COMPOSITION

Field of the Invention

This invention relates to a composition. It also relates to methods for preparing the composition and its use in a number of applications, especially tissue regeneration and/or replacement.

Background to the Invention Bone has a remarkable capacity to heal. However, in some instances the amount of bone which is needed to heal exceeds its healing capacity. These cases arise following accidents, infection or surgery to remove cancerous tissue.

Bone is the second most frequently transplanted tissue in humans. There are 2.2 million bone graft procedures annually, which have several disadvantages such as: donor site morbidity, risk of disease transmission and a limited supply of bone. While several bone substitute materials exist, none fulfil all the requirements of an ideal bone substitute material, which should mimic as closely as possible natural bone. Hoctor et al. Australian Journal of Basic and Applied Sciences, 2013, 7(5): 140-149, describes the preparation of nanocomposite scaffolds based on chitosan and hydroxyapatite for use in bone tissue engineering.

Certain recombinant proteins and synthetic peptides, known as growth factors, mimic bone growth substances normally found in the body. It is known to add such compounds to a carrier or scaffold to be used as bone graft substitutes. Once combined, these products are surgically implanted in a patient with a bone defect to promote new bone growth or to replace or heal existing bone. However, the use of growth factors in bone healing or regeneration can be associated with potentially serious adverse events, such as excess bone growth, fluid accumulation, inhibited bone healing, and swelling. Such adverse events are of particular concern in patients under age 18 because of their overall smaller size and because their bones are still growing. For this reason, the US FDA issued a Safety Communication in January 2015, informing that bone graft substitutes containing recombinant proteins or synthetic peptides should not be the first treatment considered for patients under age 18 with significant bone defects or rare bone disorders. WO 2012/142533 relates to a structure and system for growth factor incorporation which can improve the osteogenic differentiation of hMSCs, for potential bone regeneration and bone growth applications or used alone for bone repair or growth applications. The system described in this document comprises a biodegradable polyester, a hydrophilic polymer, a growth factor and optionally a bioceramic. The biodegradable scaffold is electrospun for bone tissue engineering. However, the proteins are not covalently linked to the scaffold: in contrast, they are incorporated within the melt spun fibres and are released into media through diffusion.

US 2011/229970 Al relates to a perfusion bioreactor device, and to porous hydrogel or 3D scaffolds that are capable of supporting growth and/or differentiation of a cell. The principal objective of the disclosure is to protect a bioreactor, the construct described being for use with the bioreactor in vitro. However, in this construct the proteins are not covalently linked to the scaffold: in contrast, chitosan nanoparticles are used to encapsulate protein, the bonds being weak ionic bonds between the positively charged amine groups on the chitosan and negatively charged phosphate groups on the DNA.

US 2011/256203 Al relates to a method for manufacturing a porous ceramic scaffold having an organic/inorganic hybrid coating layer containing a bioactive factor, comprising: (a) forming a porous ceramic scaffold, (b) mixing a silica xerogel and a physiologically active organic substance to prepare an organic/inorganic hybrid composite, (c) adding a bioactive factor to the organic/inorganic hybrid composite, and (d) coating the porous ceramic scaffold with the organic/inorganic composite containing the bioactive factor added thereto. However, in this construct the proteins are not covalently linked to the scaffold; in contrast, the porous ceramic containing organic/inorganic hybrid layer containing a bioactive factor is prepared by freeze thawing which is a physical crosslinking method. Due to the physically crosslinked nature of scaffold, bioactive agent release is controlled through diffusion only. Moreover, the constructs contain up to 100 μg of BMP2: this would be considered a relatively high dose and may be associated with an increased incidence of adverse events.

US 2006/149392 Al relates to compositions comprising a scaffold with growth factors chemically immobilised thereto for inducing chondrogenesis and/or osteogenesis when implanted in vivo or osteogenesis or chondrogenesis in cultures in vitro. The compositions described therein are used to enhance bone and cartilage growth. However the only examples given in this document, chemically immobilisation was performed by placing chitosan rods into a coupling buffer containing the growth factor and EDC and sulfo-NHS and stirring the solution for 72hours, followed by washing. Moreover, the growth factors as described in this application are typically bonded either directly to the scaffold or via a short linking group typically less than 10 atoms and typically having a maximum molecular weight of 130. This results in smaller and less controllable pore size, and reduced control of the diffusion of the growth factor when the bond is broken to release the growth factor, and therefore inferior bioavailability of the growth factor.

Summary of the Invention According to one aspect of the invention, there is provided composition comprising: (a) a composite material comprising constituents:

(i) an organic polymer or a mixture thereof; and optionally

(ii) an inorganic ceramic material or an inorganic glass material; or a mixture thereof; and

(b) an active ingredient covalently bonded to said composite material;

wherein the active ingredient comprises:

(i) a growth factor or a combination thereof; and/or

(ii) an active pharmaceutical ingredient or a combination thereof. In some embodiments, the active ingredient (b) is covalently bonded to the organic polymer (i). Therefore, according to one aspect of the invention, there is provided a composition comprising:

(a) a composite material comprising constituents:

(i) an organic polymer or a mixture thereof; and optionally (ii) an inorganic ceramic material or an inorganic glass material; or a mixture thereof; and

(b) an active ingredient covalently bonded to said organic polymer;

wherein the active ingredient comprises:

(i) a growth factor or a combination thereof; and/or

(ii) an active pharmaceutical ingredient or a combination thereof.

In some embodiments, the active ingredient (b) is covalently bonded to the organic polymer (i) via a linker group, in particular a linker group having a molecular weight of at least 150 and/or at least 10 atoms in length. Therefore, according to one aspect of the invention, there is provided a composition comprising:

(a) a composite material comprising constituents:

(i) an organic polymer or a mixture thereof; and optionally

(ii) an inorganic ceramic material or an inorganic glass material; or a mixture thereof; and

(b) an active ingredient covalently bonded to said composite material;

wherein the active ingredient comprises:

(i) a growth factor or a combination thereof; and/or

(ii) an active pharmaceutical ingredient or a combination thereof;

wherein the active ingredient is covalently bonded to the composite material via a linker group having a molecular weight of at least 150.

There is also provided a composition comprising:

(a) a composite material comprising constituents:

(i) an organic polymer or a mixture thereof; and optionally

(ii) an inorganic ceramic material or an inorganic glass material; or a mixture thereof; and

(b) an active ingredient covalently bonded to said composite material;

wherein the active ingredient comprises:

(i) a growth factor or a combination thereof; and/or

(ii) an active pharmaceutical ingredient or a combination thereof;

wherein the active ingredient is covalently bonded to the composite material via a linker group having at least 10 atoms. In some embodiments, the scaffold of the composite material comprises only the organic polymer (i) without the inorganic ceramic or inorganic glass material (ii). Therefore, according to one aspect of the invention, there is provided a composition comprising:

(a) a composite material comprising an organic polymer or a mixture thereof; and (b) an active ingredient covalently bonded to said organic polymer;

wherein the active ingredient comprises:

(i) a growth factor or a combination thereof; and/or

(ii) an active pharmaceutical ingredient or a combination thereof.

According to one aspect of the invention, there is provided a composition comprising:

(a) a composite material comprising constituents:

(i) an organic polymer or a mixture thereof; and

(ii) an inorganic ceramic material, an inorganic glass material; or a mixture of any thereof; and

(b) an active ingredient bonded to said composite material;

wherein the active ingredient comprises:

(i) a growth factor or a combination thereof; and/or

(ii) an active pharmaceutical ingredient or a combination thereof.

