WO2012094708A1

WO2012094708A1 - Bone graft biomaterial

Info

Publication number: WO2012094708A1
Application number: PCT/AU2012/000022
Authority: WO
Inventors: Justin Cooper-White; Jane KENNA; David Little; Aaron SCHINDELER; Nicole YU
Original assignee: The University Of Queensland; The Sydney Children's Hospitals Network (Randwick And Westmead)
Priority date: 2011-01-12
Filing date: 2012-01-12
Publication date: 2012-07-19

Abstract

The present invention discloses biomaterials useful in treating bone injuries or defects. More particularly, this invention relates to biomaterials comprising a porous polymer, an osteoinductive compound and a bone anti-catabolic compound.

Description

TITLE

BONE GRAFT BIOMATERIAL

FIELD OF THE INVENTION THIS INVENTION relates to biomaterials useful in tissue engineering applications involving bones. More particularly, this invention relates to biomaterials comprising a porous polymer, an osteoinductive compound and a bone anti-catabolic compound.

BACKGROUND TO THE INVENTION

Currently, autografts provide the best material to treat bone defects, such as those from disease or trauma, because they are biocompatible and there is little risk of disease transfer. However, the downside of autografts is that a separate operation must be performed to remove the patient's own bone. Allografts, which consist of bone from another person (including cadavers), are also available but carry the risk of disease transfer and an adverse immune response by the recipient that could lead to failure.

In order to address some of the problems associated with bone grafts, many researchers have tried to develop artificial substances for use in treating bone defects. These artificial substances should possess several qualities in order to be successful. First, the material should be degradable to allow room for new bone to grow into the implant site. Second, it should maintain mechanical strength similar to native bone. Finally, the artificial substance needs to be osteoconductive, that is, it should allow bone cells to attach and propagate on its surface.

Some of the materials that have shown promise as bone grafts include calcium phosphate compounds, such as hydroxyapatite. Calcium phosphate compounds are biocompatible because they have characteristics similar to native bone mineral. However, they are hard to shape and do not possess the same mechanical properties as bone. Other types of material that have shown promise are degradable polymers. However, while they are easily formed and have good mechanical strength, degradable polymers alone are not ideal for bone grafts because they are not very osteoconductive. Therefore, there remains a need for compositions and methods for improving and/or enhancing outcomes in treating bone defects. SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods for treating bone defects.

In one aspect, the invention provides a biomaterial, comprising a porous polymer, an osteoinductive compound and a bone anti-catabolic compound.

In one embodiment, the porous polymer is poly(lactic-co-glycolic) acid).

In another embodiment, the osteoinductive compound includes a bone morphogenetic protein.

In yet another embodiment, the bone anti-catabolic compound includes a bisphosphonate and/or an ΙκΒ kinase inhibitor.

In another aspect, the biomaterial further comprises a calcium phosphate compound.

In one embodiment, the calcium phosphate compound is hydroxyapatite.

In yet another aspect, the invention provides an implantable structure, comprising an adherent, uniform coating of the biomaterial according to any one of the preceding aspects on the surface of the implantable structure.

In one embodiment, the implantable structure is a joint replacement device, a rod, a plate, or a fixator.

In a further aspect, the invention provides a method of treating a bbne injury or defect in a subject, including the step of implanting into the subject at the site of the injury or defect a biomaterial according to any one of the preceding aspects.

In one embodiment, the bone injury or defect is a bone fracture.

In another embodiment, the bone injury or defect is a joint replacement.

In yet a further aspect, the invention provides a method of treating a dental injury or defect in a subject, including the step of applying to the site of the injury or defect in the subject a biomaterial according to any one of the preceding aspects.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Bone formation (volume) with PLGA and rhBMP-2.

Bone formation analysis with CT shows increased bone volume with PLGA compared to PDLLA control with the same rhBMP-2 dose. No statistical difference was observed between PLGA SQ and PLGA QQ scaffolds with the same rhBMP-2 dose. PDLLA: poly(D,L-lactic acid); QQ: quick quench; SQ: slow quench; BMP: bone morphogenetic protein.

Figure 2. Three dimensional representative μΟΤ images of axial sections (35 slices).

PGLA scaffolds show increased thickness of trabecular-like bone. PDLLA: poIy(D,L-lactic acid); PLGA: poly(lactic-co-glycolic acid); QQ: quick quench; SQ: slow quench; BMP: bone morphogenetic protein.

Figure 3. Bone formation (volume) with PLGA, rhBMP-2 and ZA.

(Top Panel) Bone formation analysis with μΟΤ shows that a locally delivered dose of 2 μg of zoledronic acid (ZA) increases net bone formation 5 -fold over rhBMP-2 alone. BMP: bone morphogenetic protein; ZA: zoledronic acid; HAp: hydroxyapatite.

(Bottom Panel) Bone ingrowth into the space occupied by the porous scaffold as seen on μ€Ύ images.

Figure 4. Bone formation (density) with PLGA, rhBMP-2 and ZA.

