WO2008079983A1 - Protein release from tissue graft materials - Google Patents

Abstract

Disclosed herein are alloplast graft materials capable of releasing a bioactive agent. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Description

PROTEIN RELEASE FROM TISSUE GRAFT MATERIALS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 60/871,376, filed December 21, 2006, which is hereby incorporated herein by reference in its entirety.

BACKGROUND

[0002] In the healing arts, there is often a need for an implant or graft material to replace, repair, or reconstruct tissues, in particular, hard tissues such as bone. For example, hard-tissue implant materials have been used in medicine and veterinary medicine as prosthetic bone materials to repair injured or diseased bone. Hard tissue implant materials are also used in the construction of prosthetic joints to fix the prosthetic joints to bones. In the dental art, hard tissue implant materials are used in the reconstruction of jaw bone damages caused by trauma, disease, or tooth loss; in the replacement or augmentation of the edentulous ridge; in the prevention of jaw bone loss by socket grafting; and in the treatment of periodontal bone void defects. In orthopedics, hard tissue implant materials are used in the reconstruction of bone structure caused by trauma, disease, or surgery. For surgical procedures such as intervertebral diskectomy, the intervertebral disk is removed to provide access in removing the offending tissue, or bone osteophytes. In a spinal fusion procedure, it may be required to fix the vertebrae together to prevent movement and maintain a space originally occupied by the intervertebral disk.

[0003] Graft materials, such as bone to be use for spinal fusion following a diskectomy, can be removed from another portion of the patient's body, termed an autograft. The use of bone taken from the patient's body has the important advantage of avoiding rejection of the implant, but has several shortcomings. There is always a risk in opening a second surgical site in obtaining the implant, which can lead to infection or pain for the patient, and the site of the implant is weakened by the removal of bony material. The bone implant may not be perfectly shaped and placed, leading to slippage or absorption of the implant, or failure of the implant to fuse with the vertebrae. [0004] Other options for a graft source of the implant are bone removed from cadavers, termed allograft, or from other species, termed a xenograft. In these cases while there is the benefit of not having a second surgical site as a possible source of infection or pain, there is increased difficulty of the graft rejection and the risk of transmitting communicable diseases.

[0005] An alternative approach is the use of a synthetic graft material that is biologically compatible with the body and the target tissue. For example, over the last decade, polymeric materials have been used widely as bone graft materials. These materials are bio -inert, biocompatible, can serve as a temporary scaffold to be replaced by host tissue over time, and can be degraded by hydrolysis or by other means to non-toxic products. However, these bio- inert scaffolds lack the ability to release bioactive agents such as proteins that are osteoinductive, for example. Needed in the art are compositions and methods for modifying the surface of graft materials to allow binding and release of bioactive agents.

SUMMARY

[0006] In accordance with the purpose of this invention, as embodied and broadly described herein, this invention relates to tissue graft materials, for example alloplast graft materials, capable of releasing a bioactive agent.

[0007] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

[0008] The accompanying figures are incorporated in and constitute a part of this specification. [0009] Figure 1 illustrates SEM images of the HTR particles: uncoated (1) and coated 0.5; 1 :1 and 2:1 (2, 3 and 4). The 0.5:1 protein:HTR group showed a more uniform coating (2).

[0010] Figure 2 illustrates BSA cumulative release in 8 days [0.5:1, 1 :1 and 2:1 w/w (BSA:LW): (HTR) proportion].

[0011] Figure 3 illustrates BSA cumulative release from BSA:LW:HTRP [0.5 : 1 , 1 : 1 and 2: 1 w/w (BSA:LW): (HTR) proportion].

[0012] Figure 4 illustrates cumulative BSA release from HTR particles. X axis: time (hours); Y axis: amount (mg).

[0013] Figure 5 shows HRP cumulative release in 30 days (0.5:1, 1 :1, 2:1 w/w HRP/HTR ratio). The yellow data is the control group. The binder used was 10% PVA.

[0014] Figures 6A and 6B show BSA cumulative release from coated HTR particles (20mg coated HTR).

[0015] Figures 7 A and 7B show HRP cumulative release from coated HTR particles (20mg coated HTR).

[0016] Figures 8A and 8B show HRP cumulative release from m-CPP:m-SA:PEGDA matrix using PVA (Figure 8A) and Pluronic F 127 (PLU) (Figure 8B) as the polymeric binder.

[0017] Figure 9 A shows FGF-2 cumulative release from coated HTR particles (lOOmg coated HTR). Figure 9B shows FGF-2 cumulative release from coated HTR particles dispersed in PEGDA matrix (lOOmg coated HTR).

DETAILED DESCRIPTION

[0018] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0019] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a polymer is disclosed and discussed and a number of modifications that can be made to a number of molecules including the polymer are discussed, each and every combination and permutation of polymer and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0020] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

[0021] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0022] The present invention can be understood more readily by reference to the following detailed description of aspects of the invention and the Examples included therein and to the Figures and their previous and following description.

[0023] Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. DEFINITIONS

[0024] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which may need to be independently confirmed.

[0025] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component," "a polymer," or "a residue" includes mixtures of two or more such components, polymers, or residues, and the like.

[0026] Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0027] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more -OCH₂CH₂O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more -CO(CH₂)₈CO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

[0028] As used herein, the terms "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0029] As used herein, the term "polymer" refers to a relatively high molecular weight organic compound, natural or synthetic (e.g., polyethylene, rubber, cellulose), whose structure can be represented by a repeated small unit, the monomer (e.g., ethane, isoprene, • -glucose). Synthetic polymers are typically formed by addition or condensation polymerization of monomers.

[0030] As used herein, the term "oligomer" refers to a relatively low molecular weight polymer in which the number of repeating units is between two and ten, for example, from two to eight, from two to six, or form two to four. In one aspect, a collection of oligomers can have an average number of repeating units of from about two to about ten, for example, from about two to about eight, from about two to about six, or form about two to about four.

[0031] As used herein, the term "copolymer" refers to a polymer formed from two or more different repeating units (monomer residues). By way of example and without limitation, a copolymer can be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. It is also contemplated that, in certain aspects, various block segments of a block copolymer can themselves comprise copolymers.

[0032] As used herein, the terms "comprising" or "comprises" mean "including but not limited to" and are inclusive or open-ended transitional terms and do not exclude additional, unrecited elements or method steps. In one aspect, these terms are synonymous with "including," "containing," or "characterized by."

[0033] As used herein, the terms "consisting essentially of or "consists essentially of are generally open-ended transitional terms, but limit the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0034] As used herein, the term "effective amount" refers to such amount as is capable of performing the function of the compound or property for which an effective amount is expressed. As will be pointed out below, the exact amount required will vary from process to process, depending on recognized variables such as the compounds employed and the processing conditions observed. Thus, it is not typically possible to specify an exact "effective amount." However, an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation. In various aspects, an amount can be therapeutically effective; that is, effective to treat an existing disease or condition. In further various aspects, a preparation can be prophylactically effective; that is, effective for prevention of a disease or condition. In a further aspect, a compound or moiety can be provided in an amount effective to perform an imaging function.