The invention also comprises methods for preparing the composition. Therefore, in one aspect, there is provided a method of preparing the composition of the invention, comprising mixing the organic polymer, the active ingredient or combination thereof and, if required, the inorganic ceramic material or inorganic glass material such that the active ingredient or combination thereof is bonded to the composite material. In one embodiment, mixing is carried out such that the active ingredient or combination thereof is covalently bonded to the composite material. In one embodiment, mixing is carried out such that the active ingredient or combination thereof is covalently bonded to the organic polymer.

In some embodiments, the mixing is carried out such that the active ingredient or combination thereof is bonded to the organic polymer. In one embodiment, mixing is carried out such that the active ingredient or combination thereof is covalently bonded to the organic polymer. Typically, the above process is carried out by a method initiated by external excitation, such as using a photoinitiator and/or ultraviolet or visible light. Such methods typically follow a free-radical mechanism, in contrast to the polar (typically amide-bond) linking processes described in the prior art which use activating compounds such as carbodiimides.

Therefore, in one aspect, there is provided a method of preparing the composition of the invention, comprising mixing the organic polymer, the active ingredient or combination thereof and, if required, the inorganic ceramic material or inorganic glass material such that the active ingredient or combination thereof is covalently bonded to the composite material, wherein the covalent bond is formed using a free-radical mechanism. Therefore, in one aspect, there is provided a method of preparing the composition of the invention, comprising mixing the organic polymer, the active ingredient or combination thereof and, if required, the inorganic ceramic material or inorganic glass material such that the active ingredient or combination thereof is bonded (in particular, covalently bonded) to the organic polymer.

In one embodiment, there is provided a method of preparing the composition of the invention, comprising mixing the organic polymer and the active ingredient or combination thereof such that the active ingredient or combination thereof is covalently bonded to the organic polymer.

According to another aspect of the invention, there is provided a method of preparing the composition of the invention, comprising mixing the inorganic ceramic material or inorganic glass material, the organic polymer and the active ingredient such that the active ingredient or combination thereof is covalently bonded to the composite material.

According to another aspect of the invention, there is provided a method of preparing the composition of the invention, comprising:

(a) contacting the inorganic ceramic material or inorganic glass material with the organic polymer to form the composite material; and

(b) contacting the composite material with the active ingredient such that the active ingredient is covalently bonded to the composite material. According to another aspect of the invention, there is provided use of the composition of the invention for regenerating musculoskeletal tissue, such as bone, muscle and tendon.

According to another aspect of the invention, there is provided the composition of the invention for use in regenerating musculoskeletal tissue.

According to another aspect of the invention, there is provided a method of treating a musculoskeletal disease or injury, comprising applying the composition of the invention to a site affected by said musculoskeletal disease or injury.

According to another aspect of the invention, there is provided use of the composition of the invention for regenerating muscle tissue.

According to another aspect of the invention, there is provided the composition of the invention for use in regenerating muscle tissue.

According to another aspect of the invention, there is provided a method of treating a muscle disease or injury, comprising applying the composition of the invention to a site affected by said muscle disease or injury.

According to another aspect of the invention, there is provided use of the composition of the invention as a tissue replacement and/or tissue regeneration material.

According to another aspect of the invention, there is provided the composition of the invention for use as a tissue replacement and/or tissue regeneration material.

According to another aspect of the invention, there is provided a method of treating a tissue disease or injury, comprising applying the composition of the invention to a site affected by said tissue disease or injury.

According to another aspect of the invention, there is provided use of the composition of the invention as a bone replacement and/or bone regeneration material.

According to another aspect of the invention, there is provided the composition of the invention for use as a bone replacement and/or bone regeneration material.

According to another aspect of the invention, there is provided a method of treating a bone disease or injury, comprising applying the composition of the invention to a site affected by said bone disease or injury.

According to another aspect of the invention, there is provided use of the composition of the invention as a cartilage replacement and/or cartilage regeneration material.

According to another aspect of the invention, there is provided the composition of the invention for use as a cartilage replacement and/or cartilage regeneration material.

According to another aspect of the invention, there is provided a method of treating a cartilage disease or injury, comprising applying the composition of the invention to a site affected by said cartilage disease or injury.

According to another aspect of the invention, there is provided use of the composition of the invention as a nerve replacement and/or nerve regeneration material.

According to another aspect of the invention, there is provided the composition of the invention for use as a nerve replacement and/or nerve regeneration material.

According to another aspect of the invention, there is provided a method of treating a nerve disease or injury, comprising applying the composition of the invention to a site affected by said nerve disease or injury. Brief Description of the Figures

Figure 1 illustrates the mechanical stability of the scaffold at various ratios of chitosan to hydroxyapatite;

Figure 2 illustrates the surface wettability (measured by contact angle) of the scaffold at various ratios of chitosan to hydroxyapatite;

Figure 3 illustrates the release rate of Bovine Serum Albumin (BSA) protein as a function of time from various forms of the composition of the invention;

Figure 4 illustrates the release rate of bone morphogenetic protein 4 (BMP-4) as a function of time from a composition of the invention containing 2μg BMP-4;

Figure 5 illustrates the release rate of BMP-4 as a function of time from a composition of the invention containing 2μg BMP-4 and 500 ng vascular endothelial growth-factor A (VEGF-A);

Figure 6 illustrates the release rate of VEGF-A as a function of time from a composition of the invention containing 2μg BMP-4 and 500 ng vascular endothelial growth-factor A (VEGF-A);

Figure 7 illustrates the viability of the composition without the incorporation of growth factors of the invention in a murine MC3t3-El pre-osteoblastic cell line at various chitosan : hydroxyapatite ratios;

Figure 8 illustrates the controlled release of an antimicrobial (ciprofloxacin, 6mg based on a 120mg sample) from a composition of the invention using the Kirby-Bauer test method utilised with Staphylococcus aureus;

Figure 9 illustrates FT-IR spectra of crosslmked and uncrosslinked samples of the composition of the invention; and

Figure 10 illustrates the results of ex vivo micro computed tomography ^CT) tests for bone volume using the composition of the invention compared with the scaffold alone.

Detailed Description

In one embodiment, the composition of the present invention comprises:

(a) a composite material comprising constituents:

(i) an organic polymer or a mixture thereof; and optionally

(ii) an inorganic ceramic material or an inorganic glass material; or a mixture thereof; and

(b) an active ingredient covalently bonded to said composite material;

wherein the active ingredient comprises:

(i) a growth factor or a combination thereof; and/or

(ii) an active pharmaceutical ingredient or a combination thereof.

In one embodiment, the composition of the present invention comprises:

(a) a composite material comprising constituents:

(i) an organic polymer or a mixture thereof; and

(ii) an inorganic ceramic material, an inorganic glass material; or a mixture of any thereof; and

(b) an active ingredient bonded to said composite material;

wherein the active ingredient comprises:

(i) a growth factor or a combination thereof; and/or

(ii) an active pharmaceutical ingredient or a combination thereof.

Advantages and Surprising Findings

It has surprisingly been found by the present inventors that in the composition of the invention in which the active ingredient (especially a growth factor, and particularly BMP-4 and/or VEGF-A) is covalently bonded to a scaffold (comprising an organic polymer and, optionally, an inorganic ceramic material or inorganic bioglass), the release rate of the active ingredient from the scaffold is controlled by the degradation rate of the scaffold, rather than by diffusion as in the composite materials of the prior art (particularly although not exclusively those described in WO 2012/142533 and US 2011/0256203). This means the scaffold is capable of retaining the active ingredient on the scaffold for an extended period of time, and controlling the release of the active ingredient, better than was possible in the prior art. This confers significant advantages over the prior art in that the risk of over-dosage of the active ingredient and potential adverse events can be reduced. In particular, when the active ingredient is a growth factor, the composition has the potential to be introduced into a tissue injury site (especially bone) and allow the growth factor to exert a therapeutic effect while minimising the risk of adverse events or complications known to be associated with growth factors, particularly when administered to children.