Bone formation analysis with μ€Τ shows that a locally delivered dose of 2 μg of zoledronic acid increases bone mineral density by approximately 9% over rhBMP- 2 alone. BMP: bone morphogenetic protein; ZA: zoledronic acid; HAp: hydroxyapatite.

Figure 5. Bone formation (volume) with PLGA, rhBMP-2 and PS 1 145.

Bone formation analysis with μΟΤ shows that a locally delivered dose of 40 μg of PS1 145 increases net bone formation 2-fold over rhBMP-2 alone.

Figure 6. Schematic of porous scaffold inserted into 6 mm femoral defect of rat.

New bone formation quantified six weeks post-operatively. BMP: bone morphogenetic protein.

Figure 7. Bone formation quantification of healed femoral critical-sized bone. Co-treatment with local IKK-inhibitor and with local zoledronic acid plus hydroxyapatite increased bone healing by 48.8% (PO.05) and 82% (p<0.05), respectively, compared to BMP-alone PLGA TIPS control. BMP: bone morphogenetic protein; ZA: zoledronic acid; HAp: hydroxyapatite; ACS col: control collagen sponge carrier; TIPS: thermally induced phase separation. +p<0.05, decrease vs. BMP (ACS col); *p<0.05, increase vs. BMP (TIPS); #p<0.05, increase vs. BMP (ACS col).

Figure 8. Representative 3D micro-computed tomography images of critical-sized defects at six weeks post-surgery. BMP: bone morphogenetic protein; ZA: zoledronic acid; HAp: hydroxyapatite.

Bottom panel illustrates denser regenerate region with local zoledronic acid co-treatment groups. A bone sheath is shown in BMP controls. Local IKK-inhibitor demonstrates a bone sheath with bone in-growth.

Figure 9. Bone formation quantification of ectopic bone nodules after three weeks implantation of BMP ± ZA scaffolds or BMP ± ZA/HAp scaffolds. *p<0.05 vs. low ZA.

A significant increase in bone formation is seen in the BMP + ZA + HAp group compared with the BMP + low ZA group. In pure PLGA TIPS scaffolds, significant increases in bone formation was found between the medium and high ZA co-treatment groups compared to the low ZA co-treatment group. BMP: bone morphogenetic protein; ZA: zoledronic acid; HAp: hydroxyapatite.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for treating bone defects.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

By "biomaterial" is meant any nondrug material that can be used to treat, replace or enhance any tissue, organ, or function in a subject.

The term "porous polymer" as used herein is intended to refer to a polymer (i. e. , a molecule with repeating subunits) having a collection of voids or pores. Such voids or pores can range from 5 μπι to 400 μπι in diameter. The preferred range is 100-400 μπι in diameter. The polymer material per se which forms the porous polymer may itself also be permeable, for example to liquids and/or gases.

Examples of suitable polymers that can be used in the compositions and methods of the invention include, but are not limited to, aliphatic or aliphatic-co- aromatic polyesters including poly(a-hydroxyesters) and copolymers thereof, such as polylactic acid (PLA), polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (all stereo-isomeric forms thereof); polydioxanone; polyalkanoates_¾ such as poly(hydroxy butyrate) (PHB), poly(hydroxy valerate) (PHV) and co-polymers thereof (i.e. , PHBV); and polyethylene oxide/polyethylene terephthalate, as disclosed by Reed et al. {Trans. Am. Soc. Artif. Intern. Organs 23:109-15, 1977). Other suitable polymers include biodegradable and biocompatible polycaprolactones, and copolymers of polyesters, polycarbonates, polyanhydrides, poly(ortho esters), and copolymers of polyethylene oxide/polyethylene terphthalate.

Bisphenol-A based polyphosphoesters have also been suggested for use in biodegradable porous polymer design. Such polymers include poly(bisphenol-A phenylphosphate), poly(bisphenol-A ethylphosphate), poly(bisphenol-A ethylphosphonate), poly(bisphenol-A phenylphosphonate), poly[bis(2- ethoxy)hydrophosphonic terephthalate], and copolymers of bisphenol-A based poly(phosphoesters), as described in US Pat. No. 5,686,091.

Other polymers suitable for use in the compositions and methods of the invention include polymers of tyrosine-derived diphenol compounds. Methods for preparing tyrosine-derived diphenol monomers are disclosed in US Pat. Nos. 5,587,507 and 5,670,602. Preferred diphenol monomers are des-aminotyrosyl- tyrosine (DT) esters. These monomers have a free carboxylic acid group that can be used to attach a pendent chain. Usually, various alkyl ester pendent chains are employed, for example, ethyl ester, butyl ester, hexyl ester, octyl ester and benzyl ester pendant chains.

The tyrosine-derived diphenol compounds are used as monomeric starting materials for polycarbonates, polyiminocarbonates, polyarylates, polyurethanes, polyethers, and the like. Polycarbonates, polyiminocarbonates and methods of their preparation are disclosed in US Pat. Nos. 5,099,060 and 5,198,507. Polyarylates and methods of their preparation are disclosed in US Pat. No. 5,216,115. Block copolymers of polycarbonates and polyarylates with poly(alkylene oxides) and methods of their preparation are disclosed in US Pat. No. 5,658,995. Strictly alternating poly(alkylene oxide ether) copolymers and methods of their preparation are disclosed in International Application No. PCT/US98/23737.