[0035] "Optional" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0036] As used herein, the term "subject" means any target of administration. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term "patient" includes human and veterinary subjects.

[0037] As used herein, the term "biocompatible" refers to materials, or by-products thereof, that are non-toxic and do not elicit a strong immunological reaction against the material. However, the term "biocompatible" does not necessarily exclude materials that elicit an immunogenic response such that the reaction is not adverse.

[0038] As used herein, the term "biodegradable" refers to materials which are enzymatically or chemically degraded, or degraded by dissociative processes such as unlinking of an ionically cross-linked material, or dissociation of physically cross-linked structures in vivo into simpler chemical species or species that can be processed by the body through excretory mechanism's.

[0039] As used herein, the terms "implanting" or "implantation" refer to any method of introducing a composition, for example a graft material, into a subject. Such methods are well known to those skilled in the art and include, but are not limited to, surgical implantation or endoscopic implantation. The term can include both sutured and bound implantation.

[0040] As used herein, the term "alloplast" refers to one example of a tissue graft material. In one aspect, an alloplast is an inert material. In a further aspect, an alloplast is a degradable material, for example a material that absorbs slowly within a subject. In a yet further aspect, an alloplast is a non-degradable material, for example a material that does not substantially absorbs within a subject.

B. COMPOSITION

[0041] Provided herein is an implantable composition comprising a graft material, a bioactive agent, and a soluble polymeric binder.

1. GRAFT MATERIALS

[0042] The graft material of the provided implantable composition can be any biocompatible material suitable for implantation into a subject. For example, the graft material can be a polymeric particle, ceramic, metal, orthopaedic or dental implant, endovascular device, stent, balloon catheter, barrier membrane, surgical mesh, wound dressing, or tissue engineering scaffold. In one aspect, the graft material is a bone graft material.

[0043] There are three main types of bone graft materials: autogenous bone that is naturally osteogenic, osteoinductive, and osteoconductive; allografts (cortical or trabecular), that can be osteoinductive and osteoconductive; and alloplasts (synthetic or natural), which are generally osteoconductive only. The graft material can be an osteogenic, osteoinductive, or osteoconductive material. Thus, in another aspect of the provided implantable composition, the graft material is a natural or synthetic alloplast bone grafting material.

[0044] There are numerous examples of natural and synthetic alloplast bone grafting materials known in the art, some of which are specifically disclosed herein. Any of these known or newly discovered materials are contemplated for use in the herein disclosed compositions and methods.

[0045] For example, the graft material can comprise a porous matrix of biologically- compatible polymeric particles. In one aspect, calcium hydroxide is distributed in the pores of the matrix. Examples of polymeric particles for use in implants are known and disclosed herein. [0046] U.S. Pat. Nos. 4,535,485 and 4,536,158 are incorporated by reference for the teaching of polymer-based implantable porous prostheses for use as bone or other hard tissue replacement which are composed generally of polymeric particles.

[0047] U.S. Pat. No. 4,728,570 is incorporated by reference for the teaching of porous implant material which induces the growth of hard tissue. Based on the '570 patent, Bioplant Inc. (South Norwalk, Conn.) manufactures a very slowly absorbable product called Bioplant® HTR®.

[0048] Medical devices made with degradable polyesters such poly (L-lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid) are approved for human use by the Food and Drug Administration, and have been used in many medical applications, for example, in sutures.

[0049] U.S. Patent No. 6,511 ,510 is incorporated herein by reference for the teaching of implantable ceramic materials. Suitable examples of ceramic materials include coral, calcium phosphates, glass ceramics and materials containing calcium phosphates and/or glass ceramics. Examples of calcium phosphates are octacalcium phosphate, apatites, such as hydroxyapatite and carbonate apatite, whitlockites, such as • -tricalcium phosphate and • -tricalcium phosphate, and combinations thereof.

[0050] The graft material can comprise both macropores and micropores. The total porosity can range from about 0.1% to about 99.99%, including about 20% to about 90%, including from about 40% to about 70%. The macropores of the graft material can have a size of from about 0.1 mm to 1.5 mm, including from about 0.2 mm and 1 mm. The micropores of the graft material can have a size of from about 0.05 μm to about 20 μm, including from about 0.5 μm to about 10 μm. Preferably, the micropores are at least located in the macropores. In accordance with this embodiment, the formation of bone tissue is highly promoted. The micropores can be at least present in the surface of the macropores. The microporosity of the material's surface can lie between about 40% and about 60%. 2. SOLUBLE POLYMERIC BINDER

[0051] One aspect of the herein disclosed implantable composition is the use of a soluble polymer to bind the bioactive agent to the graft material in a manner suitable for controlled release of the bioactive agent after implantation.

[0052] In one aspect, the soluble binder can be a binder that is water soluble (e.g., aqueous). In a further aspect, soluble can refer soluble binders that are soluble in solvents or solvent systems that are miscible with water, for example, alcohols (e.g., methanol, ethanol, and propanol), glycols, and polyglycols, tetrahydrofuran (THF), acetone, N-methyl pyrolidone (NMP), dimethylsufoxide (DMSO), dimethylformamide (DMF), and mixtures thereof (e.g., NMP/water, NMP/water/ethanol, NMP/ethanol, water/acetone, NMP/acetone,

NMP/acetone/ethanol, NMP/acetone/ethano I/water). In a further aspect, soluble can refer soluble binders that are soluble in solvents or solvent systems that are polar organic in nature, for example, alcohols (e.g., methanol, ethanol, and propanol), glycols, tetrahydrofuran (THF), acetone, methylethyl ketone (MEK), N-methyl pyrolidone (NMP), dimethylsufoxide (DMSO), dimethylformamide (DMF), and mixtures thereof (e.g., NMP/water, NMP/water/ethanol, NMP/ethanol, water/acetone, NMP/acetone, NMP/acetone/ethanol, NMP/acetone/ethano I/water) .

[0053] Solubility of the binder can be based on conditions prior to implantation of the disclosed implant or after implantation. For example, the binder can be soluble ex- vivo. Alternatively, the binder can be soluble after biodegradation of the implant in vivo. Thus, in one aspect, the binder can be soluble prior to implantation. In a further aspect, the binder can be soluble after implantation. In a further aspect, the binder can be soluble after degradation of the binder. In a further aspect, the binder can be soluble after enzymatic degradation. In a further aspect, the binder can be soluble after denaturation. In a further aspect, the binder can be soluble after alteration of pH. In a further aspect, the binder can be soluble after heating. In a further aspect, the binder can be soluble after chelation. For example, the binder can be soluble after the addition of EDTA.

[0054] The binder can be soluble in the solvent or solvent system at a solubility of at least 1 mg/ml, for example, 2 mg/ml, 3 mg/ml, 5 mg/ml, 10 mg/ml, 25 mg/ml, 50 mg/ml or 100 mg/ml. In a further aspect, the binder can be soluble in the solvent or solvent system at a solubility of from about 1 mg/ml to about 50 mg/ml, for example, from about 1 mg/ml to about 25 mg/ml, from about 1 mg/ml to about 10 mg/ml, from about 5 mg/ml to about 25 mg/ml, from about 5 mg/ml to about 50 mg/ml, or from about 5 mg/ml to about 100 mg/ml.