In addition, it has surprisingly been found by the present inventors that the

composition of one embodiment of the invention comprising a scaffold which is a composite material (especially an inorganic ceramic material, particularly

hydroxyapatite, and a natural polymer, especially chitosan) to which is covalently bonded an active ingredient (especially a growth factor, and particularly BMP-4 and/or VEGF-A) is capable of retaining the active ingredient on the scaffold for an extended period of time, such that the active ingredient is released in a controlled manner. This makes the composition particularly suitable for a number of

applications, particularly tissue regeneration and/or replacement, such as

musculoskeletal tissue regeneration and/or replacement, cartilage tissue regeneration and/or replacement , nerve tissue regeneration and/or replacement and especially bone regeneration and/or replacement.

In one embodiment of the present invention, the active ingredient is bonded to the polymer via a long linker group (typically a linker having a molecular weight of at least 150; and/or a linker group providing a spacing of at least 10 atoms). Without wishing to be bound by theory, in contrast to the short linker groups (or direct bond between the active and the polymer) described in US 2006/0149392, it is believed that a longer and/or higher molecular weight linker group can result in the composite material having increased pore size, and improved control of pore size, leading to more controlled diffusion of the active ingredient through the membrane and better control of the bioavailability of the active ingredient.

In particular, it has been found that when the scaffold is a composite material comprising an inorganic ceramic material, particularly hydroxyapatite, bonded to an organic polymer, the ceramic phase of the scaffold is electrically conductive, thereby supporting its use in muscle and nerve regeneration.

The above compositions are typically formed using methods initiated by external excitation, such as using a photoinitiator and/or ultraviolet or visible light. Such methods typically follow a free-radical mechanism and involve the formation of carbon-carbon bonds. In contrast to the polar (typically amide bond or ester bond formation) processes found in the prior art, especially that described in

US 2006/0149392, forming the linker using external excitation, such as UV crosslinking according to the present invention, takes both a shorter time (typically 40 minutes as opposed to 3 days) and enables improved control of the pore size of the composite material leading to more controlled diffusion of the active ingredient through the membrane and better control of the bioavailability of the active ingredient. Composite Material

Constituent (a) of the composition of the invention comprises a composite material. The composite material forms a scaffold to which the active ingredient is covalently linked.

In one embodiment, the composite material which forms the scaffold comprises (i) an organic polymer or a mixture thereof; and (ii) an inorganic ceramic material or an inorganic glass material; or a mixture thereof. The active ingredient is covalently linked to this scaffold, preferably to the organic polymer part of the scaffold.

In another embodiment, the composite material which forms the scaffold comprises only an organic polymer or a mixture thereof; without the inorganic ceramic material or inorganic glass material. The active ingredient is covalently linked to the organic polymer which forms the scaffold.

The constituent (ii) of the composite material, where present, comprises an inorganic ceramic material or an inorganic glass material; or a mixture thereof. The function of constituent (ii) of the composite material, where present, is to provide support for the scaffold to which the active ingredient is covalently linked.

In one embodiment, constituent (ii) of the composite material (a) comprises a ceramic material. In this specification the term "ceramic" in its broadest sense means an inorganic, nonmetallic, crystalline solid, typically prepared by the action of heat (preferably to any temperature from room temperature to 2000°C, such as 40°C to 1000°C, such as 50°C to 500°C, such as 50°C to 100°C) and subsequent cooling. A ceramic according to the present invention may also prepared by a precipitation method. Examples of ceramics are well known to those skilled in the art, and include metal and semimetal oxides such as silica, alumina, ceria, and zirconia; metal and semimetal carbides such as silicon carbide, tungsten carbide, boron carbide and titanium carbide, borides such as silicon boride; nitrides such as boron nitride, zirconium nitride, tungsten nitride, vanadium nitride, tantalum nitride, and niobium nitride, phosphate minerals such as those defined and exemplified below, and combinations thereof. The ceramic may be particulate reinforced and/or fibre reinforced.

In one embodiment, the ceramic is a phosphate mineral. Examples of phosphate minerals are known to those skilled in the art, and include triphylite, monazite, erythrite, amblygonite, lazulite, wavellite, turquoise, phosphophyllite, struvite, and calcium phosphate minerals including those of the apatite group such as those defined and exemplified below.

In one embodiment, the constituent (ii) of the composite material (a) comprises a calcium phosphate mineral. In this specification the term "calcium phosphate

2_ _| _

mineral" means a minerals containing calcium ions (Ca ) together with

orthophosphates (P0 ₄ ^3~ ), metaphosphates or pyrophosphates (P ₂ 0 ₇ ^4" ) and optionally one or more further ions selected from hydrogen ions, hydroxide ions or halide (fluoride, chloride, bromide or iodide) ions. Examples of calcium phosphate minerals include monocalcium phosphate, (Ca(H ₂ P0 ₄ ) ₂ ), dicalcium phosphate (dibasic calcium phosphate, CaHP0 ₄ ), tricalcium phosphate (tribasic calcium phosphate or tricalcic phosphate, Ca ₃ (P0 ₄ ) ₂ ), hydroxyapatite (Ca ₅ (P0 ₄ ) ₃ OH), fluorapatite (Ca ₅ (P0 ₄ ) ₃ F), chlorapatite (Ca _s (P0 ₄ ) ₃ Cl), bromapatite (Ca ₅ (P0 ₄ ) ₃ Br), apatite Ca ₁₀ (PO ₄ ) ₆ (OH, F, CI, Br) ₂ , Octacalcium phosphate Ca ₈ H ₂ (P0 ₄ ) ₆ .5H ₂ 0 and biphasic calcium phosphate.

In one embodiment, constituent (ii) of the composite material (a) comprises hydroxyapatite or tricalcium phosphate. In one embodiment, constituent (ii) of the composite material (a) comprises hydroxyapatite. In one embodiment, the particle size of hydroxyapatite is in the nanometre range (defined herein as from lnm to less than 1 μηι). In one embodiment, constituent (ii) of the composite material (a) comprises tricalcium phosphate. In one embodiment, constituent (ii) of the composite material (a) comprises beta-tricalcium phosphate. In one embodiment, constituent (ii) of the composite material (a) comprises an inorganic glass material. In this specification the term "inorganic glass material" means an inorganic solid that possesses a non-crystalline (i.e. amorphous) structure and exhibits a glass transition when heated towards the liquid state. Examples of inorganic glasses are well known to those skilled in the art, and include silicate glasses, borosilicate glasses and bioactive glasses.

In one embodiment, constituent (ii) of the composite material (a) comprises bioactive glass. Bioactive glasses are a group of surface reactive glass-ceramic biocompatible glasses commonly used as implant materials in the human body to repair and replace diseased or damaged bone. Examples of bioactive glasses include Bio glass™ 45 S 5 (comprising 46.1 mol% Si0 ₂ , 26.9 mol% CaO, 24.4 mol% Na ₂ 0 and 2.5 mol% P ₂ 0 ₅ ), Bioglass™ 58S (comprising 60 mol% Si0 ₂ , 36 mol% CaO and 4 mol% P ₂ 0 ₅ ) and Bioglass™ 70S30C (comprising 70 mol% Si0 ₂ and 30 mol% CaO). Constituent (i) of the composite material (a) comprises an organic polymer. In one embodiment the organic polymer is the sole material which forms the scaffold to which the active ingredient is covalently linked.. In another embodiment the organic polymer and the inorganic ceramic or inorganic bioactive glass material together form the scaffold to which the active ingredient is covalently linked.