Additional polymers suitable for use in the compositions and methods of the invention include the polycarbonates, polyimino-carbonates, polyarylates, polyurethanes, strictly alternating poly(alkylene oxide ethers) and poly(alkylene oxide) block copolymers polymerised from dihydroxy monomers prepared from α-, β- and γ-hydroxy acids, and derivatives of tyrosine. The preparation of the dihydroxy monomers and methods of their polymerisation are disclosed in International Patent Application No. PCT/US98/036013.

Polycarbonates, polyimino carbonates, polyarylates, poly(alkylene oxide) block copolymers, and polyethers of the diphenol and dihydroxy tyrosine monomers that contain iodine atoms or that contain free carboxylic acid pendent chains may also be employed. Iodine-containing polymers are radio-opaque. These polymers and methods of preparation are disclosed in International Patent Application No. PCT/US98/23777.

Preferred polymers that can be used in accordance with the invention to prepare biomaterials for use in treating bone defects include poly(a-hydroxyesters) and copolymers thereof, particularly PGA, PLA and PLGA.

The PLGA copolymer can include the PLGA copolymer or a salt thereof, for example, a salt or a complex salt of the PLGA copolymer with an inorganic base including an alkali metal, such as sodium or potassium, and an alkaline earth metal such as calcium or magnesium, an organic base including an organic amine such as triethylamine and basic amines such as arginine, or a transition metal such as zinc, iron, or copper can be used. The PLGA copolymer can have a compositional molar ratio of about 90:10 to 40:60, preferably about 70:30 to 80:20 of polylactic acid to poly gly colic acid.

The biomaterial of the invention comprising a porous polymer can be conveniently prepared using conventional polymer processing techniques, such as extrusion and injection moulding. Moulds used to form the biomaterial into a desired shape can be made from various materials, such as glass, metal, ceramic, and plastic.

As used herein, "osteoinductive compound" describes a compound that induces or promotes bone formation. For example, osteoinductive compounds can be polypeptide or polynucleotide compositions. Polypeptide compositions of the osteoinductive compounds include, but are not limited to, bone morphogenetic protein (BMP), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), osteoprotegerin, growth differentiation factors (GDFs), cartilage derived morphogenic proteins (CDMPs), lim mineralization proteins (LMPs), platelet derived growth factor, (PDGF), insulin-like growth factor (IGF), or transforming growth factor beta (TGF-beta) proteins, including recombinant proteins (particularly recombinant human osteoinductive proteins).

Polynucleotide compositions of the osteoinductive compounds include, but are not limited to, gene therapy vectors harbouring polynucleotides encoding a osteoinductive polypeptide of interest. Such gerie therapy vectors may utilise a polynucleotide that codes for the osteoinductive polypeptide operatively linked or associated to a promoter or any other genetic elements necessary for the expression of the osteoinductive polypeptide by the target tissue. Suitable gene therapy vectors include vectors that do not integrate into the host genome. Alternatively, suitable gene therapy vectors include vectors that integrate into the host genome.

Variants of the osteoinductive proteins include, but are not limited to, polypeptide variants that are designed to increase the duration of activity of the osteoinductive protein in vivo. Preferred embodiments of variant osteoinductive proteins include full length proteins or fragments thereof that are conjugated to polyethylene glycol (PEG) moieties to increase their half-life in vivo (also known as pegylation). Methods of pegylating polypeptides are well known in the art (see, e.g., US Pat. No. 6,552,170). In some embodiments, the osteoinductive proteins are provided as fusion proteins. In one embodiment, the osteoinductive proteins are available as fusion proteins. Examples of fusion proteins include fusions between osteoinductive proteins or fragments thereof and the Fc portion of human Immunoglobulin G (IgG). In another embodiment, the osteoinductive proteins are available as hetero- or homodimers ormultimers. Methods of making fusion proteins and constructs encoding the same are well known in the art.

Osteoinductive proteins suitable for use in the present invention can also be identified by means of routine experimentation, for example, using the art-recognised bioassay described by Reddi and Sampath (see, Sampath et al. , Proc. Natl. Acad. Sei. USA 84:7109-13, 1987).

The term "bone morphogenic protein" refers to a protein belonging to the BMP family of the TGF-β superfamily of proteins, based on DNA and amino acid sequence homology. A protein belongs to the BMP family according to the invention when it has at least 50% amino acid sequence identity with at least one known BMP family member within the conserved C-terminal cysteine-rich domain, which characterizes the BMP protein family. Preferably, the protein has at least 70% amino acid sequence identity with at least one known BMP family member within the conserved C-terminal cysteine rich domain. Members of the BMP family may have less than 50% DNA or amino acid sequence identity overall.

BMPs utilised as osteoinductive compounds include one or more of BMP- 1 , BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP- 11 , BMP-12, BMP-13, BMP-15, BMP-16, BMP-17, or BMP-18, as well as any combination of one or more of these BMPs, including full length BMPs or fragments thereof, or combinations thereof, either as polypeptides or polynucleotides encoding the polypeptide fragments of all of the recited BMPs. The isolated BMP osteoinductive compounds may be administered as polynucleotides, polypeptides, full-length protein, fragments thereof, or combinations thereof.