[0055] It is also understood that, while a soluble binder can be soluble in a solvent or solvent system, this can be used to describe the physiochemical properties of the binder, and the binder can be provided in the absence or substantial absence of the solvent or solvent system.

[0056] In one aspect, by soluble polymer, it is meant a polymer that exhibits a solubility of at least about 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.1%, 0.2%, 0.3%, 0.4% w/v, 0.5% w/v, 0.6% w/v, 0.7% w/v, 0.8% w/v, 0.9% w/v, 1.0% w/v.

[0057] For example, the soluble polymeric binder can comprise poly(vinyl alcohol), poly(acrylic acid), poly(acryl amide), polaxomers (Pluronics®; e.g., Pluronic F127, or a derivative thereof.), polyethyelenoxide, polyethylene glycol, poly(propylene glycol), poly(propylene oxide), poly(saccharides), starch, cellulose, cellulose acetate, or poly(beta- amino esters).

3. BlOACTIVE AGENT

[0058] The bioactive agent of the disclosed implantable composition can be any agent such as a molecule, protein, nucleic acid, transfecting agent (vector), therapeutic agent, or diagnostic agent, that is suitable for release into tissue by the disclosed implantable composition. Preferred bioactive agents either promote tissue growth and/or healing, prevent infection, prevent inflammation, or aid in diagnosis. Other known or newly discovered bioactive agents suitable for release from an implant are considered for use herein.

4. BONE PROMOTING AGENTS

[0059] For example, the bioactive agent can comprise one or more growth factors, cytokines, and/or hormones. The agents can include, for example, proteins originating from various animals including humans, microorganisms and plants, as well as those produced by chemical synthesis and using genetic engineering techniques. As used herein, a "growth factor" includes any soluble factor that regulates or mediates cell proliferation, cell differentiation, tissue regeneration, cell attraction, wound repair and/or any developmental or proliferative process. For example, the bioactive agent can comprise fibroblast growth factor-2 (FGF-2), fibroblast growth factor- 1 (FGF-I), epidermal growth factor (EGF), heparin binding growth factor (HBGF), Placental Growth Factor (PlGF), vascular endothelial growth factor (VEGF), transforming growth factor-alpha (TGF-* ), transforming growth factor-beta (TGF-* ), insulin- like growth factor (IGF-I, IGF-II), platelet derived growth factor (PDGF), leukemia inhibitory factor (LIF), and/or platelet rich plasma (PRP). For example, the bioactive agent can comprise various interferons, including interferon-* , -• , and •, and/or interleukin-2 and -3. For example, the bioactive agent can comprise insulin, growth hormone-releasing factor, and/or calcitonin.

[0060] In one aspect, the agents can promote and/or induce bone formation. Non-limiting examples of suitable bone promoting materials include growth factors such as BMP (Sulzer Orthopedics), BMP-2 (Medtronic/Sofamor Danek), bFGF (Orquest/Anika Therapeutics), Epogen (Amgen), granulocyte colony-stimulating factor (G-CSF) (Amgen), Interleukin growth factor (IGF)-I (Celtrix Pharmaceuticals), osteogenic protein (OP)-I (Creative BioMolecules/Stryker Biotec), platelet-derived growth factor (PDGF) (Chiron), stem cell proliferation factor (SCPF) (University of Florida/Advanced Tissue Sciences), recombinant human interleukin (rhIL) (Genetics Institute), transforming growth factor beta (TGF-* ) (Collagen Corporation/Zimmer Integra Life Sciences), and TGF-* -3 (OSI Pharmaceuticals). Bone formation may be reduced from several months to several weeks. In orthopedic and dental applications, bone regenerating molecules, seeding cells, and/or tissue can be incorporated into the compositions. For example bone morphogenic proteins such as those described in U.S. Pat. No. 5,011,691, the disclosure of which is incorporated herein by reference, can be used in these applications.

[0061] TGF-* superfamily proteins are expressed during bone and joint formation and have been implicated as endogenous regulators of skeletal development. They are also able to induce ectopic bone and cartilage formation and play a role in joint and cartilage development (Storm E E, Kingsley D M. Dev Biol. 1999 May l;209(l): 1-27; Shimaoka et al, J Biomed Mater Res A. 200468(1): 168-76; Lee et al., J Periodontol. 2003 74(6):865-72). The BMP proteins that may be used include, but are not limited to, BMP-I or a protein from one of the three subfamilies. BMP-2 (and the recombinant form rhBMP2) and BMP-4 have 80% amino acid sequence homology. BMP-5, -6, and -7 have 78% amino acid sequence homology. BMP- 3 is in a subfamily of its own. Normal bone contains approximately 0.002 mg BMP/kg bone. For BMP addition to induce bone growth at an osseous defect, 3 to 3.5 mg BMP has been found to be sufficient, although this number may vary depending upon the size of the defect and the length of time it will take for the BMP to release. Additional carriers for the BMP may be added, and include, for example, inorganic salts such as a calcium phosphate or CaO₄S. (Rengachary, SS, Neurosurg. Focus, 13(6), 2 (2002)). Particular GDFs useful in the present compositions include, but are not limited to GDF-I; GDF-3 (also known asVgr-2); the subgroup of related factors: GDF-5, GDF-6, and GDF-7; GDF-8 and highly related GDF-11; GDF-9 and -9B; GDF-IO; and GDF- 15 (also known as prostate-derived factor and placental bone morphogenetic protein).

5. THERAPEUTIC AGENTS

[0062] The bioactive agent of the provided implantable composition can comprise one or more pharmaceutically active agents. As used herein, the term "pharmaceutically active agent" includes a "drug" and means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. This term includes human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like. This term may also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a bioactive effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or mixtures or combinations thereof, including, for example, DNA nanoplexes, antisense molecules, aptamers, ribozymes, triplex forming molecules, RNAi, and external guide sequences. Pharmaceutically active agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the invention.

[0063] Thus, the bioactive agent of the provided implantable composition can comprise one or more preventive or therapeutic active agents and salts or esters thereof, including but not limited to: antipyretic analgesic anti-inflammatory agents, including non-steroidal antiinflammatory drugs (NSAIDs) such as indomethacin, aspirin, diclofenac sodium, ketoprofen, ibuprofen, mefenamic acid, azulene, phenacetin, isopropylantipyrin, acetaminophen, benzydamine hydrochloride, phenylbutazone, flufenamic acid, mefenamic acid, sodium salicylate, choline salicylate, sasapyrine, clofezone or etodolac; and steroidal drugs such as dexamethasone, dexamethasone sodium sulfate, hydrocortisone, or prednisolone; antibacterial and antifungal agents such as penicillin, ampicillin, amoxicillin, cephalexin, erythromycin ethylsuccinate, bacampicillin hydrochloride, minocycline hydrochloride, chloramphericol, tetracycline, erythromycin, fluconazole, itraconazole, ketoconazole, miconazole, terbinafme; nlidixic acid, piromidic acid, pipemidic acid trihydrate, enoxacin, cinoxacin, ofloxacin, norfloxacin, ciprofloxacin hydrochloride, sulfamethoxazole, or trimethoprim; anti- viral agents such as trisodium phosphonoformate, didanosine, dideoxycytidine, azido-deoxythymidine, didehydro-deoxythymidine, adefovir dipivoxil, abacavir, amprenavir, delavirdine, efavirenz, indinavir, lamivudine, nelfϊnavir, nevirapine, ritonavir, saquinavir or stavudine; high potency analgesics such as codeine, dihydrocodeine, hydrocodone, morphine, dilandid, demoral, fentanyl, pentazocine, oxycodone, pentazocine or propoxyphene; anti-proliferative agent such as taxol; and salicylates which can be used to treat heart conditions or as an anti-inflammatory.