In one embodiment constituent (i) of the composite material (a) comprises a single type of organic polymer. In another embodiment constituent (i) of the composite material (a) comprises a mixture of different types of organic polymer. The organic polymer may be a natural organic polymer or a synthetic organic polymer. Examples of organic polymers are well known to those skilled in the art, and include, but are not limited to, polyalkenes (polyolefins), substituted polyolefins, polyamides (nylons), polyesters, polycarbonates, polyimides and mixtures thereof. Examples of suitable polyolefins and substituted polyolefins include, but are not limited to: polyethylenes; polypropylenes; poly(l-butene); poly(methyl pentene); poly(vinyl chloride); poly(acrylonitrile); poly(tetrafluoroethyiene) (PTFE - Teflon ^® ), poly(vinyl acetate); polystyrene; poly(methyl methacrylate); ethylene-vinyl acetate copolymer; ethylene methyl acrylate copolymer; styrene-acrylonitrile copolymers; cycloolefm polymers and copolymers; and mixtures and derivatives thereof.

Examples of suitable polyethylenes include, but are not limited to, low density polyethylene, linear low density polyethylene, high density polyethylene, ultra-high molecular weight polyethylene, and derivatives thereof. Examples of suitable polyamides include nylon 6-6, nylon 6-12 and nylon 6. Examples of suitable polyesters include polyethylene terephthalate, polybutylene terephthalate,

polytrimethylene terephthalate, polyethylene adipate, polycaprolactone, polylactic acid, polyhydroxybutyrate, polyglycolic acid, polylacticglycolic acid, and mixtures and derivatives thereof. In one embodiment constituent (i) of the composite material (a) comprises a natural organic polymer. In one embodiment constituent (i) of the composite material (a) comprises chitosan, collagen, chitin or alginate, or a mixture of any thereof. In one embodiment constituent (ii) of the composite material (a) comprises chitosan or collagen.

In one embodiment constituent (i) of the composite material (a) comprises chitosan. Chitosan is a linear polysaccharide composed of randomly distributed P-(l-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is typically made by treating shrimp and other crustacean shells with the alkali sodium hydroxide.

Typically, the molecular weight of the chitosan is between 1000 and 1,000,000, such as 3000 to 500,000, such as 5000 to 300,000, such as 10,000 to 200,000, such as 20,000 to 150,000, such as 50,000 to 125,000. In a particularly preferred

embodiment, the molecular weight of the chitosan is about 100,000.

In one embodiment, constituent (i) of the composite material (a) comprises a vinyl lactam homopolymer or copolymer. The vinyl lactam polymers which may form constituent (i) of the composite material (a) have a repeating unit of the following general formula:

wherein n is 0 to 6. In the above general formula, preferably n is 1, 2 or 3, more preferably 1 or 3, most preferably 1. When n is i, the repeating unit is a vinyl pyrrolidone repeating unit. When n is 3, the repeating unit is a vinyl caprolactam repeating unit. The vinyl lactam repeating units may be the same or different. In one embodiment, the vinyl lactam polymer is a homopolymer (i.e. it contains only one type of vinyl lactam repeating unit). Examples of such vinyl lactam

homopolymer s include polyvinyl pyrrolidone (PVP, wherein n = 1) and polyvinyl caprolactam (wherein n = 3). In another embodiment, the vinyl lactam polymer is a copolymer including one or more other vinyl repeating unit in addition to the vinyl lactam repeating unit. The other repeating unit may be another vinyl lactam repeating unit, such as those described and exemplified above. Alternatively, the other repeating unit may be a vinyl repeating unit, examples of which include ethylene; propylene; 1-butene; 2- methylpentene; acrylonitrile; vinyl acetate; styrene; vinyl imidazole; methacrylic acid; alkyl methacrylate; acrylic acid, N-isopropylacrylamide; and mixtures thereof.

In one embodiment, one or more stimuli sensitive components may be incorporated into the scaffold to enhance the release rate of the active ingredient from the composite material, typically the organic polymer. Examples of such stimuli sensitive components include poly(acrylic acid) or acrylic acid, which confer pH

responsiveness, and poly(N-isopropylacrylamide) or N-isopropylacrylamide, which confer temperature responsiveness. Covalent Link

In the composition of the present invention, the active ingredient is covalently linlced to the composite material. Typically, the active ingredient is covalently linked to the organic polymer. The covalent bonding of the active to the composite, typically the polymer, allows the active to be released at a rate controlled by degradation of the polymer rather than the poorly controlled diffusion mechanisms of the prior art, thereby reducing the potential for overdosage and adverse events. In one embodiment, the active ingredient forms a direct covalent bond to the organic polymer. In one embodiment, the active ingredient is covalently linked to the organic polymer by means of a linker group. Typically, such linker groups are derived from crosslinking agents such as those defined and exemplified below. The linker group may contain any atoms typically found in organic chemistry, such as C, N, O or S. Preferably the linker group contains at least one carbon atom.

In one embodiment the linker group comprises or consists of an alkylene group. In this specification the term "alkylene group" when used to define the linker group means a saturated, divalent, hydrocarbon moiety. The alkylene group is typically a C _1-30 alkylene group, such as a CMO alkylene group, such as a C _1-6 alkylene group, such as a C _1-4 alkylene group, such as a methylene, ethylene, methylmethylene, propylene or butylene group, and especially an ethylene group. The alkylene group may be substituted with one or more (typically only one) substituent, examples of which include halogen (especially fluorine or chlorine), hydroxy, nitrile (-CN), carboxylic acid (-C0 ₂ H) and carboxylic ester (-C0 ₂ R) where R is hydrogen or a substituent, typically a Ci _-6 alkyl group or a benzyl group. In one embodiment, the substituent on the alkylene group links the alkylene group to the rest of the linker group, such as those defined and exemplified below.

In another embodiment the linker comprises or consists of an oxyalkylene or polyoxyalkylene group. An oxyalkylene group has the formula:

-[CH(R -CH(R ₂ )-O]-„ wherein i and R ₂ are hydrogen or a C _1-4 alkyl group, such as a methyl group, and n is typically 1 to 350, such as 1 to 100, such as 1 to 50, such as 1 to 20. such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. When n is 1, the linker comprises an oxyalkylene group: when n is 2 or more, the linker comprises a poly oxyalkylene group. Typically the linker group is a oxyethylene or polyoxyethylene group (i.e. wherein and R ₂ are hydrogen).

In another embodiment the linker comprises or consists of an ester (-C(=0)-0-) group. In another embodiment the linker comprises or consists of an amide (-C(=0)- N(R)-) group, where R is hydrogen or a substituent, typically a C _1-6 alkyl group or a benzyl group. In another embodiment the linker comprises or consists of an ether (-0-) group.

In one embodiment, the linker group comprises both an alkylene group (as defined and exemplified above) and an oxyalkylene or polyoxyalkylene group (as defined above). The linker group may comprise an oxyalkylene or polyoxyalkylene group having two alkylene termini. In this embodiment, the oxyalkylene or polyoxyalkylene group may be bonded directly to the two alkylene termini or may be bonded via a linker group, typically an ester group.

In one embodiment, the linker group is derived from a diacrylate or dimethacrylate. It will be understood by the skilled person such a linker group is typically (although not exclusively) formed by free-radical polymerisation method, whereby one terminal olefin of the diacryl ate or dimethacrylate reacts with a reactive site (typically formed by hydrogen abstraction) on the organic polymer which forms the scaffold and the other terminal olefin group reacts with a reactive site on the active ingredient to covalently link this group to the active ingredient. The linker group thereby formed comprises ethylene (or methylethylene) groups at either end, linked by the backbone of the acrylate or methacrylate molecule. The backbone of the acrylate or

methacrylate molecule may comprise an alkylene group (as defined and exemplified above) and/or an oxyalkylene or polyoxyalkylene group (as defined above).

