel) sponge coupons shown in FIGS. 20A and 20B 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.

In a separate experiment, cell binding peptide SEQ ID NO: 4 was covalently attached to collagen as described herein and tested for its ability to bind hMSCs using an experimental setup similar to that described above. Briefly, collagen with covalently attached SEQ ID NO: 4 peptide was serial diluted (starting from 25 μM) and mixed with 20,000 hMSCs in 2% FBS/PBS. After 1 hr incubation on ice, cells were washed twice with 2% FBS/PBS and cells bound to the collagen with covalently attached SEQ ID NO: 4 peptide were detected with Neutravidin-PE. Cells were washed, transferred into 96-well plates for FACS analysis. An approximate 100-fold increase in fluorescence signal was observed for the collagen having covalently attached SEQ ID NO: 4 peptide compared to control. In addition to the increased cell binding observed for the collagen having attached cell binding peptide, it was also observed that the hMSCs bound to the collagen having attached cell binding peptide showed enhanced adherence to polypropylene relative to the cells not exposed to the binding peptide-modified collagen. This result was verified showing that ˜80% of hMSCs were retained on the walls of polypropylene plates after binding in the presence of collagen having attached SEQ ID NO: 4 cell binding peptide, but not in the presence of unmodified collagen or SEQ ID NO: 4 cell binding peptide.

Example 9 Capture of Endothelial Cells with a Cell Binding Peptide Covalently Attached to Collagen

This experiment measured the ability of collagen having covalently attached cell binding peptide SEQ ID NO: 4 (demonstrated to bind to stem cells, fibroblasts, and endothelial cells in Example 3 to capture and retain HUVECs. FIG. 21A shows HUVEC capture by collagen having covalently attached cell binding peptide SEQ ID NO: 4 (generated according to Example 7 using BS3 reagent). Briefly, 96-well plates were coated with increasing amounts of binding peptide-modified collagen over night at 4° C. Unbound collagen was removed and the plates were blocked in tris buffered saline containing 1% BSA for 1 hr at room temperature. After washing, 10,000 HUVECs were added per well in serum free medium for 30 min at 37° C. The plates were washed 3 times with PBS containing 2% FBS, and captured cells were detected with CELLTITER-GLO (INVITROGEN) using a luminometer and a standard curve. FIG. 21A shows a peptide dependent increase in cell retention on peptide-modified collagen, with a 3-fold increase in cell retention for the peptide-modified collagen over unmodified collagen. The 96 well plates were coated with 25 μg of collagen and the data in FIG. 21B show that the collagen coating was similar for all the samples shown in FIG. 21A. For FIG. 21B, the collagen was extracted in gel loading buffer, separated by PAGE, and total protein was stained with SIMPLYBLUE (INVITROGEN). FIG. 21C shows evaluation of HUVEC retention by peptide-modified collagen in the plate format. Plates with captured HUVECs were prepared as described in FIG. 20A. After washing they were incubated with agitation at RT for 1 hour in PBS containing 1% FBS, washed and then retained cells were detected with CELLTITER-GLO. The graph in FIG. 21C shows that after 1 hour with agitation in PBS containing 2% FBS, peptide-modified collagen retained 7-fold more HUVECs than unmodified collagen.

Example 10 Cell Binding Peptide Modified-Collagen Binds Endothelial Cells without Altering Phenotype

Once immobilized on a vascular graft, endothelial cells must retain their characteristic properties of promoting endothelialization. Therefore, the phenotype, viability, and anti-thrombogenic protein expression profiles were evaluated for endothelial cells in the presence of collagen modified with the endothelial cell binding peptide SEQ ID NO: 4 (generated according to Example 7 using BS3 reagent). Cells cultured in the presence of peptide-modified collagen, collagen with free peptide or collagen alone for 24 hours maintained their endothelial cell phenotype and their ability to secrete anti-thrombogenic proteins. Specifically, HUVECs were plated on SEQ ID NO: 4 peptide-modified collagen, collagen with 10 μM free SEQ ID NO: 4 peptide or collagen alone for 24 hours. Flow cytometry was performed using a BD FACSARRAY, and ELISAs were performed according to manufacturers instructions. (FIGS. 22A & 22B). Tissue-type Plasminogen Activator (t-PA) and 6-keto Prostaglandin F1a protein expression levels secreted in this assay are aligned with other reports on cultured HUVECS (Kimura and Yokoi-Hayashi, Biochim Biophys Acta, 1996, 1310:1-4; Merhi-Soussi et al., J Leukoc Biol, 2000, 68:881-9).