Recombinant human BMPs can be used, and can be commercially obtained or prepared as described and known in the art, for example, in US Pat. Nos. 5,187,076; 5,366,875; 4,877,864; 5,108,932; 5,116,738; 5,013,649; and 5,106,748.

The osteoinductive compounds can be isolated from tissue sources, such as bone. Methods for isolating osteoinductive compounds (e.g., BMP) from bone are described, for example, in US Pat. No. 4,294,753.

By "bone anti-catabolic compound" is meant a compound that lessens or prevents bone catabolism, such as by osteoclasts. For example, bone anti-catabolic compounds can be small molecules. Bone anti-catabolic compounds include, but are not limited to, bisphosphonates, such as etidronic acid, clodronic acid, tiludronic acid, pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, risedronic acid, zoledronic acid, and salts thereof.

Bone anti-catabolic compounds also include cathepsin K inhibitors, vacuolar ATPase inhibitors, calcitonin,, and antibodies to receptor activator of NF-κΒ ligand (RANKL).

Bone anti-catabolic compounds further include ΙκΒ kinase (IKK) inhibitors, such as, for example, parthenolide, BMS345541 (4(2'-aminoethyl)amino-l,8- dimethylimidazo(l ,2-a)quinoxaline), IMD0354 (N-[3,5-bis(trifluoromethyl)phenyl]- 5-chloro-2-hydroxybenzamide), ML120B (N-(6-chloro-7-methoxy-9H-b-carbolin-8- yl)-2-methylnicotinamide), TPCA1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3- thiophenecarboxamide), and PS1 145 (5-bromo-6-methoxy-b-carboline), as are well known in the art (see, e.g., Strnad and Burke, TRENDS Pharmacol Sci. 28:142-48, 2007). Preferably, the inhibitor should inhibit ΙΚΚ-β.

In certain embodiments, the biomaterial comprising a porous polymer, an osteoinductive compound and a bone anti-catabolic compound further comprises a calcium phosphate compound.

By "calcium phosphate compound" is meant a compound containing inorganic calcium and phosphate, such as, for example, amorphous calcium phosphate, dicalcium phosphate (CaHP0₄), tricalcium phosphate (Ca₃(P0₄)₂; both a and β forms), tetracalcium phosphate monoxide (Ca40(P0₄)2), and hydroxyapatite (HAp) (Ca₁₀(PO₄)₆(OH)₂).

The biomaterial of the invention can also include additional biologically active compounds, including pharmaceutically active compounds, as well as additives, such as dyes, pigments and stabilisers. The incorporation of such additional additives in the biomaterial of the invention can be particularly advantageous when the biomaterial is to be used in tissue engineering applications, such as for treating bone defects. For example, the biomaterial can include bioactive substances that function as receptors or chemoattractors for a desired population of cells.

The incorporation of compounds and/or additives in the biomaterial of the invention may occur in a liquid composition prior to the formation of the porous polymer structure. Generally it is preferred that the compounds/additives are incorporated in the porous polymer structure by this approach such that the compounds/additives are at least initially incorporated within the polymer matrix of the porous polymer structure (i.e., not simply located within the pores or on the surface of the porous polymer structure). In this case, it may be desirable to select the solvent or solvent/non-solvent used in accordance with the invention to not only dissolve the one or more polymers but also the one or more compounds/additives.

It may be desired that one or more of the compounds/additives of the biomaterial be incorporated for subsequent release in a controlled fashion. The compounds/additives may be released by bioerosion of the porous polymer, or by diffusion from the porous polymer. Alternatively, the compounds/additives may migrate to the porous polymer surface to become active.

The compounds/additives of the biomaterial can be provided in a physiological acceptable carrier, excipient, stabiliser, etc., and may be provided in sustained release or timed release formulations. The compounds/additives may also incorporate agents to facilitate their delivery, such as antibodies, antibody fragments, growth factors, hormones, or other targeting moieties, to which the compounds/additives are coupled.

Acceptable pharmaceutical carriers for therapeutic use are well known in the pharmaceutical field, and are described, for example, in Remington's Pharmaceutical Science, Mac Publishing Co. (A.R. Gennaro ed., 1985). Such materials are generally non-toxic to recipients at the dosages and concentrations employed, and include diluents, solubilizers, lubricants, suspending agents, encapsulating materials, solvents, thickeners, dispersants, buffers such as phosphate, citrate, acetate and other organic acid salts, anti-oxidants such as ascorbic acid, preservatives, low molecular weight (less than about 10 residues) peptides such as polyarginine, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as poly(vinylpyrrolindinone), amino acids such as glycine, glutamic acid, aspartic acid or arginine, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrines, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counter-ions such as sodium, and/or non-ionic surfactants such as tween, pluronics or PEG.