Examples of such linker groups are those derived from alkylene diacrylate or dimethacrylates (especially ethylene glycol diacrylate or dimethacrylate) and polyalkylene glycol diacrylates and polyalkylene glycol dimethacrylates, especially polyethylene glycol dimethacrylate. The polyethylene glycol moiety of polyethylene glycol diacrylates and polyethylene glycol dimethacrylates typically has an average molecular weight ranging from 200 to 20,000, typically 200 to 1000, suitably from 200 to 800, more preferably 200 to 600. In one embodiment, the linker group is derived from polyethylene glycol 600 dimethacrylate (i.e. wherein the polyethylene glycol moiety of the polyethylene glycol dimethacrylate has a molecular weight of about 600, typically from 570 to 630).

In one embodiment, the linker group has a molecular weight of at least 150. Without wishing to be bound by theory, it is believed that a higher molecular weight linker group can result in the composite material having increased pore size, and improved control of pore size, leading to more controlled diffusion of the active ingredient through the membrane and better control of the bioavailability of the active ingredient.

In this specification, the molecular weight of the linker group is typically calculated from the point of attachment of the linker to the composite material (especially the organic polymer) to the point of attachment of the linker to the active ingredient, but excluding any linker atoms normally found on the backbone of the polymer and the active ingredient (e.g. excluding any oxygen atoms where the linker is via an ether or ester group and the organic polymer and/or the active ingredient normally has a hydroxy group at the point of attachment of the linker group).

In one embodiment, the linker group provides at least 4 atoms spacing (in other words, at least 4 atoms are present in a chain between the point of attachment to the polymer residue and the point of attachment to the active ingredient, excluding atoms in a side chain). Without wishing to be bound by theory, it is believed that a longer linker group can result in the composite material having increased pore size, and improved control of pore size, leading to more controlled diffusion of the active ingredient through the membrane and better control of the bioavailability of the active ingredient.

Typically, the linker group provides a spacing of at least 8 atoms, such as at least 9 atoms, such as at least 10 atoms, such as at least 12 atoms, such as at least 15 atoms, such as at least 18 atoms, such as at least 20 atoms, such as at least 25 atoms, such as at least 30 atoms, such as at least 35 atoms, such as at least 40 atoms, such as at least 50 atoms, such as at least 60 atoms, such as at least 70 atoms, such as at least 80 atoms, such as at least 90 atoms, such as at least 100 atoms, such as at least 125 atoms, such as at least 150 atoms, such as at least 200 atoms, such as at least 250 atoms, such as at least 300 atoms, such as at least 350 atoms. In this specification the term "provides a spacing of x atoms" means that there are x linker atoms in the chain which separates the organic polymer residue at its point of attachment to the linker group from the active ingredient at its point of attachment to the linker group (i.e. excluding any atoms in a side chain).

Typically, the linker group provides a spacing of a maximum of 500 atoms, such as 400 atoms, such as 300 atoms, such as 200 atoms, such as 150 atoms, such as 100 atoms, such as 50 atoms, such as 40 atoms, such as 30 atoms, such as 25 atoms, such as 20 atoms, such as 15 atoms.

In one embodiment, the linker group provides a spacing of 9 to 50 atoms. Such a linker group can be formed when the cross-linking agent is a an ethylene glycol diacrylate or dimethacrylate) and polyalkylene glycol diacrylates and polyalkylene glycol dimethacrylates, especially polyethylene glycol dimethacrylate where the polyethylene glycol moiety has a molecular weight ranging from 200 to 1000, suitably from 200 to 800, more preferably 200 to 600.

The covalent linking of the active ingredient to the composite material, typically the organic polymer, may be carried out by a number of processes well known to those skilled in the polymer arts. Typically, any process which permits a hydrogen atom to be abstracted from an organic polymer may be used. Typically, the covalent linking is carried out using a free-radical polymerisation method. For example, the covalent- linking can be initiated by heat, pressure, change in pH (acid or alkaline), or electromagnetic radiation (particularly visible or ultraviolet light). Preferably, the covalent linking is initiated by using ultraviolet light or other photoexcitation, especially UV excitation. In one embodiment, the covalent linking of the active ingredient to the composite material, typically the organic polymer, is carried out using a cross-linking agent. Typically, the cross-linking agent contains one or more (preferably at least two) carbon-carbon double bonds capable of reacting with either (preferably both) of a radical derived by abstracting a hydrogen atom from a polymer and an appropriate reactive site on the active ingredient. Examples of cross-linking agents are well known to those skilled in the art.

In one embodiment, the cross-linking agent is a diolefin (when referring to cross- linking agents, the term "diolefin" means any organic compound having more than one carbon-carbon double bond, preferably at the terminal carbon atoms). Examples of diolefms include diacrylates and dimethacrylates, such as ethylene glycol diacrylate or dimethacrylate and polyalkylene glycol diacrylates and polyalkylene glycol dimethacrylates, especially polyethylene glycol dimethacrylate. The polyethylene glycol moiety of polyethylene glycol diacrylates and polyethylene glycol

dimethacrylates typically has an average molecular weight ranging from 200 to 20,000, typically 200 to 1000, suitably from 200 to 800, more preferably 200 to 600. In this specification the average molecular weight of the polyalkylene glycol moiety typically has a tolerance range of ±10%, especially ±5%, so that for example a molecular weight stated 500 typically comprises values in the range 450 to 550, such as 475 to 525.

In one embodiment, the cross-linking agent comprises polyethylene glycol 600 dimethacrylate (i.e. wherein the polyethylene glycol moiety of the polyethylene glycol dimethacrylates has an average molecular weight of 540 to 660, typically from 570 to 630).

In one embodiment, the cross-linking agent is present in an amount of 10 ng to 1 mg, such as 100 ng to 500 μg, such as 1 μg to 200 g, such as 10 μg to 100 μg, per g of the composite material. In this specification the expression "per g of the composite material" means the total weight of the specified ingredient per g of the total weight of all constituents (i) and (ii) of the composite material (a), excluding the weight contribution of the active ingredient (b). In one embodiment, the covalent linking of the active ingredient to the composite material, typically the organic polymer, is carried out using a photoinitiator. A photoinitiator is a chemical compound that decomposes into free radicals when exposed to light. The photoinitiator may be a Type I or Type II photoinitiator. Type I photoinitiators undergo cleavage upon irradiation to generate two free radicals in which only one is reactive and proceeds to initiate polymerization. Type II

photoinitiators form an excited state (e.g. a triplet state) upon irradiation but must abstract an atom or electron from a donor synergist, which then acts as the initiator for polymerization.

Examples of photoinitiators are well known to those skilled in the art. Examples of Type I photoinitiators include azobis(isobutyronitrile) (AIBN), peroxides such as benzoyl peroxide, benzoin ethers, benzil ketals, -dialkoxyacetophenones, - aminoalkylphenones, a-hydroxyacetophenones, and acyl phosphine oxides. Examples of Type II photoinitiators include diaryl ketones (benzophenones) such as

benzophenone and substituted benzophenones, thioxanthones such as isopropyl thioxanthone and 2,4-diethylthioxanthone, and quinones such as benzoquinone, camphorquinone and anthraquinone. Type II photoinitiators are particularly preferred for use in the present invention. Type II photoinitiators abstract a hydrogen atom from the surface of the organic polymer to form a radical on the polymer surface, thereby enabling the polymer to react with the active ingredient to form a covalent linkage between the polymer and the active.Type II photoinitiators are especially preferred when the organic polymer is chitosan, as they are capable of abstracting a hydrogen atom from the backbone chain of the chitosan phase of the composite material and allows the attachment of an unsaturated crosslinking agent.