In addition, the following experiment was performed to show that HUVECs can colonize and proliferate on cell binding peptide SEQ ID NO: 4-modified collagen. In 96-well plates 7,000 HUVECs were plated on peptide-modified soluble collagen or unmodified collagen for 48 hours at 37 C. Plates were removed and washed and the remaining attached cells were detected using CELLTITER-GLO on a luminomenter. The graph in FIG. 23 shows that HUVECs colonize and proliferate equally on unmodified and cell binding peptide-modified collagen (FIG. 23).

The presentation of cell binding peptide-modified collagen to endothelial cells in a plate format may be very different than an acellular, collagen-rich artery. Therefore, cell binding peptide-modified HELISTAT collagen sponges were used to better model the proposed vascular graft application. The cell binding peptide-modified HELISTAT collagen sponges were generated according to Example 7. HUVEC retention was compared for cell binding peptide-modified- and unmodified-collagen sponges. Briefly, peptide-modified or unmodified collagen sponges were rehydrated in serum free medium containing 50,000 CELLTRACKER GREEN labeled HUVECs. The sponges were incubated at RT rotating in PBS containing 2% FBS. At 1, 2, and 20 h the samples were placed in a new tube with fresh buffer. After the 20 h incubation, the sponges were digested with 1000 collagenase for 16 h and the remaining cells enumerated. After 20 hours with agitation, the cell binding peptide-modified sponge retained 5-fold more cells than the unmodified collagen sponge (FIG. 24).

The ability of the collagen modified with endothelial cell binding peptide SEQ ID NO: 4 to capture ECFCs in flow was analyzed as follows. Glass coverslips were coated with unmodified collagen or cell binding peptide modified-collagen and mounted in a polycarbonate parallel plate flow chamber. PBS/10% FBS was circulated through a closed 150 ml system to block the coverslips. Approximately 2×106 ECFC were injected into the system and were circulated for 2 h at 37° C. Coverslips were washed in PBS to remove loosely adhering cells, the remaining cells on the coverslips were lysed in CELLTITERGLO (INVITROGEN, CA), and the cells enumerated by luminescence against a standard curve. The data in FIG. 25A show that the peptide modified collagen captured about 20-fold more cells than the unmodified collagen.

The ability of the collagen modified with endothelial cell binding peptide SEQ ID NO: 4 to retain HUVECs under shear stress was analyzed as follows. Glass cover-slips were coated with peptide conjugated collagen or unmodified collagen and seeded with HUVECs. Coverslips were secured in a polycarbonate slide with six 1 cm diameter wells enabling simultaneous testing of materials. A closed loop peristaltic pump was used to expose the samples to flow conditions for 30 minutes at laminar flow rates up to 12 dynes/cm2 in PBS containing 2% FBS. The number of cells retained on the cover slip was determined using CELLTITER-GLO (INVITORGEN) and a luminometer. The data in FIG. 25B show that the peptide modified-collagen captured about 3-fold more cells than the unmodified collagen, and the application of shear stress did not significantly affect cell capture by peptide modified- or unmodified-collagen.

Example 11 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. 26, panels A (undifferentiated MSCs) and panel B (adipocyte differentiated MSCs)). The image of the adipose differentiated cells (FIG. 26, panel B) contains a larger magnification inset where the lipid vacuoles are clearly visible. The results shown in panels A and B of FIG. 26 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 R-glycerophosphate, and 1% antibiotic/antimycotic. Calcium mineralization was measured by Alizarin Red S (SIGMA) staining to reveal mineralizing osteoblasts (see FIG. 26, panel C (undifferentiated MSCs) and panel D (osteoblast differentiated MSCs)). The results shown in panels C and D of FIG. 26 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. 27, 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 12 Enhanced Fibroblast Binding to Cell Binding Peptide-Modified Collagen