The compounds/additives may be covalently attached to porous polymers having pendent free carboxylic acid groups. Detailed chemical procedures for the attachment of various moieties to polymer bound free carboxylic acid groups have been described in the literature (see, for example, US Pat. Nos. 5,219,564 and 5,660,822; Nathan et al, Bio. Cong. Chem. 4:54-62, 1992; Nathan, Macromolecules 25: 4476, 1992). These publications disclose procedures by which polymers having pendent free carboxylic acid groups are reacted with moieties having reactive functional groups, or that are derivatized to contain active functional groups, to form a polymer conjugate.

Hydrolytically stable conjugates may be utilised when the compounds/additives are active in conjugated form. Hydrolysable conjugates may be utilised when the compounds/additives are inactive in conjugated form.

The amount of a given compound/additive incorporated into the porous polymer structure will of course vary depending upon its nature and intended function. Those skilled in the art will readily appreciate such dosage requirements. Likewise, the dose and method of administration will vary from subject to subject and be dependent upon such factors as the type of subject being treated, its sex, weight, diet, concurrent medication, overall clinical condition, the particular compounds/additives employed, the specific use for which the compounds/additives are employed, and other factors which those skilled in the art will recognise.

The biomaterial can be utilised in vivo as bone engineering and bone guided regeneration scaffolds in mammals, such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, rats, and mice, or in vitro. Examples of pharmaceutically active compounds that may be included in the biomaterial of the invention include, but are not limited to, acyclovir, cephradine, malfalen, procaine, ephedrine, adriomycin, daunomycin, plumbagin, atropine, quanine, digoxin, quinidine, chlorin e₆, cephalothin, proline and proline analogues such as cis-hydroxy-L-proline, penicillin V, aspirin, ibuprofen, steroids, nicotinic acid, chemodeoxycholic acid, chlorambucil, and the like. Therapeutically effective dosages may be determined by either in vitro or in vivo methods.

For each particular compound or additive, individual determinations may be made to determine the optimal dosage required. The determination of effective dosage levels, that is, the dosage levels necessary to achieve the desired result, will be within the ambit of one skilled in the art. The release rate of the compounds/additives may also be varied within the routine skill in the art to determine an advantageous profile, depending on the condition to be treated.

A typical compound or additive dosage might range from about 0.001 mg/kg to about 1000 mg/kg, preferably from about 0.01 mg/kg to about 100 mg/kg, and more preferably from about 0.10 mg/kg to about 20 mg/kg, relative to the weight of the subject.

In another aspect, the invention provides an implantable structure, comprising an adherent, uniform coating of the biomaterial according to the above aspect on the surface of the structure. The coating may be applied through absorption, adsorption and/or chemical bonding, as will be well known to one of ordinary skill in the art.

By "implantable structure" is meant a device for insertion within the body of a subject. Implantable structures include, but are not limited to, joint replacement devices, dental prostheses, rods, plates, and fixators, such as screws and the like.

In yet another aspect, the invention provides a method of treating a bone injury or defect in a subject, including the step of implanting into the subject at the site of the injury or defect the biomaterial according to the above aspect.

As used herein, "bone injury" includes, but is not limited to, a bone fracture and a joint replacement.

In still another aspect, the invention provides a method of treating a dental injury or defect in a subject, including the step of applying to the site of the injury or defect in the subject the biomaterial according to the above aspect.

So that the invention maybe readily understood and put into practical effect, the following non-limiting Examples are provided.

EXAMPLES

Example 1 - Ectopic Bone Formation

Experimental Procedures

Scaffold fabrication

Poly(D,L-lactic acid) (PDLLA) scaffold (solvent cast method): A 17% (w/v) solution of PDLLA was prepared by dissolving PDLLA in ethyl acetate solvent. Stock solutions for 5, 10, and 20 μg recombinant human (rh) BMP -2 groups were made by addition of rhBMP-2. A control group without rhBMP-2 was also prepared. Following sonication and vortex mixing, the solution was transferred to eppendorf tubes, and the solvent was vacuum evaporated. The resulting semi-solid was compressed and moulded into disks (φ 3 mm; 1 mm height), and the residual solvent was removed.

PLGA scaffold (thermally induced phase separation, TIPS method): A 10% (w/v) solution of PLGA (lactide:glycolide ratio 50:50) was prepared by dissolving polymer in 1 , 4-dioxane (DO) solvent. Stock solutions for 5, 10, and 20 μg rhBMP-2 groups were made by addition of rhBMP-2. A control group without rhBMP-2 was also prepared. Following sonication and vortex mixing, solutions were transferred to glass moulds, and cooled with two quenching regimes: (1) slow quench (SQ): 25°C to -10°C (cooling rate of 0.5°C/minute; (2) quick quench (QQ): -80°C freezer. The DO was removed from the phase-separated mixture by high pressure vacuum (7.3 x 10-4 for 8 hours), resulting in a highly a porous scaffold. Cylindrical sections of the scaffold were cut into disks (φ 3 mm; 1 mm height).