In one embodiment, the photoinitiator is benzophenone. In another embodiment, the photoinitiator is a water soluble Type II photoinitiator. Examples of water soluble Type II photoinitiators include sulfonate and carboxylate salts of quinones. A particular example is anthraquinone 2-sulfonic acid sodium salt. In one embodiment, the photoinitiator is present in an amount of 10 pg to 1 mg, such as 100 pg to 100 μg, such as 1 ng to 10μg, such as 10 ng to 1 μg, per g of the composite material. In one embodiment the covalent link is formed using both a photoinitiator (as defined and exemplified above) and a crosslinking agent (such as those defined and exemplified above). In this embodiment, the composite material, typically the organic polymer, forms a covalent bond with one reactive site on the cross-linking agent (typically by a free- radical mechanism where a radical formed on the surface of the polymer by hydrogen abstraction reacting with a carbon-carbon double bond on the cross-linking agent) and the active ingredient forms another covalent bond with another reactive site on the cross-linking agent.

Preferred Polymer Mixtures

In one embodiment constituent (i) of the composite material (a) comprises a mixture of chitosan and a vinyl lactam polymer. In one embodiment constituent (i) of the composite material (a) comprises a mixture of chitosan and polyvinyl pyrrolidone. In one embodiment constituent (i) of the composite material (a) comprises a mixture of chitosan and cross-linked polyvinyl pyrrolidone. In one embodiment constituent (i) of the composite material (a) comprises a mixture of chitosan and polyvinyl pyrrolidone cross-linked using acrylic acid and/or a polyethylene glycol diacrylates or

polyethylene glycol dimethacrylate having a molecular weight ranging from 200 to 1000. In each of the above embodiments, the chitosan may comprise 1 to 99%, such as 5 to 95%), such as 10 to 90%, such as 20 to 50%, by weight of the total weight of chitosan and vinyl lactam polymer which together forms the constituent (i). In each of the above embodiments, the vinyl lactam polymer may comprise 1 to 99%, such as 5 to 95%, such as 10 to 90%, such as 50 to 80%, by weight of the total weight of chitosan and vinyl lactam polymer which together forms the constituent (i).

In one embodiment, constituent (ii) of the composite material (a) comprises from 1% to 99% by weight of the composite material. In one embodiment, constituent (ii) of the composite material (a) comprises from 5% to 95%> by weight of the composite material. In one embodiment, constituent (ii) of the composite material (a) comprises from 10% to 90% by weight of the composite material. In one embodiment, constituent (ii) of the composite material (a) comprises from 5% to 50% by weight of the composite material. In one embodiment, constituent (ii) of the composite material (a) comprises from 10% to 30% by weight of the composite material. In this specification the expression "by weight of the composite material" means the total weight of all constituents (i) and (ii) of the composite material (a), excluding the weight contribution of the active ingredient (b).

In one embodiment, constituent (i) of the composite material (a) comprises from 0.01% to 100% by weight of the composite material. In one embodiment, constituent (i) of the composite material (a) comprises from 0.1% to 99.9% by weight of the composite material. In one embodiment, constituent (i) of the composite material (a) comprises from 0.5% to 99.5 % by weight of the composite material. In one embodiment, constituent (i) of the composite material (a) comprises from 1% to 99% by weight of the composite material. In one embodiment, constituent (i) of the composite material (a) comprises from 5% to 95% by weight of the composite material. In one embodiment, constituent (i) of the composite material (a) comprises from 10% to 90% by weight of the composite material. In one embodiment, constituent (i) of the composite material (a) comprises from 55% to 95% by weight of the composite material. In one embodiment, constituent (i) of the composite material (a) comprises from 70% to 90% by weight of the composite material.

In one embodiment constituent (ii) of the composite material (a) comprises hydroxyapatite and constituent (i) of the composite material (a) comprises chitosan. In this embodiment the hydroxyapatite may comprise from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 5% to 50%, such as 10% to 30%, by weight of the composite material. In this embodiment the chitosan may comprise in an amount from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 50% to 95%, such as 70% to 90%, by weight of the composite material. In one embodiment the hydroxyapatite and the chitosan are present in a weight ratio of 6: 1 to 1 :6. In one embodiment the hydroxyapatite and the chitosan are present in a weight ratio of 4:1 to 1 :4. In one embodiment the hydroxyapatite and the chitosan are present in a weight ratio of 2: 1 to 1 : 2. In one embodiment the hydroxyapatite and the chitosan are preferably present in a weight ratio of 6:1 to 1 :2. More preferably the hydroxyapatite and the chitosan are present in a weight ratio of 1 : 1 to 4: 1.

In one embodiment constituent (ii) of the composite material (a) comprises tri calcium phosphate and constituent (i) of the composite material (a) comprises chitosan. In this embodiment the tricalcium phosphate may comprise from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 5% to 50%, such as 10% to 30%, by weight of the composite material. In this embodiment the chitosan may comprise from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 50% to 95%, such as 70% to 90%, by weight of the composite material.

In one embodiment constituent (i) of the composite material (a) comprises hydroxyapatite and constituent (i) of the composite material (a) comprises a mixture of polyvinyl pyrrolidone and chitosan. In this embodiment the chitosan may comprise from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 15% to 50%, such as 20% to 30%, by weight of the composite material. In this embodiment the polyvinyl pyrrolidone may comprise from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 30% to 80%, such as 40% to 60%, by weight of the composite material. In this embodiment the hydroxyapatite may comprise from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 15% to 50%, such as 20% to 30%, by weight of the composite material.

In one embodiment constituent (ii) of the composite material (a) comprises tricalcium phosphate and constituent (i) of the composite material (a) comprises a mixture of polyvinyl pyrrolidone and chitosan. In this embodiment the chitosan may comprise from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 15% to 50%, such as 20% to 30%), by weight of the composite material. In this embodiment the polyvinyl pyrrolidone may comprise from 1% to 99%, such as 5% to 95%, such as 10% to 90%, such as 30% to 80%, such as 40% to 60%, by weight of the composite material. In this embodiment the tricalcium phosphate may comprise from 1 % to 99%, such as 5% to 95%, such as 10% to 90%, such as 15% to 50%, such as 20% to 30%, by weight of the composite material. In one embodiment, an acid is added to the reactants during the formation of the composite material. The acid may be any acid known in the art, examples of which include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid and sulphuric acid, and organic acids such as formic acid, acetic acid, acrylic acid, citric acid, tartaric acid, methanesulfonic acid and p-toluenesulfonic acid; preferred is acetic acid. In one embodiment, the acid is present in an amount of 10 ng to 1 mg, such as 100 ng to 500 g, such as 1 μg to 200 μg, such as 10 μg to 100 μg, per g of the composite material. Active Ingredient

Component (b) of the composition of the invention comprises an active ingredient. According to the invention, the active ingredient is covalently bonded to the composite material, preferably the organic polymer,, typically in such a way that the active ingredient is able to be released from the composite material in a controlled manner or upon the degradation of the polymer phase of the composite, rather than diffusion out of the composite as in the prior art materials.

The active ingredient may be any substance capable of exerting a biological effect in a subject, especially in a mammalian subject, more especially in a human subject. In one embodiment these active ingredient may be a small molecule (typically a molecule of molecular weight less than 1000 daltons). In another embodiment the active ingredient is a protein (in this specification the term "protein" is synonymous with "amino acid sequence", "peptide" and "polypeptide").