This example demonstrates that collagen modified with cell-binding peptide (SEQ ID NO: 4) binds more fibroblasts than unmodified collagen. The experiment was performed according to the following procedure. Cell-binding peptide (SEQ ID NO: 4) was covalently attached to soluble collagen as described herein at Example 7. 96-well plates were coated with various amounts of unmodified or the peptide-modified collagen (177 μmol peptide/g collagen) over night at 4° C. Unbound collagen was removed and the plates were blocked for 1 h. After washing, 5,000 human dermal fibroblasts were added per well in serum-free medium for 30 min at 37° C. The plates were washed, and bound cells were detected with CELLTITER-GLO (PROMEGA) using a luminometer. At 0.0025 μg collagen, fibroblast binding was increased 11-fold for the peptide-modified collagen compared to unmodified collagen (see FIG. 28). Increased cell binding was also observed at higher concentrations of the SEQ ID NO: 4 peptide-modified collagen (data not shown).

Example 13 Identification of TGF-β Binding Peptides by Phage Display

Peptides that bind TGF-β3 were identified by phage display biopanning. Recombinant human TGF-β3 (rhTGF-β3) was biotinylated and immobilized on streptavidin-coated microtiter plates. Twenty-two different phage display libraries were used for the selection. Enrichment of target-binding phage in the selection was monitored using an ELISA-type assay with an HRP-conjugated anti-M13 antibody. After enrichment for TGF-β3 specific phage, individual phage were picked, propagated on E. coli, and tested for binding to TGF-β3. Over 20 individual phage were isolated that bound TGF-β3. The DNA from phage displaying peptides that displayed binding to TGF-β3 was analyzed, and the amino acid sequences of the displayed peptides were deduced from the DNA sequence. The TGF-β3-binding peptide sequences were then synthesized using standard solid-phase peptide synthesis techniques on a Symphony Peptide Synthesizer (PROTEIN TECHNOLOGIES, Tucson, Ariz.) using standard Fmoc chemistry as described herein above at Example 2. Biotinylated peptides were generated similarly, with a GSSGK(biotin) sequence added to the C-terminus of the peptide.

The TGF-β binding peptides with the biotin moiety on their C-terminus were tested for their ability to bind the target growth factor using the following methods. Streptavidin-coated plates were first prepared by generating a 1:100 dilution of streptavidin (1 mg/mL) in 0.1M NaHCO3 buffer. In each well of a 96-well Immulon 4HBX plate, 50 μL of this solution was added (0.5 μg of streptavidin per well). Plates were incubated overnight at 4° C. Wells were blocked with 150 μL/well of bovine serum albumin (1%) in 0.1M NaHCO3 buffer. Plates were either stored at 4° C. or incubated 1 hr at room temperature, if plates were to be used immediately. Prior to use plates were washed three times in TBS-T (TBS+0.05% Tween-20). Peptides were diluted to 0.2 μM in 100 μL of TBS-T. Peptide solution was added to each well and incubated for 45 min at room temperature. Remaining streptavidin binding sites were blocked with free biotin (0.5 mM). Plates were washed three times in TBS-T to remove unbound peptide. Growth factor solution (TGF-β3), starting at 1 μM was run through a 2-fold dilution series to generate a range of concentrations (1 μM-1 nM) in a 50 μL volume in each well. Growth factor was added to each well and incubated for 40 min at room temperature. Plates were washed five times in TBS-T to remove unbound growth factor. Antibodies against growth factor TGF-βwere added (R & D SYSTEMS) for 30 min at room temperature. Plates were washed with TBS-T and an alkaline phosphatase-conjugated secondary antibody (anti-mouse, 1:1000 dilution) was added to each well. To reveal immunoreactivity, 100 μL of para-nitrophenyl phosphate solution was added to each well, and optical density was recorded on a spectrophotometer. The peptide having the following sequence: SSCNNLSVCTFDKIIERNRSSR (SEQ ID NO: 21) was observed to have the best binding affinity for TGF-β3.