Scaffold preparation

To prepare the implant groups, four stock solutions were prepared. Firstly, PLGA polymer stock solution (0.125 mg/ml) was prepared by mixing PLGA polymer in dioxane solvent. ZA stock solutions were prepared by addition of ZA in dioxane solvent, at low and medium concentrations 0.283 mg/ml (0.2 g ZA group) and 2.83 mg/ml (2 μg ZA group). Furthermore, for the preparation of ZA-doped ceramic HAp particles, 1 mg/ml of ZA in distilled water was prepared, and HAp particles were added at a ZA:HAp ratio of 1 :70. Each pellet contained 0.707 mg PLGA and 7.069 total dioxane solvent which was then evaporated. The protocol to prepare one pellet of each group was as follows:

1. BMP alone: 10 g rhBMP added to 5.655 μΐ PLGA polymer stock, 1.414 μΐ dioxane added.

2. BMP + low ZA (0.2 μg ZA): 10 μg rhBMP added to 5.655 μΐ PLGA polymer stock, 0.707 μΐ low ZA stock (0.283 mg/ml) added, 0.707 μΐ. dioxane added.

3. BMP + medium ZA (2 μg ZA): 10 μg rhBMP added to 5.655 μΐ PLGA polymer stock, 0.707 μΐ medium ZA stock (2.83 mg/ml) added, 0.707 μΐυ dioxane added.

4. BMP + low ZA (0.2 μg ZA) via HAp: 10 μg rhBMP added to 5.655 μΐ, PLGA polymer stock, 0.707 μ ΐονν ZA/HAp stock (0.02 g/ml of HAp in dioxane), added, 0.707 μΐ, dioxane added.

The final PLGA/dioxane concentration was 0.1 mg/ml and final BMP/dioxane concentration was 1.414 mg/ml for all groups. Following the addition of rhBMP -2 to polymer stock, the mixture was mixed by sonication for 5 minutes. Following the addition of dioxane, ZA or ZA/HAp solutions, the final mixture was mixed by vortexing.

The mixtures were then transferred to 3 mm diameter glass moulds and quenched to induce thermal phase separation between dioxane and polymer. The mixture was quenched from 25 °C to -10 °C at a cooling rate of 0.5 °C/min using a Programmable Refrigerating Circulator, model 91 12 (Polyscience, Niles, IL, USA). Following phase separation, glass tubes containing polymer rods were transferred to a liquid nitrogen bath for cooling. Polymer rods were released from moulds and to a glass vacuum chamber, and dioxane solvent was removed by high pressure vacuum (- 7.3 x 10^-4 bar for a minimum of 8 hours) using a Primary Vacuum Pump, T series DD300 (Javac, Vitoria, Australia), and Turbo Vacuum Pump, TPH 062 (Pfeiffer, West Sussex, United Kingdom). Following dioxane vacuum removal, a porous PLGA 3 mm diameter-rod remained. Polymer rods were stored in a vacuum desiccator until implant preparation.

To prepare implants for surgery, polymer rods were cut to 1 mm height with a stainless steel microtome blade (N35; Feather Safety Razor Co. Ltd., Osaka, Japan), height checked with veneer callipers and weighed. The disks were UV sterilised in a tissue culture hood (2 minutes).

Surgical model

Four dorsal muscle pouches were created in back musculature of 8-10 week old female C57BL6/J wild-type mice. Pellets corresponding to each rhBMP-2 dose (0, 5, 10, and 20 μg rhBMP-2) were introduced into each mouse. Following scaffold insertion, the muscle and skin were sutured closed. The different delivery systems are summarized in Table 1.

Analysis

Three weeks post-operatively, animals were euthanized and the ectopic bone was harvested with the surrounding muscles. Samples were fixed in 4% paraformaldehyde, and stored in 70% ethanol. Radiographs and micro-computed tomography (BCT) were used to assess bone formation. BCT was used to quantify bone volume (BV, mm3) and generate three-dimensional representative images. Histological analysis was performed on decalcified samples using haematoxylin and eosin (H&E)-, and alcian blue/picrosirius red-staining.

For statistical analysis, group sizes <10 necessitated non-parametric statistical tests. Kruskal WalHs and Mann Whitney U tests were performed using SPSS Statistics (SPSS Inc., Chicago, IL, USA). Statistical significance was set at a cut-off ofP<0.05.

Results^'

rhBMP-2 delivered via a porous scaffold

MicroCT analysis showed that bone formation in the porous PLGA scaffold samples was significantly greater than in the non-porous PDLLA scaffold (Figure 1). Both the slow and quick quench methods generated comparable amounts of new bone, which was up to 3-fold greater than the PDLLA controls. This was consistent at all (5, 10, 20 μg) rhBMP-2 doses. Further, the porous scaffold generated similar amounts of bone with 5 μg rhBMP-2 as the non-porous scaffold did with 20 μg rhBMP-2. Morphologically, porous PLGA scaffold produced a thicker cortical shell and increased interior trabecular-like bone compared with the PDLLA controls (Figure 2).

Histological analysis at three weeks showed advanced resorption of the porous polymer with an established marrow cavity. The interior of the non-porous PDLLA polymer samples contained non-degraded polymer.