[0064] The agents can be incorporated in the disclosed composition directly, or can be incorporated in microparticles or nanoparticles which are then incorporated in the composition. Incorporating the agents in microparticles or nanoparticles can be advantageous for those agents that are reactive with one or more of the components of the composition. 6. DIAGNOSTIC AGENTS

[0065] The bioactive agent of the provided implantable composition can comprise one or more diagnostic agents. Diagnostic/imaging agents can be used which allow one to monitor bone repair following implantation of the compositions in a patient. Suitable agents include commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, X-ray, fluoroscopy, and magnetic resonance imaging (MRI).

[0066] Examples of suitable agents useful in MRI include the gadolinium chelates currently available, such as diethylene triamine pentaacetic acid (DTPA) and gadopentotate dimeglumine, as well as iron, magnesium, manganese, copper and chromium gadolinium chelates.

[0067] Examples of suitable agents useful for CAT and X-rays include iodine based materials, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexol, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte.

[0068] These agents can be detected using standard techniques available in the art and commercially available equipment.

7. BIOACTIVE AGENT PROTECTION

[0069] It is important for the bone promoting agent to remain active through the polymerization process. For example, many enzymes, cytokines, etc. are sensitive to the radiation used to cure polymers during photopolymerization. The method provided in Baroli et al., J. Pharmaceutical Sci. 92:6 1186-1195 (2003) can be used to protect sensitive molecules from light-induced polymerization. This method provides protection using a gelatin-based wet granulation. This technique may be used to protect the bone promoting agent incorporated into the polymer composition. [0070] In some aspects of the provided implantable composition, the bioactive agent is incorporated into a polymeric composition. Thus, the in one aspect, the bioactive agent can be protected from polymerization-induced damage.

[0071] In addition to possible light-induced alterations such as photo -oxidation during photopolymerization, sensitive molecules may be chemically altered upon reacting with monomers, matrix components, and polymerizing species. See Davies M J, Truscott R J W, "Photo -oxidation of proteins and its role in cataractogenesis," J Photochem Photobio B: Biology 63, 114-125 (2001), herein incorporated by reference. Denaturation reactions are of significance, because entrapped drugs may lose their activity or trigger an immune response. See McNally E J, editor, "Protein formulation and delivery," New York: Marcell Dekker, Inc. (2000); Cleland J L, Powell M F, Shire S J, "The development of stable protein formulations: A close look at protein aggregation, deamination, and oxidation," Crit Rev Ther Drug Carrier Syst 10(4), 307-377 (1993); all herein incorporated by reference. Although some studies have shown that proteins can be released from photopolymerized matrices, there are few reports of enzyme entrapment. See Mellot M B, Searchy C, Pishko M V, "Release of protein from highly cross-linked hydrogels of poly(ethylene glycol)diacrylate fabricated by UV polymerization," Biomaterials 22, 929-941 (2001); Elisseeff J, Mclntosh W, Anseth K, Langer R, "Cogelation of hydrolysable cross-linkers and poly(ethylene oxide) dimethacrylate and their use as controlled release vehicles," in Dinh S M, DeNuzzio J D, Comfort A R, editors, "Intelligent materials for controlled release," Washington D.C. : ACS, 1-13 (1999); An Y, Hubbell J A, "Intraarterial protein delivery via intimally-adherent bilayer hydrogels," J Controlled Release 64, 205-215 (2000); Elisseeff J, Mclntosh W, Fu K, Blunk T, Langer R, "Controlled-release of IGF-I and TGF-* 1 in a photopolymerizing hydrogel for cartilage tissue engineering," J Orthop Res 19(6), 1098-1104 (2001); all herein incorporated by reference. Nevertheless, in these latter cases, no quantitative assessment was made regarding the extent of enzyme inactivation or enzyme structure modification.

[0072] U.S. Pat. No. 5,902,599 is incorporated by reference for the teaching of methods for protecting sensitive therapeutic agents from light-induced polymerization when incorporated in a polymer composition. [0073] As disclosed herein, the bioactive molecules of the implantable composition can be admixed with a photopolymerizable monomer. Thus, in one aspect, the bioactive molecules can be shielded from the monomers by an insoluble material that undergoes a solid-gel transition at body temperature. Thus, the insoluble material can be insoluble in the monomer. Upon polymerization, the monomers produce a cross-linked structure and the shielded bioactive molecules are protected from attack in the polymerizing environment.

[0074] The photopolymerizable monomer may belong to any class of compounds, may be of any molecular weight, and may react directly or indirectly to any electromagnetic radiation by polymerizing. In certain embodiments, electromagnetic radiation is comprised under UV, Visible or IR spectrum. When reacting indirectly, a suitable system of one, or a mixture of, photoinitiators and accelerators may be responsible of the radiation energy transfer to the monomer. In certain other embodiments, photoinitiators may include radical polymerization by either photoclevage or hydrogen abstraction, or cationic photopolymerization.

[0075] The insoluble material may be a gelatin, collagen, natural polymer or synthetic polymer. The bioactive molecules can be shielded by the insoluble material by granulation, spray drying, spray chilling, lyophilization, coating vapor deposition (CVD), compression, microencapsulation, coating, subcoating, sealing, coacervation, suspension, precipitation, cogelation, gelation, inclusion in pre-formed delivering systems, inclusion into matrix and micromatrix, or evaporation.

[0076] Thus, the bioactive agent can be admixed with photo -polymerizable monomers, wherein the bioactive agent is shielded from the monomers by an insoluble material that undergoes a solid-gel transition at body temperature, wherein upon polymerization, the monomers produce a cross-linked structure and the shielded bioactive molecules are protected from attack in the polymerized environment.

8. BIOACTIVE AGENT RELEASE

[0077] The bioactive agent can be released from the composition over a period of hours or days. For example, 50% of the bioactive agent can be release from the composition in at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer.

C. DELIVERY VEHICLE

[0078] In one aspect of the disclosed implantable composition, the biocompatible graft is a biocompatible polymeric matrix with desired mechanical properties for implantation. However, in other aspects, the biocompatible graft lacks the desired mechanical properties for implantation.