Isolated

In one aspect, the active ingredient, such as a protein, used in the present invention is in an isolated form. The term "isolated" means that the active ingredient, such as a protein, is at least substantially free from at least one other component with which the active ingredient, such as a protein, is naturally associated in nature and as found in nature. The active ingredient, such as a protein used in the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. Thus, for example it may be substantially free of one or more potentially contaminating polypeptides and/or nucleic acid molecules.

Purified

In one aspect, preferably the active ingredient, such as a protein, according to the present invention is in a purified form. The term "purified" means that the given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 90%, or at least about 95% or at least about 98%, said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.

Sequence Identity or Sequence Homology

The present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology with amino acid sequence(s) of a

polypeptide having the specific properties defined herein or of any nucleotide sequence encoding such a polypeptide (hereinafter referred to as a "homologous sequence(s)"). Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".

The homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the protein.

In the present context, in some embodiments a homologous sequence is taken to include an amino acid or a nucleotide sequence which may be at least 97.7% identical, preferably at least 98 or 99% identical to the subject sequence. In some embodiments a homologous sequence is taken to include an amino acid or a nucleotide sequence which may be at least 85% identical, preferably at least 90 or 95% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence for instance. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

In one embodiment, a homologous sequence is taken to include an amino acid sequence or nucleotide sequence which has one or several additions, deletions and/or substitutions compared with the subject sequence.

In the present context, "the subject sequence" relates to the nucleotide sequence or polypeptide/amino acid sequence according to the invention.

A "parent nucleic acid" or "parent amino acid" means a nucleic acid sequence or amino acid sequence, encoding or coding for the parent polypeptide, respectively.

In one embodiment the present invention relates to a protein whose amino acid sequence is represented herein or a protein derived from this (parent) protein by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and having the activity of the parent protein.

Suitably, the degree of identity with regard to an amino acid sequence is determined over at least 20 contiguous amino acids, preferably over at least 30 contiguous amino acids, preferably over at least 40 contiguous amino acids, preferably over at least 50 contiguous amino acids, preferably over at least 60 contiguous amino acids, preferably over at least 100 contiguous amino acids, preferably over at least 200 contiguous amino acids.

In one embodiment the present invention relates to a nucleic acid sequence (or gene) encoding a protein whose amino acid sequence is represented herein or encoding a protein derived from this (parent) protein by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and having the activity of the parent protein.

In the present context, in one embodiment a homologous sequence or foreign sequence is taken to include a nucleotide sequence which may be at least 97.7% identical, preferably at least 98 or 99% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence).

In another embodiment, a homologous sequence is taken to include a nucleotide sequence which may be at least 85% identical, preferably at least 90 or 95% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence).

Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology or % identity between two or more sequences. % homology or % identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology or % identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons.

Calculation of maximum % homology or % identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST 2 (see FEMS

Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov), FASTA (Altschul et al 1990 J. Mol. Biol. 403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60), such as for example in the GenomeQuest search tool (www.genomequest.com). Although the final % homology or % identity can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI package.

Alternatively, percentage homologies may be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244). Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

Should Gap Penalties be used when determining sequence identity, then preferably the following parameters are used for pairwise alignment:

In one embodiment, CLUSTAL may be used with the gap penalty and gap extension set as defined above.

Suitably, the degree of identity with regard to a nucleotide sequence or protein sequence is determined over at least 20 contiguous nucleotides/amino acids, preferably over at least 30 contiguous nucleotides/amino acids, preferably over at least 40 contiguous nucleotides/amino acids, preferably over at least 50 contiguous nucleotides/amino acids, preferably over at least 60 contiguous nucleotides/amino acids, preferably over at least 100 contiguous nucleotides/amino acids.

Suitably, the degree of identity with regard to a nucleotide sequence is determined over at least 100 contiguous nucleotides, preferably over at least 200 contiguous nucleotides, preferably over at least 300 contiguous nucleotides, preferably over at least 400 contiguous nucleotides, preferably over at least 500 contiguous nucleotides, preferably over at least 600 contiguous nucleotides, preferably over at least 700 contiguous nucleotides, preferably over at least 800 contiguous nucleotides.

Suitably, the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence taught herein.

Suitably, the degree of identity with regard to a protein (amino acid) sequence is determined over at least 100 contiguous amino acids, preferably over at least 200 contiguous amino acids, preferably over at least 300 contiguous amino acids.

Suitably, the degree of identity with regard to an amino acid or protein sequence may be determined over the whole sequence taught herein.

In the present context, the term "query sequence" means a homologous sequence or a foreign sequence, which is aligned with a subject sequence in order to see if it falls within the scope of the present invention. Accordingly, such query sequence can for example be a prior art sequence or a third party sequence.

In one preferred embodiment, the sequences are aligned by a global alignment program and the sequence identity is calculated by identifying the number of exact matches identified by the program divided by the length of the subject sequence.

In one embodiment, the degree of sequence identity between a query sequence and a subject sequence is determined by 1) aligning the two sequences by any suitable alignment program using the default scoring matrix and default gap penalty, 2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical amino acid or nucleotide in the two aligned sequences on a given position in the alignment and 3) dividing the number of exact matches with the length of the subject sequence.

In yet a further preferred embodiment, the global alignment program is selected from the group consisting of CLUSTAL and BLAST (preferably BLAST) and the sequence identity is calculated by identifying the number of exact matches identified by the program divided by the length of the subject sequence.

The sequences may also have deletions, insertions or substitutions of amino acid residues result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthyl alanine and phenylglycine.

Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-a-amino butyric acid*, L-y-amino butyric acid*, L-a-amino isobutyric acid*, L-s-amino caproic acid", 7-amino heptanoic acid*, L-methionine sulfone* ^* , L-norleucine*, L-norvaline*, p- nitro-L-phenylalanine*, L-hydroxyproline , L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamefhyl-Phe*, L-Phe (4-amino) ^# , L- Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (l,2,3,4-tetrahydroisoquinoline-3- carboxyl acid)*, L-diaminopropionic acid ^# and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β- alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue's nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al, PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134. Suitably there may be at least 2 conservative substitutions, such as at least 3 or at least 4 or at least 5. Suitably there may be less than 15 conservative substitutions, such as less than 12, less than 10, or less than 8 or less than 5. Growth Factor

In one embodiment, the active ingredient (b) comprises a growth factor or a combination thereof. A growth factor is a substance (which may be naturally occurring, synthetic or semi-synthetic) capable of stimulating biological mechanisms such as cellular growth proliferation and cellular differentiation.

In one embodiment the growth factor is a protein. In another embodiment the growth factor is a steroid hormone. The active ingredient (b) may comprise one growth factor or a combination of growth factors. In one embodiment the active ingredient (b) comprises a single growth factor. In another embodiment the active ingredient (b) comprises a combination of growth factors, preferably 2 to 5 growth factors, more preferably 2 or 3 growth factors, and most preferably 2 growth factors.

In one embodiment the active ingredient (b) comprises a growth factor selected from an angiogenic growth factor (AGF), an osteogenic growth factor (OGF), a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), a platelet-derived growth factor (PDGF), a transforming growth factor (TGF), an angiopoietin, a nerve growth factor (NGF), a cartilage-derived retinoic acid protein (CDRAP), an insulin like growth factor (IGF) or a receptor to any thereof, or any combination of said growth factors and/or said receptors.

In one embodiment the active ingredient (b) comprises a growth factor selected from an angiogenic growth factor (AGF), an osteogenic growth factor (OGF), or any combination thereof. In one embodiment the active ingredient (b) comprises a combination of an angiogenic growth factor and an osteogenic growth factor.