Compared to the non-porous PDLLA implants, rhBMP-2 delivered via the porous PLGA polymer generated significantly increased bone formation and tissue ingrowth, and showed higher biodegradation.

rhBMP-2 and zoledronic acid delivered via a porous scaffold

After demonstrating that TIPS manufactured PLGA polymers showed superior bone formation at lower doses, co-delivery of 10 μg rhBMP-2 as an anabolic agent with the potent bisphosphonate zoledronic acid was tested.

A locally delivered dose of 2 μg of zoledronic acid increased net bone formation 5-fold over rhBMP-2 alone (Figure 3, Top Panel). This was comparable to giving 50 times the total dose of systemic zoledronic acid (dosed thrice weekly), while avoiding unnecessary systemic exposure. Ingrowth of bone into the space occupied by the porous scaffold was seen on μCT images (Figure 3, Bottom Panel). Further, the introduction of hydroxyapatite as a delivery vehicle for zoledronic acid increased bone volume almost 3 -fold at low doses.

The locally delivered dose of 2 μg of zoledronic acid increased bone mineral density (BMD, mg/cm³) by approximately 9% over rhBMP-2 alone (Figure 4). Ingrowth of bone into the space occupied by the porous scaffold was seen on μΟΤ images. Further, the introduction of hydroxyapatite as a delivery vehicle for 0.2 μg zoledronic acid increased bone volume by approximately 11%.

Local ZA delivery of 2 μg Z A or 0.2 μg ZA +HAp were comparable to giving 100 μg ZA by systemic delivery (dosed thrice weekly). rhBMP-2 and PS1145 delivered via a porous scaffold

Co-delivery of 10 μg rhBMP-2 as an anabolic agent with the IKK inhibitor PS 1145 was also tested.

Local delivery of the IKK inhibitor PS 1145 at a dose of 4(^g produced a 2- fold increase in net bone formation over rhBMP-2 alone (Figure 5).

Example 2 - Bone Regeneration in Critical-Sized Bone Defect

Materials and Methods

Porous PLGA scaffolds were used to locally deliver anabolic rhBMP-2 and various combinations of anti-catabolic drugs. Porous scaffolds were prepared by the TIPS method with modifications with drug solution combinations. The scaffold group details were as per Table 2. Scaffolds with dimensions of φ 3 mm; 5 mm height were prepared for insertion into rat critical-sized femoral defect. Anabolic rhBMP-2 was used to locally induce bone healing of rat critical-size bone defects. Anti-catabolic drugs were used to locally modulate BMP-induced bone healing, and included the IKK inhibitor PS 1 145 and the bisphosphonate zoledronic acid. In addition hydroxyapatite particles (2% w/v) were added with zoledronic acid to further improve bone healing. Scaffolds loaded with BMP and anti-catabolic drugs were inserted into a critical-sized (6 mm) rat femoral defect (Figure 6). The defect was stabilised with a polylacetyl plate and secured with wires. Bone formation and healing was quantified six weeks post-operatively with micro-computed tomography.

Results

Comparing bone formation between porous PLGA TIPS scaffolds loaded with BMP ± anti-catabolic drugs, co-treatment with local IKK-inhibitor and with local zoledronic acid plus hydroxyapatite increased bone healing by 48.8% (PO.05) and 82% (p<0.05), respectively, compared to BMP-alone PLGA TIPS control (Figure 7). Furthermore, BMP and zoledronic acid/hydroxyapatite co-treatment via PLGA TIPS scaffold induced 36.8% (P<0.05) greater bone formation than commercially used BMP-alone via ACS collagen. Increased density of regenerate region was also found with zoledronic acid co-treatment (Figure 8). In a six week rat critical-sized bone defect, BMP-bone formation in the regenerate region was modulated with local anti-catabolic agents IKK inhibitor and zoledronic acid with hydroxyapatite delivered via porous PLGA TIPS scaffolds. Co- treatment of BMP + zoledronic acid/hydroxyapatite led to greatest amount of bone formation, which was superior to BMP -alone PLGA control and BMP-ACS collagen control (commercially available alternative).

Example 3 - Additional Ectopic Bone Formation

Materials and Methods

Porous PLGA scaffolds were used to locally deliver anabolic rhBMP-2 and various doses of the anti-catabolic drug zoledronic acid. Porous scaffolds were prepared by the TIPS method with modifications with drug solution combinations. The scaffold group details were as per Table 3. Scaffolds with dimensions of φ 3 mm; 1 mm height were prepared for insertion into mouse muscle pouch to induce ectopic bone formation. Anabolic rhBMP-2 was used to locally induce ectopic bone formation over three weeks. The anti-catabolic drug zoledronic acid was used at various doses (6, 30 and 150 μg) to locally modulate BMP-induced bone healing. Additionally, hydroxyapatite was added at 2% w/v to BMP and zoledronic acid scaffolds to further modulate bone formation.