[0079] Thus, the herein disclosed implantable composition comprising a graft material, bioactive agent, and polymeric binder can be admixed with a delivery vehicle such as a biocompatible polymeric matrix to modify the mechanical properties of the implantable composition. For example, the biocompatible polymeric matrix can be injectable, in situ formable, malleable, or curable. The biocompatible polymeric matrix can also be biodegradable. Examples of biocompatible polymeric matrices for use as delivery vehicles of graft materials are known in the art and disclosed herein.

[0080] It is understood that each of the delivery vehicles disclosed herein can in some aspects additionally or alternatively be used as a graft material in the disclosed compositions and methods. Thus, reference herein to a composition as a delivery vehicle or biocompatible polymeric matrix is also reference to that same composition as a graft material.

[0081] In some aspects, the disclosed delivery vehicle such as a biocompatible polymeric matrix can be a semi-interpenetrating polymer network ("semi-IPN") composition. U.S. Pat. No. 5,837,752 is incorporated by reference for the teaching of a semi-IPN composition for bone repair comprising (1) a linear polymer selected from the group consisting of linear, hydrophobic biodegradable polymers and linear non-biodegradable hydrophilic polymers; and (2) one or more crosslinkable monomers or macromers containing at least one free radical polymerizable group, wherein at least one of the monomers or macromers includes an anhydride linkage and a polymerizable group selected from the group consisting of acrylate or methacrylate. [0082] In some aspects, the disclosed delivery vehicle such as a biocompatible polymeric matrix can comprise polymerizing anhydride prepolymers. U.S. Pat. No. 5,902,599 is incorporated by reference for the teaching of biodegradable polymer networks formed by polymerizing anhydride prepolymers containing crosslinkable groups, such as unsaturated moieties. The anhydride prepolymers can be crosslinked, for example in a photopolymerization reaction by irradiation of the prepolymer with light in the presence of a photosensitive free radical initiator.

[0083] U.S. Patent Publications 2006/0148923 Al and 2006/0052471 Al are hereby incorporated herein by reference for the teaching of crosslinkable polymeric materials. For example, the delivery vehicle such as a biocompatible polymeric matrix can comprise a crosslinkable anhydride prepolymer comprising monomers and/or oligomers having polymerizable groups, such as radically polymerizable groups, which crosslink to form a polymer network. Suitable polymerizable groups include unsaturated alkenes (i.e., vinyl groups) such as vinyl ethers, allyl groups, unsaturated monocarboxylic acids, unsaturated dicarboxylic acids, and unsaturated tricarboxylic acids. Unsaturated monocarboxylic acids include acrylic acid, methacrylic acid, and crotonic acid. Unsaturated dicarboxylic acids include maleic, fumaric, itaconic, mesaconic or citraconic acid. The polymerizable groups can be acrylates, diacrylates, oligoacrylates, dimethacrylates, oligomethacrylates, and other biologically acceptable polymerizable groups, such as (Meth)acrylates.

[0084] In some aspects, the disclosed delivery vehicle such as a biocompatible polymeric matrix can comprise a thermoplastic system. For example, U.S. Pat. No. 5,278,202 is incorporated by reference for the teaching of a thermoplastic system in which a solid, linear- chain, biodegradable polymer is dissolved in a biocompatible solvent to form a liquid, which can then be administered via a syringe and needle. Examples of biodegradable polymers which can be used in the thermoplastic system include polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials. The polymers can have a lower degree of crystallization and are more hydrophobic. These polymers and copolymers are more soluble in the biocompatible solvents than the highly crystalline polymers such as polyglycolide and chitin which also have a high degree of hydrogen-bonding. Examples of materials with the desired solubility parameters are the polylactides, polycaprolactones, and copolymers of these with glycolide in which there are more amorphous regions to enhance solubility.

[0085] In some aspects, the disclosed delivery vehicle such as a biocompatible polymeric matrix can comprise a ceramic composite material. For example, U.S. Pat. No. 6,027,742 is incorporated by reference for the teaching of a bioactive ceramic composite material that is biocompatible, bioresorbable and possesses high strength and/or other desirable mechanical properties. The disclosed bioactive ceramic composite material can be formed at low temperatures, is readily formable and/or injectable, and yet can harden to high strength upon further reaction. The disclosed bioactive ceramic composite material can contain a nano-size, poorly crystalline apatitic calcium phosphate solids with Ca/P ratios comparable to naturally occurring bone minerals and having stiffness and fracture toughness similar to natural bone. The disclosed bioactive ceramic composite material is strongly bioresorbable and its mechanical properties can be adjusted to meet the demands of the particular therapy and/or implant site. The disclosed composite material can be obtained by providing an amorphous calcium phosphate in the presence of a limited quantity of water to produce a hydrated precursor in the form of a putty or paste and promoting the conversion of the amorphous calcium phosphate to a poorly crystalline apatitic calcium phosphate. The conversion is associated with hardening of the paste and can produce a poorly crystalline apatitic calcium phosphate.

[0086] The delivery vehicle such as a biocompatible polymeric matrix can comprises cross- linked sodium alginate. Sodium alginate is biocompatibile and in vivo biodegradabe. A sterile and low endotoxin form of sodium alginate is available under product number K8P569 from Monsanto, 800 N. Lindbargh Blvd. St. Louis, Mo., or under product number UP MVG from Pro Nova, Strandveien 18, N- 1324 Lysaker, Norway. Very low endotoxin levels can be obtained in alginates by use of a highly specialized purification process. Alginates in a water gel form have the unique ability to form elastic films by reaction with calcium salts and/or magnesium salts. Once cross-linked, these films retain their shape and resist stress.

[0087] The delivery vehicle such as a biocompatible polymeric matrix can comprises a UV photo-active polymerhydrogel. Monomers that are polymerizable upon exposure to light radiation have the potential advantage of being formed in vivo at the tissue site of interest via minimally invasive procedures, and can be used as scaffolds in tissue engineering, for cell encapsulation, as drug delivery systems, and as fillers for a tissue defect. See Muggli D S, Burkoth A K, Keyser S A, Lee H R, Anseth K S, "Reaction behavior of biodegradable, photo- cross-linkable polyanhydrides," Macromolecules 3, 4120-4125 (1998); Lu S, Anseth K S, "Photopolymerization of multilaminated poly(HEMA) hydrogel for controlled release," J Controlled Release 57, 291-300 (1999); Elisseeff J, Anseth K, Sims D, Mclntosh W, Randolph M, Langer R, "Transdermal photopolymerization for minimally invasive implantation," Proc Natl Acad Sci USA 96(6), 3104-3107 (1999); Burkoth A K, Anseth K S, "A review of photo-crosslinked polyanhydrides: In situ forming degradable networks," Biomaterials 21 (23), 2395-2404 (2000); Elisseeff J, Mclntosh W, Anseth K, Riley S, Ragan P, Langer R, "Photoencapsulation of chondrocytes in poly(ethyleneoxide)-based semi- interpenetrating networks," J Biomed Mater Res 51(2), 164-171 (2000); Cruise G M, Hegre O D, Lamberti F V, Hager S R, Hill R, Scharp D S, Hubbel J A, "In vitro and in vivo performance of porcine islets encapsulated in interfacially photopolymerized poly(ethylene glycol) diacrylate membranes," Cell Transplant 8(3), 293-306 (2000); Smeds K A, Grinstaff M W, "Photocrosslinkable polysaccharides for in situ hydrogel formation," J Biomed Mat Res 54(1), 115-121 (2001); all incorporated herein by reference. For example, the polymeric matrix can comprise a polyphosphazene that can be ionically cross-linked or photocured. The polymeric matrix can be a photocurable PEG, such as poly (ethylene glycol) diacrylate (PEGDA).