In another experiment, peptides that bind TGF-β1 are identified by phage display biopanning. Recombinant human TGF-β1 (SIGMA-ALDRICH; St. Louis, Mo.) is immobilized for phage display with an antibody against the growth factor (R&D SYSTEMS, INC.; Minneapolis, Minn.). Each library is pre-screened in the absence of TGF-β1 against an uncoated plate or an antibody-coated plate to remove phage that might bind the antibody or well surface. Libraries that show enrichment of TGF-β1˜binding phage are diluted and plated onto lawns of E. coli to isolate individual phage. Based on the phage sequences, candidate peptides are synthesized as described herein above, and tested for TGF-β1-binding in an ELISA assay. For the assay, peptides are immobilized on a streptavidin-coated plate, and the rhTGF-β1 concentration is titrated to yield EC50 values for peptide binding to TGF-β1. The binding assay is repeated with serial dilutions of TGF-β3, PDGF, and IGF-1 to determine the TGF-β1 binding peptides cross-reactivity for other growth factors. Peptide cross-reactivity with platelets is determined. Washed platelets are prepared from human plasma (AMERICAN RED CROSS) as previously described (Arthur et al., Thromb Haemost, 2005, 93:716-23) and incubated with biotinylated peptide and fluorescently tagged neutravidin (INVITROGEN) and then acquired on a BD FACSARRAY. Peptide reactivity is measured as the fraction of cells staining with NEUTRAVIDIN-PE relative to a control without the addition of biotinylated peptide.

Example 14 Covalent Attachment of TGF-β Binding Peptide to Polyester Suture for Meniscus Repair

In this experiment, a TGF-β binding peptide is covalently attached to a polyethylene terephthalate (PET) polyester suture for use in meniscus repair surgery. The TGF-β binding peptide-modified suture provides an improved method for delivering TGF-β to the site of healing by prolonging the retention of TGF-β and increasing its local concentration at the site of healing. The localized retention of the growth factor would allow for long-term signaling and accelerated healing. In one embodiment, the TGF-β binding peptide is a TGF-β1 binding peptide. In one embodiment, the peptide-modified suture is loaded with TGF-β prior to implantation, by mixing the peptide-modified suture with platelet-rich plasma (PRP). PRB has been shown to contain high concentrations of TGF-β (Weibrich et al., Growth Factors, 2002, 20:93-97). The peptide-modified suture loaded with TGF-β would not only improve the therapeutic efficacy of TGF-β by prolonging its release at the site of healing, but also would obviate the need for use of recombinant TGF-β proteins. PRP alone has been shown to enhance healing in the avascular zone of the meniscus in both in vitro and in vivo experiments (Lopez-Vidriero et al., Arthroscopy, 2010, 26: 269-78; Ishida et al., Tissue Eng, 2007, 13: 1103-12). Clinically, the combination of meniscal repair with PRP has also produced successful results (Lopez-Vidriero et al., Arthroscopy, 2010, 26:269-78). In another embodiment, the peptide-modified suture is mixed with synovial fluid prior to implantation.

The TGF-β binding peptide is covalently coupled to a PET suture according to the following methods. A PET polyester suture was chosen because it is non-absorbable, which is optimal for the slow healing rate of the meniscus. In addition, PET is used in currently available meniscal repair devices (e.g., RAPIDLOC Device; Kocabey et al., Arthroscopy, 2006, 22:406-13), and its surface can be functionalized to covalently link peptides to the suture (Chollet et al., Biomol Eng, 2007, 24:477-82; Lieb et al., Biomaterials, 2005, 26:2333-41; Chen, W. and T. J. McCarthy, Macromolecules, 1998, 31:3648-3655). Modification of PET involves chain cleavage that leads to surface functionality at the new chain ends but also causes the degradation of the sample. In this Example two separate chemistries are used to generate functional groups on the PET surface.