Results

Bone formation quantification of ectopic bone nodules with micro-computed tomography showed significant increase in bone formation in the BMP + local low zoledronic acid (6 μg) + hydroxyapatite group compared with the BMP + low zoledronic acid (6 μg) group (Figure 9), indicating that the additive osteogenic effect of hydroxyapatite addition was apparent in the BMP + low zoledronic acid groups, but not in the medium or high zoledronic acid groups. In pure PLGA TIPS scaffolds (i.e., no hydroxyapatite addition), significant increases in bone formation was found between the medium and high zoledronic acid co-treatment groups compared to the low zoledronic acid co-treatment group. There was no significant difference in bone formation between the PLGA hydroxyapatite scaffold groups. The additive osteogenic effect of hydroxyapatite to a BMP and zojedronic acid scaffold was apparent at low local doses of zoledronic acid, but not at medium and high doses. The local zoledronic acid dosage effect was apparent in pure porous PLGA scaffolds, but not in composite PLGA and hydroxyapatite scaffolds.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

ble 1. Experimental groups

p Delivery system rhBMP-2 dose/scaffold n

PDLLA (solvent cast) 0, 5, 10, 20 μg 6

PLGA (TIPS, SQ) 0, 5, 10, 20 μ 6

PLGA (TIPS, QQ) 0, 5, 10, 20 μg 6 ble 2. Scaffold groups for rat femoral defect insertion

} [D Anabolic agent Anti-catabolic agent

BMP Medtronic control 20 Mg rhBMP-2

BMP TIPS control 20 Mg rhBMP-2

BMP TIPS + IKK inhibitor 20 pg rhBMP-2 100 Mg IKK inhibitor (PS 1145) BMP TIPS + ZA 20 Mg rhBMP-2 5 Mg ZA

BMP TIPS + ZA/HAp 20 Mg rhBMP-2 5 Mg ZA + 2% w/v HAp TIPS control

ble 3. Scaffold groups: BMP and ZA ± HAp particles

} \D_ Anabolic agent ZA HAp

BMP . 10 g rhBMP-2 — —

BMP + low ZA 10 Mg rhBMP-2 6 M ZA —

BMP + med ZA 10 Mg rhBMP-2 30 Mg ZA —

BMP + high ZA 10 M rhBMP-2 150 Mg ZA -

BMP + low ZA + HAp 10 Mg rhBMP-2 6 Mg ZA 2% w/v

BMP + med ZA + HAp 10 Mg rhBMP-2 30 Mg ZA 2% w/v

BMP + high ZA + HAp 10 Mg rhBMP-2 150 Mg ZA 2% w/v

Claims

1. A biomaterial, comprising:

(a) a porous polymer;

(b) an osteoinductive compound; and

(c) a bone anti-catabolic compound.

2. The biomaterial according to claim 1, further comprising a calcium phosphate compound.

3. The biomaterial according to claim 1 or claim 2, wherein said porous polymer comprises polylactic acid, polyglycolic acid or poly(lactic-co-glycolic acid).

4. The biomaterial according to claim 3, wherein said porous polymer is poly(lactic- co-glycolic acid).

5. The biomaterial according to claim 4, wherein said poly(lactic-co-glycolic acid) polymer has a compositional molar ratio of from about 90:10 to about .40:60 of polylactic acid to polyglycolic acid.

6. The biomaterial according to claim 5, wherein said poly(lactic-co-glycolic acid) polymer has a compositional molar ratio of from about 70:30 to about 80:20 of polylactic acid to polyglycolic acid.

7. The biomaterial according to any one of claims 1 -6, wherein said osteoinductive compound comprises a bone morphogenetic protein (BMP).

8. The biomaterial according to claim 7, wherein said BMP is selected from the group consisting of BMP -2 and BMP-7.

9. The biomaterial' according to any one of claims 1-8, wherein said bone anti- catabolic compound comprises a bisphosphonate.

10. The biomaterial according to claim 9, wherein said bisphosphonate is selected from the group consisting of etidronic acid, clodronic acid, tiludronic acid, pamidronic acid, neridronic acid, olpadronic acid, alendronic acid, ibandronic acid, risedronic acid, zoledronic acid, and salts thereof.

1 1. The biomaterial according to any one of claims 1-8, wherein said bone anti- catabolic compound comprises an ΙκΒ kinase (IKK) inhibitor.

12. The biomaterial according to claim 1 1 , wherein said IKK inhibitor is selected from the group consisting of BMS345541, IMD0354, ML120B, TPCA1, and PS1 145.

13. The biomaterial according to any one of claims 2-12, wherein said calcium phosphate compound is selected from the group consisting of amorphous calcium phosphate, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate monoxide, and hydroxyapatite.

14. The biomaterial according to any one of the preceding claims, wherein said porous polymer contains pores sized 200 μηι or less.

15. An implantable structure, comprising an adherent, uniform coating of the biomaterial according to any one of the preceding claims on the surface of said implantable structure.

16. The implantable structure according to claim 15, wherein said structure is selected from the group consisting of a joint replacement device, a rod, a plate, or a fixator.

17. A method of treating a bone injury or defect in a subject, including the step of implanting into said subject at the site of said injury or defect the biomaterial according to any one of the preceding claims.

18. The method according to claim 17, wherein said bone injury or defect is a bone fracture.

19. The method according to claim 17, wherein said bone injury or defect is a joint replacement.

20. A method of treating a dental injury or defect in a subject, including the step of applying to the site of said injury or defect in said subject the biomaterial according to any one of the preceding claims.