[0088] The delivery vehicle such as a biocompatible polymeric matrix can comprise a hydrogel. The polymeric matrix can be a biocompatible hydrogel comprising at least one polymer. A "hydrogel," as used herein, refers to a network of polymer chains that are water- soluble, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels can be superabsorbent natural or synthetic polymers. For example, hydrogels can contain over 99% water. Hydrogels can also possess a degree of flexibility very similar to natural tissue, due to their significant water content. However, it is also understood that in one aspect, the disclosed hydrogels can comprise water or water mixed with other miscible liquids, for example, alcohols. Hydrogels can comprise positively charged, negatively charged, and neutral hydrogels that can be saturated or unsaturated. Examples of hydrogels are

TETRONICS™ and POLOXAMINES™, which are poly(oxyethylene)-poly(oxypropylene) block copolymers of ethylene diamine; polysaccharides, chitosan, poly(vinyl amines), poly( vinyl pyridine), poly(vinyl imidazole), polyethylenimine, poly-L-lysine, growth factor binding or cell adhesion molecule binding derivatives, derivatised versions of the above (e.g. polyanions, polycations, peptides, polysaccharides, lipids, nucleic acids or blends, block- copolymers or combinations of the above or copolymers of the corresponding monomers); agarose, methylcellulose, hydroxyproylmethylcellulose, xyloglucan, acetan, carrageenan, xanthan gum/ocust beangum, gelatine, collagen particularly Type 1), PLURONICS™, POLOXAMERS™, POLY(N-isopropylacrylmide) and N-isopropylacrylmide copolymers. Thus, for example, the at least one polymer can comprise a saccharide residue, an ethylene oxide residue, a propylene oxide residue, an acrylamide residue, or a blend or copolymer thereof. Theus, the at least one polymer can be agarose. The at least one polymer can be a polaxomers, or a derivative thereof. The at least one polymer can be a polyacrylamides, or a derivative thereof. The at least one polymer can be N-isopropylacrylamide (NIPAM), or a derivative thereof. The at least one polymer can be Pluronic F 127, or a derivative thereof.

[0089] Thus, in some aspects, the graft material and the delivery vehicle can be the same material. In further aspects, the graft material and the delivery vehicle can be the same materials.

D. METHOD

[0090] Also provided herein is a method of preparing an implantable composition, the method comprising the steps of coating a graft material with a soluble polymeric binder, and linking a bioactive agent to the graft material, thereby providing a coated graft material. The method can further comprise the step of admixing the coated graft material with a polymeric matrix as disclosed herein. [0091] As disclosed herein, linking can comprise non-covalent bonding, adsorption, absorption, physical association, or electrostatic association.

[0092] In one aspect of the disclosed method, the coating step is performed before the linking step. In another aspect of the disclosed method, the linking step is performed before the coating step. In another aspect of the disclosed method, the coating step is performed simultaneously with the linking step.

[0093] Also provided is a method of administering to a subject an implantable composition disclosed herein.

[0094] Also provided is a method of treating a tissue disorder in a subject comprising administering to the subject a herein disclosed implantable composition, wherein a bioactive agent is released from the composition and promotes tissue growth, repair, regeneration, or a combination thereof.

[0095] Also provided is a method of delivering a bioactive agent to a tissue in a subject, the steps comprising: coating a graft material with a soluble polymeric binder, linking the bioactive agent to the graft material, admixing the bioactive agent in a biocompatible polymeric matrix, and implanting the graft material into the tissue of the subject.

[0096] The subject of the disclosed methods can be a mammal. The subject can be human. The subject can be a patient. The tissue disorder can be a bone injury. For example, the composition can be implanted in or adjacent to a bone or joint of the subject.

[0097] As another example, the composition of the disclosed methods can be used for bladder bulking. Urethral bulking to treat urinary incontinence involves injecting material around the urethra. This may be done to close a hole in the urethra through which urine leaks out or build up the thickness of the wall of the urethra so it seals tightly when you hold back urine. Most bulking materials are injected around the urethra just outside the muscle of the urethra at the bladder outlet. Injecting the bulking material can be done through the skin, through the urethra, or, in women, through the vagina. Needle placement is guided by the use of a cystoscope inserted into the urethra. For example, U.S. Pat. No. 6,129,761 is incorporated by reference for the teaching of a method of treatment of vesicoureteral reflux, incontinence and other defects comprising injecting a liquid polymeric material injected into the area of the defect, for example, which provides the required control over the passage of urine.

[0098] As further examples, the composition of the disclosed methods can be used for strengthening sphincter muscles for urinary incontinence, craniofacial reconstruction, reconstruction of trachea, breast augmentation, abdominal wall reconstruction potentially, penile tissue. Other uses of the disclosed implantable composition wherein release of a bioactive agent is desired are known in the art and can be appreciated based on the present disclosure.

E. KITS

[0099] The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits for preparing a biocompatible implant, the kit comprising a graft material, a polymeric binder, and a bioactive agent. The kits also can contain a delivery vehicle.

F. EXAMPLES

1. EXAMPLE 1:

[00100] Disclosed is a surface coating constituted of a model protein (bovine serum albumin, BSA) associated with a binding polymeric hydrophilic agent [poly (vinyl alcohol), PVA] that can be released in a controlled way, for a FDA approved alloplast bone grafting material, the HTR™ (Hard Tissue Replacement™, Syntetic Bone™, Bioplant Inc., New York, NY; by United States Surgical Corporation, Norwalk, CT, USA), (that works in a osteoconductive way).

[00101] HTR has a history of clinical success in bone grafting procedures (Yukna,

1990, Yukna, 1994) and showed bone formation around its particles (Yukna and Greer Jr., 1992; Calogne et al., 2001). However, these results are not universal. In other clinical studies (Stahl et al.; 1990; Shahmiri et al., 1992) the bone formation in periodontal defects was not superior than in control groups and bone regeneration was seen occasionally in some sites.

[00102] In order to improve the ability of HTR to regenerate bone, disclosed herein is the modification of its surface in order to allow binding and release of a bioactive agent (e.g. a protein), and use a photo-curable polymer matrix as a delivery vehicle for this preparation in vitro.