In the first scheme in which the chemistry is depicted in FIG. 35A, PET is modified to create carboxylic acid functions on the PET surface by either hydrolysis or UV irradiation, and the amount of CO2H grafted onto the surface is determined using the toluidine blue-O method, in which the dye stains the deprotonated acid groups through ionic interaction. Reaction conditions is fine-tuned to optimize functional group concentration and minimize degradation. The most commonly used techniques of PET surface modification are hydrolysis and reduction (Chollet et al., Biomol Eng, 2007, 24:477-82; Chen, W. and T. J. McCarthy, Macromolecules, 1998, 31:3648-3655). The chemistry for the surface modification of PET and covalent attachment of the peptide after functionalization is depicted in FIG. 35A. PET-CO2H is mmersed in a solution of dimethylaminopropyl-3-ethyl carbodiimide hydrochloride (EDC) and NHS in (2-(N-morpholino)-ethanesulfonic acid. The activated samples are treated with peptide solution, and the dried conjugates are analyzed for peptide loading. Elmann's test or tryptic digestion is used to determine peptide loading if the peptide contains a disulfide bond or lysine, respectively. In the second scheme, in which the chemistry is depicted in FIG. 35B, PET is modified to create hydroxyl functions on the PET surface. PET-OH is activated with p-toluenesulfonyl chloride and pyridine in dry acetone and heating for 1 h at 50° C. under argon atmosphere.

Additional methods for PET functionalization include plasma (Wang et al., J. App. Pol. Sci., 1993, 50:585-599), corona discharge (Strobel et al., J. Adhesion Sci. Technol., 1992, 6:429-443), and ion beam treatment (Bertrand, Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms, 1987, 19-20(Part 2):887-890).

To demonstrate the ability of the peptide-modified suture to bind TGF-β, a binding assay is performed. Briefly, varying concentrations of TGF-β1 or TGF-β3 is added to wells containing peptide-modified suture pieces (0.5 cm) and incubated for 30 min at RT. Sutures are washed, and the amount of bound growth factor is measured by ELISA assay. The ability of the peptide modified sutures to retain bound TGF-β is measured as follows. Briefly, peptide-modified sutures of varying peptide densities or unmodified suture are loaded with TGF-β1 or TGF-β3 and transferred to wells containing a 3 mg/mL solution of hyaluronic acid (HA). The sutures are incubated for 6 weeks with regular media changes (1, 6, 24, 48, and 72 h, and then weekly). The amount of TGF-β released into solution is measured after each change by ELISA. The concentration of HA was chosen because it falls in the range of HA in healthy synovial fluid (Balazs et al., Arthritis Rheum, 1967, 10: 357-76; Bagga et al., J Rheumatol, 2006, 33: 946-50).

The ability of the peptide modified suture to capture TGF-β from complex biological fluids is measured as follows. PRP is prepared from human plasma (AMERICAN RED CROSS) using the Curasan protocol, as previously described (Weibrich et al., Int J Oral Maxillofac Implants, 2002, 17:184-90), and activated with thrombin (140 U/mL) to release growth factors (He et al., Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2009, 108:707-13). Thrombin activation should release 80% of TGF-β within 24 h (Tsay et al., J Oral Maxillofac Surg, 2005, 63:521-8). Multiple suture pieces (0.5 cm) are placed into wells containing the PRP for 24 h with shaking at 37° C. Sutures are removed and washed at various time-points (0, 1, 3, 10, 24 h), and the amount of TGF-β bound to the suture is determined by ELISA.

To test the bioactivity of peptide-bound TGF-β, chondrocytes are cultured in media containing peptide-bound TGF-β and proteoglycan synthesis, cell survival, and IL-1 inhibition is assessed as described previously (Chubinskaya et al., Growth Factors, 2008, 26:275-83). Controls groups include 1) peptide without TGF-β, 2) TGF-β without peptide, and 3) no peptide or TGF-β. For testing of IL-1 inhibition, groups with IL-1β are included using the above experimental conditions. A concentration of IL-1β is used that inhibits proteoglycan synthesis 50% (Chubinskaya et al., Growth Factors, 2008, 26:275-83). Chondrocytes are isolated from healthy human cartilage (NDRl) by enzymatic digestion and combined with alginate beads. Beads are recovered and cultured in wells for nine days with media changes every two days, at which time anabolic and anti-catabolic effects on the chondrocytes are measured. Anabolic effects of the bound TGF-β1 is determined by measuring proteoglycan content by the DMMB assay (Chandrasekhar et al., Anal Biochem, 1987, 161:103-8) and synthesis by addition of radiolabeled sulfate during the final 4 h of culture and measurement of sulfate incorporation using the Alcian blue precipitation method (Masuda et al., Anal Biochem, 1994, 217:167-75). Proteoglycan content and synthesis is normalized to DNA content assessed by Hoechst dye. To determine cell survival, cells are stained with LIVE/DEAD Viability/Cytotoxicity Reagent (MOLECULAR PROBES/INVITROGEN) to quantify viable and apoptotic cells. Anti-catabolic effects of the bound TGF-β are determined by measuring proteoglycan synthesis and content after culture with IL-1.