[00103] Protein therapeutic agents are being developed for pharmaceutical applications such as for oral and transdermal use. Although these molecules are very attractive as new therapeutic agents for a practical point of view they have a very low bioavailability when they are used with these traditional ways of administration. Protein controlled delivery formulations are an attractive way to overcome these problems creating a dosage form that can deliver the protein for a longer period of time at the therapeutic window. Using this approach the bioactive agent is protected from degradation and elimination and are targeted to a specific body site, lowering systemic exposure and increasing patient compliance (Putney, S.D., et al. 1998).

[00104] As a matrix, the chosen material was a light-curable matrix of poly (ethylene glycol) diacrylate (PEGDA) that have the potential advantage of being formed in a mold or in vivo for filling bone defects (Baroli, 2002; Baroli, 2006).

[00105] The protein was formulated using lactose (L) as an excipient in a proportion of 1 :10 w/w BSA:L proportion and tested for the uniformity of the sample content. This formulation was then protected with the wet granulation process (BSA:LW).

[00106] The Bioplant HTR™ (HTR) surface was modified using a 10% PVA coating in order to bind it to BSA:LW preparation. The protein formulation then was linked to the HTR PVA changed surface (HTRP). The release studies of this preparation in 48 hours shows that the BSA is released in controlled way (Fig. 3).

[00107] The BSA:LW:HTRP particles were added in the PEGDA monomers (1:3 w/w ratio). Benzoyl peroxide, camphorquinone, N, N-Dimethyl-p-toluidine and ethyl 4- dimethylamino-benzoate initiator were then added and mixed to the samples and photo-cured for 3 minutes with a dental curing light (400-500nm wavelength and 45OmW of intensity).

[00108] The BSA release curves from the PEGDA:BSA:LW:HTRP show that the release increased to seven days using these crosslinked matrices (Figure 2).

[00109] This strategy allowed an FDA approved efficient graft material, HTR, to release a bioactive molecule represented as a model molecule, e.g., BSA. By incorporating this formulation into a photocurable biodegradable matrix permits it to be molded into device of various shapes and directly in the dental cavity as needed during restorative dentistry procedures, where it can not only serve as the matrix for new bone formation and implant stabilization but also as a controlled release vehicle for the delivery of many bioactives including small molecules (analgesics, NSAID), proteins and growth factors.

2. EXAMPLE 2: ENGINEERING BIOMATERIAL SURFACES FOR CONTROLLED RELEASE OF THERAPEUTICS

[00110] Disclosed is a general approach for non-covalent immobilization of proteins onto polymeric and ceramic particulate surfaces. The surface chosen for the present study was Bioplant®-HTR® (Bioplant Inc., New York, NY; by United States Surgical Corporation, Norwalk, CT), an FDA approved hard tissue replacement material that is currently used as a socket filler in dentistry. Bioplant-HTR is derived from poly(methymethacrylate) as a test substrate and has been shown to promote bone formation in dental osseous environment. Additionally, Bioplant-HTR can be dispersed into a photo-curable carrier for minimally invasive maxillo-facial reconstruction (Yukna RA J Periodontol. 1994; 65:342-349).

a. METHODS

[00111] Bovine serum albumin (BSA) was mixed with lactose and the uniformity of the composition was verified. Formulated protein was mixed with a gelatin binder and forced through a sieve to yield granules (Baroli B. J Pharm Sci. 2002; 92:1186-1195). The protein granules were immobilized onto the HTR surface using water-soluble polymeric binders. In this study two such binders were explored: polyvinyl alcohol (PVA, Aldrich); and the poloxamer (Pluronic^®, BASF Corporation, Mount Olive, NJ). Both PVA and Pluronic have sufficient biocompatibility in low concentrations and are deemed safe for in vivo use. In the first step, the HTR particles were coated with the binder and, then, following a brief drying step, the protein powder was immobilized by physical mixing in a pestle and mortar. Three different protein:binder (PB) ratios (0.5 : 1 ; 1 :1 and 2:1 w/w) were studied. Protein release from HTR and dispersed into a hydrophilic photo-crosslinked poly(ethylene glycol)-diacrylate (PEGDA) network were assessed. The protein release studies were carried out as follows. Briefly, HTR with PVA coating (HPV), HTR with Pluronic coating (HP), and HPV-PEGDA matrices were incubated in 10 ml PBS in a scintillation vial and the release buffer was removed periodically at pre-determined time-points, replaced with fresh release buffer, and protein content assayed using the Coomassie protein assay.

b. RESULTS

[00112] The process yielded binding of 3.11+0.58, 4.07+0.88 and 9.05+1.99mg of

BSA/lOOmg HTR in PVA system (HPV), and 2.84+0.83, 3.33+1.05 and 4.94+0.47mg of BSA/100mg HTR in the Pluronic system (HP), for 0.5:1, 1 :1 and 2:1 PB ratios. In general, increase in PB ratio resulted in increased BSA association with the HTR. HPV exhibited sustained release over 36-hours with 32, 28 and 20% released in 30 minutes for 0.5:1, 1:1 and 2:1, respectively; and 100% release at 36 hours. In the HP system, the release was of 25, 26 and 16% in 30 minutes with 100% released by 72 hours. In the HPV system upon incorporation into the PEGDA matrix, the initial burst at 24 hours was directly proportional to the amount of protein on the HTR grafting material, with a release of 93, 89 and 88% respectively. BSA release was observed up to one week, at which time 100% release was attained for all formulations.

[00113] Coated particles exhibited sustained release over 24-hours with 32, 28 and 20% released in 30 minutes with 100% released at 36 hours (BSA PVA) and 25, 26 and 16% in 30 minutes with 100% released by 48 hours (Figure 6; BSA PLU).

[00114] The HRP release from the particles within the groups reached 100% in 24 hours, with 34, 36 and 43% release (PVA) and 15, 10 and 11% (PLU) at the first half hour in each group respectively and it continued for up to 30 days when included in the polymeric matrix (Figure 7).

[00115] Also demonstrated was the release of protein from the surface modified HTR dispersed in a photo-curable polymeric matrix (Figures 8 and 9). Further disclosed is the ability of coated HTR to release immunoreactive FGF-2 in a controlled manner from the particle itself and from a photo cross-linked PEGDA matrix suggesting retention of its activity (Figure 9).

c. CONCLUSIONS

[00116] A non-covalent strategy to immobilize proteins onto a biomaterial surface has been developed. Short term sustained release of a model protein from modified HTR surface has been demonstrated. Furthermore, enhancement of sustained release has been achieved by dispersing the HTR in a photocurable matrix. HTR surface modified with the appropriate osteogenic factors can offer superior clinical outcomes with respect to new-bone formation in reconstructive dentistry.

[00117] This strategy allowed an FDA approved efficient graft material, HTR, to release a bioactive molecule represented as a model molecule, e.g., BSA. Incorporating this formulation into a photocurable biodegradable matrix permits it to be molded into device of various shapes and directly in the dental cavity as needed during restorative dentistry procedures, where it can not only serve as the matrix for new bone formation and implant stabilization but also as a controlled release vehicle for the delivery of many bioactives including small molecules (analgesics, NSAID), proteins and growth factors

G. REFERENCES

1. Putney, S. D., Burke, P. A. (1998) Improving protein therapeutics with sustained- release formulations. Nature Biotechnology 16, 153-157.