Example 15 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 NaCNBH3 was 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).

Example 16 Cell Binding Peptide Modified Collagen Coated on Peek

To test whether collagen having a covalently attached cell binding peptide could increase cell adhesion to PEEK discs, PEEK discs were coated with collagen and hMSC attachment was measured. Industrial-grade PEEK discs were obtained from VERTEC POLYMERS, Inc Houston, Tex. The discs were machined with a steel punch for a 0.5 cm diameter (thickness of 0.125). Discs (n=2 per group) were coated by two methods. In the first method, the discs were incubated overnight with varying concentrations of cell binding peptide SEQ ID NO: 4-modified collagen (88.5 μmol peptide/g of collagen). The discs were washed to remove unattached peptide and collagen, and the attached biotinylated peptide was detected with Neutravidin-HRP in an ELISA assay. As the concentration of collagen increased, the amount of peptide loaded on the surface of the PEEK also increased (data not shown). In the second method, uncoated PEEK discs were placed in a solution of unmodified or peptide-modified collagen and hMSCs. After 2 h at 37° C., discs were moved to a new plate and washed with PBS to remove unbound cells. The number of attached cells was counted by CELLTITER-GLO. Collagen alone had a small positive effect on cell adhesion (data not shown). On the other hand, peptide-conjugated collagen increased cell attachment relative to both uncoated and collagen-coated PEEK. At the highest concentration of peptide-modified collagen, 26% of the cells were attached, whereas only 9% of cells attached to the uncoated discs and 11% to the discs coated with unmodified collagen. These data indicate that the cell binding peptide-conjugated collagen can be passively adsorbed onto PEEK to promote cell adhesion to an inert material.

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. A method for capturing cells, comprising contacting a sample comprising cells with a cell binding peptide attached to a substrate, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16, and wherein the cells comprised in the sample are captured onto the substrate through binding to the cell binding peptide.

2. The method of claim 1, wherein the cell binding peptide binds to one or more of stem cells, fibroblasts, or endothelial cells and the sample comprising cells comprises one or more of stem cells, fibroblasts, or endothelial cells.

3. The method of claim 1, wherein the sample comprising cells comprises bone marrow, allogeneic stem 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, tissue punch, omentum, or a homogeneous or heterogeneous population of cultured cells, or combinations or derivatives thereof.

4. The method of claim 2, further comprising a step of releasing the captured cells from the substrate.

5. The method of claim 4, wherein the captured cells are culture expanded on the substrate prior to release from the substrate.

6. The method of claim 4, wherein the step of releasing the captured stem cells is a physical means comprising shaking or centrifugation.

7. The method of claim 4, wherein the step of releasing the captured stem cells is a chemical means comprising a change in pH, a change in salt concentration, or a competitive inhibition binding with molecules that compete with the captured stem cells.

8. The method of claim 1, wherein the cell binding peptide is attached to the substrate through a spacer.

9. The method of claim 8, wherein the spacer is selected from the group consisting of amino acids, 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 method of claim 1, wherein the cell binding peptide comprises one or more modifications to the peptide N-terminus, peptide C-terminus, or within the peptide amino acid sequence, to allow for attachment of the cell binding peptide to the substrate or release of the cell binding peptide from the substrate.

11. The method of claim 10, wherein the modification is selected from the group consisting of amino acids, cysteine, lysine, hydroxyl group, thiol group, aldehyde group, 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”), a group cleavable by addition of a reducing agent, an amino acid sequence cleavable by addition of an enzyme, and combinations thereof.