2. Baroli, B., Shastri, V. P., Langer, R. (2002) A method to protect sensitive molecules from a light-induced polymerizing environment. Journal of Pharmaceutical Sciences 92, 1186-1195. 3. Baroli, B. (2006) Photopolymerization of biomaterials: issues and potentialities in drug delivery, tissue engineering, cell encapsulation applications. Journal of Chemical Technology and Biotechnology 81, 491-499.

4. Anseth, K. S., Shastri, V. R., and Langer, R. (1999) Photopolymerizable degradable poly anhydrides with osteocompatibility. Nat Biotechnol 17, 156-159

5. Langer, R., Vacanti, J. P. (1993) Tissue engineering. Science 260, (920-926).

6. Trombelli, L., Heitz-Mayfeld, L., Needelman, L, Moles, D., Scabbia, A. (2002) A systematic review of graft materials and biological agents for periodontal intraosseous defects. Journal of Clinical Periodontology 29 (Suppl. 3), 117-135.

7. Garg, A. K. (1999) Grafting materials in repair and restoration. In: Lynch, S. E.,

Genco, R. J., Marx, R. E. Tissue Engineering: Applications in maxillofacial surgery and periodontics. Carol Stream: Qintessence, 83-101.

8. Calongne, K. B., Aichelmann-Reidy, M. E., Yukna, R. A., Mayer, E. T. (2001) Clinical comparison of microporous biocompatible composite of PMMA, PHEMA and calcium hydroxide grafts and expanded polytetrafluoroethylene barrier membranes in human mandibular molar Class II furcations. A case series. Journal of periodontology 72, 1451-1459.

9. Yukna, R. A. (1990) HTR polymer grafts in human periodontal osseous defects. I. 6- month clinical results. Journal of periodontology 61, 633-42.

10. Yukna, R. A. (1994) Clinical evaluation of HTR polymer bone replacement grafts in human mandibular Class II molar furcations. . Journal of periodontology 65, 342-9.

11. Yukna, R. A., Greer Jr., R. O. (1992) Human gingival response to HTR polymer. Journal of Biomedical Materials Research 26, 517-527.

12. Shahmiri, S., Singh, I. J., Stahl, S. S. (1992) Clinical response to the use of the HTR polymer implant in human intrabony lesions. The International Journal of Periodontics &

Restorative Dentistry 12, 294-9. [00118] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

CLAIMSWhat is claimed is:

1. An implantable composition comprising:

(a) a graft material,

(b) a bio active agent,

(c) a soluble polymeric binder,

2. The composition of claim 1 , wherein the graft material is an inert alloplast material.

3. The composition of claim 2, wherein the graft material comprises a porous matrix of biologically-compatible polymeric particles, wherein calcium hydroxide is distributed in the pores of the matrix.

4. The composition of claim 2, wherein the graft material is a ceramic.

5. The composition of claim 2, wherein the graft material is a metal orthopaedic or dental implant, endovascular device, stent, balloon catheter, barrier membrane, surgical mesh, wound dressing, or tissue engineering scaffold.

6. The composition of claim 1, wherein the bioactive agent is a protein.

7. The composition of claim 6, wherein the protein is a growth factor.

8. The composition of claim 7, wherein the protein is selected from the group consisting of bone morphogenic protein (BMP), TGF-beta, FGF-I, FGF-2, VEGF, PDGF, IGF, insulin, epidermal growth factor, or epithelial growth factor.

9. The composition of claim 1 , wherein the bioactive agent is protected from polymerization- induced damage.

10. The composition of claim 9, wherein the bioactive agent is admixed with photo- polymerizable monomers, wherein the bioactive agent is shielded from the monomers by an insoluble material that undergoes a solid-gel transition at body temperature, wherein upon polymerization, the monomers produce a cross-linked structure and the shielded bioactive molecules are protected from attack in the polymerized environment.

11. The composition of claim 1, wherein the soluble polymeric binder is poly(vinyl alcohol), poly(acrylic acid), poly(acryl amide), polaxomers (Pluronics®), polyethyelenoxide, polyethylene glycol, poly (ethylene glycol) diacrylate (PEGDA), poly(propylene glycol), poly(propylene oxide), poly(saccharides), starch, cellulose, cellulose acetate, or poly(beta- amino esters).

12. The composition of claim 1, further comprising a vehicle comprising a biocompatible polymeric matrix.

13. The composition of claim 12, wherein the polymeric matrix is biodegradable.

14. The composition of claim 12, wherein the polymeric matrix is an injectable biomaterial or in situ formable biomaterial.

15. The composition of claim 12, wherein the polymeric matrix is malleable and curable.

16. The composition of claim 12, wherein the polymeric matrix comprises sodium alginate.

17. The composition of claim 12, wherein the polymeric matrix comprises photocrosslinkable polysaccharides

18. The composition of claim 12, wherein the polymeric matrix comprises polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures thereof.

19. The composition of claim 12, wherein the polymeric matrix comprises apatitic calcium phosphate.

20. The composition of claim 12, wherein the polymeric matrix comprises

21. The composition of claim 12, wherein the polymeric matrix is a biocompatible hydrogel comprising at least one polymer.

22. The composition of claim 21, wherein the at least one polymer is agarose.

23. The method of claim 21, wherein the at least one polymer is a polaxomers, polyacrylamides, or a derivative thereof.

24. The method of claim 21, wherein the at least one polymer is N-isopropylacrylamide (NIPAM), or Pluronic F127.

25. A method of preparing an implantable composition, the method comprising the steps of:

(a) coating a graft material with a soluble polymeric binder, and

(b) linking a bio active agent to the graft material,

thereby providing a coated graft material.

26. The method of claim 25, further comprising the step of combining the coated graft material with a polymeric matrix.

27. The method of claim 25, wherein linking comprises covalent bonding, non-covalent bonding, adsorption, absorption, or physical association.

28. The method of claim 25, wherein the coating step is performed before the linking step.

29. A method of administering an implantable composition comprising the step of implanting the composition of claim 1 into a subject.

30. A method of treating a tissue disorder comprising implanting the composition of claim 1 into a subject, wherein the bioactive agent is released from the composition and promotes tissue growth, repair, regeneration, or a combination thereof.

31. The method of claim 30, wherein the tissue disorder is a bone injury.

32. A method of delivering a bioactive agent to a tissue in a subject, the steps comprising:

(a) coating a graft material with a soluble polymeric binder,

(b) admixing the bioactive agent in a biocompatible polymeric matrix,

(c) linking the bioactive agent to the graft material, and

(d) implanting the graft material into the tissue of the subject.

33. The method of claim 29, 30, or 32, wherein the bioactive agent is released from the composition over a period of hours or days.

34. The method of claim 29, 30, or 32, wherein the subject is a mammal.

35. The method of claim 29, 30, or 32, wherein the subject is a human.

36. The method of claim 29, 30, or 32, wherein the subject is a patient.

37. The method of claim 29, 30, or 32, wherein the composition is implanted in or adjacent to a bone or joint of the subject.