12. The method of claim 1, wherein the substrate comprises metal, glass, plastic, synthetic matrix, silica gel, polymer, polysaccharide, dextran, agarose, biopolymer, collagen, hydrogel, gel, keratin, silk, cellulose derivative, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitosan, chitin, hyaluronic acid or derivatives or combinations thereof.

13. The method of claim 1, wherein the substrate is in the form of beads, coated beads, gel, hydrogel, mesh, fibrous form, hollow fibers, or sheets.

14. The method of claim 13, wherein the cell capture is performed by an adsorption column, an adsorption membrane, or a density centrifugation.

15. A device for chromatography comprising the a cell binding peptide attached to a substrate, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16.

16. A filter apparatus for capturing cells from a sample, the filter apparatus comprising a substrate to which a cell binding peptide is attached, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, conservatively substituted variants of SEQ ID NOs: 1-16, and variants having at least 65% sequence identity to SEQ ID NOs: 1-16.

17. A method for delivering cells to a subject, comprising:

a. contacting a sample comprising cells with a cell binding peptide attached to a substrate, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, 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 cells comprised in the sample are captured onto the substrate through binding to the cell binding peptide;
b. releasing the cells from the cell binding peptide; and
c. delivering the released cells to the subject.

18. A method for delivering cells to a subject, comprising contacting a sample comprising cells with a cell binding peptide attached to a substrate in the form of a bead, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, 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 cells comprised in the sample are captured onto the bead substrate through binding to the cell binding peptide; and delivering the beads having the captured cells to the subject.

19. A method for enhancing cell attachment to a synthetic matrix comprising, contacting a sample comprising cells with a collagen having a covalently attached cell binding peptide, wherein the cell binding peptide is selected from the group consisting of: SEQ ID NOs: 1-20, 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 cells comprised in the sample are captured onto the collagen through binding to the attached cell binding peptide; and contacting the collagen having the cells bound to the attached cell binding peptide with the synthetic matrix, wherein the attachment of the cells bound to the attached cell binding peptide to the synthetic matrix is enhanced relative to free cells.

20. The method of claim 19, wherein the synthetic matrix is selected from the group consisting of plastic, polypropylene, polystyrene, polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), and polyether ether ketone (PEEK).

21. The method of claim 19, wherein the synthetic matrix is a cell culture plate.

22. The method of claim 19, wherein the cells are one or more of stem cells, fibroblasts, or endothelial cells.

23. A method for promoting tissue integration of an implantable device, comprising:

a. coating the implantable device with a polymer having a covalently attached cell binding peptide;
b. contacting the coated implantable device with a sample comprising cells, wherein the cells comprised in the sample are captured onto the collagen substrate through binding to the attached cell binding peptide; and
c. delivering the coated implantable device having the bound cells to a subject for promoting tissue integration of the implantable device.

24. The method of claim 23, wherein the polymer is selected from the group consisting of a collagen, a keratin, a silk, a polysaccharide, an agarose, a cellulose derivative, an oxidized cellulose, an oxidized regenerated cellulose, a carboxymethylcellulose, a hydroxypropylmethylcellulose, a chitosan, a chitin, a hyaluronic acid, and derivatives and combinations thereof.

25. The method of claim 23, wherein the implantable device comprises one or a combination of plastic, polypropylene, polystyrene, polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), polyether ether ketone (PEEK), or polyether ether ketone (PEEK).

26. The method of claim 23, wherein the polymer is collagen and the implantable device comprises polyether ether ketone (PEEK).

Patent History
Publication number: 20110117169
Type: Application
Filed: Nov 18, 2010
Publication Date: May 19, 2011
Applicant: AFFINERGY, INC. (Research Triangle Park, NC)
Inventors: Isaac Gilliam Sanford (Durham, NC), Dalia Isolda Juzumiene (Cary, NC), Shrikumar Ambujakshan Nair (Cary, NC), Martyn Kerry Darby (Chapel Hill, NC), Jonathan Allen Hodges (Durham, NC)
Application Number: 12/949,767