In one embodiment the growth factor is a member of the Transforming growth factor- Beta family of proteins, especially a bone morpho genetic protein (BMP). Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines and as metabologens and are capable of orchestrating tissue architecture throughout the body. In one embodiment the BMP is a BMP capable of inducing the formation of bone and cartilage. In one embodiment the BMP is selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP- 8a, BMP-8b, BMP-10 and BMP-15.

Examples of BMPs are disclosed in WO 93/09229 and US 4,619,989. In one embodiment the bone morphogenetic protein is BMP-4. Bone morphogenetic protein 4 is a protein that in humans is encoded by the BMP 4 gene found on chromosome 14q22-q23. BMP-4 is a protein belonging to the TGF-β superfamily of proteins, and is capable of stimulating in bone and cartilage development, specifically tooth and limb development and fracture repair. BMP-4 plays an important role in the onset of endochondral bone formation in humans. It has also been shown to be involved in muscle development, bone mineralization, and ureteric bud development.

In one embodiment the BMP-4 is human BMP-4. In one embodiment the BMP-4 is human recombinant BMP-4. In a particularly preferred embodiment, the BMP-4 is that available from Glenbio Ltd of Toomebridge, Co. Antrim, United Kingdom, under catalogue number RP AO 14HuO 1. This BMP-4 is human BMP-4 and typically has a molecular weight of 14.3kDa, and/or a purity exceeding 95%.

In one embodiment the BMP-4 is a BMP-4 having the amino acid sequence set out in accession number UniProt PI 2644.

In one embodiment the growth factor is a vascular endothelial growth factor (VEGF). Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate. The normal function of VEGF is to create new blood vessels.

Examples of VEGFs are disclosed in WO 96/26736 and US 6,479,654. In one embodiment, the AGF is selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PGF) and fibroblast growth factor.

In one embodiment, the vascular endothelial growth factor is VEGF-A. VEGF-A is a dimeric glycoprotein that plays a significant role in neurons and is considered to be the main, dominant inducer to the growth of blood vessels.

In one embodiment the VEGF-A is human VEGF-A. In one embodiment the VEGF-A is human recombinant VEGF-A. In a particularly preferred embodiment, the VEGF- A is that available from Glenbio Ltd of Toomebridge, Co. Antrim, United Kingdom, under Catalogue Number: RPA143Hu01. This VEGF-A is human VEGF-A and typically has a molecular weight of 17.8kDa and/or a purity exceeding 95%.

In one embodiment the VEGF-A is a VEGF-A having the amino acid sequence set out in accession number UniProt PI 5692.

In another embodiment, the active ingredient is an active pharmaceutical ingredient, typically a small molecule active pharmaceutical ingredient. Typical classes of active pharmaceutical ingredient useful in the present invention include the following:

analgesic drugs including NSAIDs, classes of which include salicylates such as salicylic acid, aspirin and diflunisal; COX-2 selective inhibitors such as celecoxib, parecoxib, firocoxib, lumiracoxib and etoricoxib; propionic acid derivatives such as ibuprofen, dexibuprofen, naproxed, fenoprofen, ketoprofen, dexketoprofen, fluribiprofen, oxaprozen and loxoprofen; acetic acid derivatives such as diclofenac, aceclofenac, keterolac; etolodac, sulindac, tolmetin, indomethacin; oxicam derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam;

opioids such as morphine, codeine, thebaine, hydromorphone, hydrocodone, oxycodone, oxymorphone, ethylmorphine, buprenorphine, fentanyl, pethidine, levorphanol, methadone and tramadol; and other analgesics such as acetaminophen (paracetamol);

antibacterials (antibiotics), typical classes of which include sulfa drugs such as sulfadiazine and sulfadimethoxine; aminoglycosides such as streptomycin, kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin and neomycin; and quinolones such as ciprofloxacin, enoxacin, fleroxacin, lomefloxacin,

nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin

levofloxacin, pazufloxacin, sparfloxacin, tosufloxacin, clinafloxacin, gemifloxacin, sitafloxacin, moxifloxacin and prulifloxacin;

antiviral drugs, examples of which include abacavir, acyclovir, amprenavir, atazanavir, darunavir, efavirenz, emtricitabine, famciclovir, fosamprenavir, ganciclovir, idoxuridine, imiquimod, indinavir, lamivudine, lopinavir, loviride, maraviroc, nelfmavir, nevirapine, oseltamivir, penciclovir, peramivir, raltegravir, ribavirin, ritonavir, saquinavir, tenofovir, tipranavir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir and zidovudine;

anti-fungal drugs (typical classes of which include polyenes such as amphotericin B, natamycin and nystatin; imidazoles such as bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole; triazoles such as albaconazole, efinaconazole, fluconazole, isavuconazole, itraconazole, posaconazole, ravuconazole, terconazole and voriconazole; thiazoles such as abafungin; allylamines such as butenafine and terbinafine; and echinocandins such as anidulafungin, caspofungin and micafungin),

anti-inflammatory drugs (typical classes of which include NSAIDs as listed above and corticosteroids, including those of the hydrocortisone type such as hydrocortisone and prednisolone, those of the acetonide type such as budesonide and mometasone, and those of the betamethasone type such as betamethasone and dexamethasone), and anti-cancer (anti-neoplastic) drugs, typical classes of which include cytotoxic drugs, including kinase inhibitors such as imatinib, ponatinib, nilotinib, dasatinib, bosutinib bafatinib and borteozmib; therapeutic antibodies such as rituximab, trastuzumab, bevacizumab, and other anti-cancer drugs such as lenalidomide, permetrexed, leuporelin and abiraterone). In one embodiment the active pharmaceutical ingredient is an antibiotic. In one embodiment the active pharmaceutical ingredient is a quinolone antibiotic. In one embodiment the active pharmaceutical ingredient is ciprofloxacin.

In one embodiment the active pharmaceutical ingredient is an analgesic. In one embodiment the active pharmaceutical ingredient is an NSAID. In one embodiment the active pharmaceutical ingredient is a salicylate. In one embodiment the active pharmaceutical ingredient is aspirin. In one embodiment the active pharmaceutical ingredient is acetaminophen.

In one embodiment the active pharmaceutical ingredient is a steroid. In one embodiment the active pharmaceutical ingredient is dexamethasone.

Dosage

The active ingredient may be present in any amount capable of exerting a biological effect. In one embodiment the active ingredient is present in an amount from 1 pg to 100 g, such as 10 pg to 50 g, such as 100 pg to 25 g, such as 1 ng to 10 g, such as 10 ng to 5 g, such as 100 ng to 2.5 g, such as 1 μg to 1 g, such as 10 g to 500 mg, such as 50 μg to 250 mg, such as 100 μg to 100 mg. When the active ingredient is an angiogenic growth factor, the angiogenic growth factor is typically present in an amount of 1 pg to 10 mg. In one embodiment the angiogenic growth factor is typically present in an amount of 1 pg to 10 μg. In one embodiment the angiogenic growth factor is present in an amount of 100 pg to 10 μg. In one embodiment, the active ingredient is a bone morphogenetic protein and the BMP is present in an amount of 1 pg to 10 mg. In one embodiment the BMP is present in an amount of 1 pg to 10 μg. In one embodiment the BMP is present in an amount of 100 pg to 10 μg. In one embodiment, the active ingredient is BMP-4 and the BMP-4 is present in an amount of 1 pg to 10 mg. In one embodiment the BMP-4 is present in an amount of 1 pg to 10 μg. In one embodiment the BMP-4 is present in an amount of 100 pg to 10 When the active ingredient is an osteogenic growth factor, the osteogenic growth factor is typically present in an amount of 1 pg to 10 mg. In one embodiment the osteogenic growth factor is present in an amount of 1 ng to 10 μg. In one

embodiment the osteogenic growth factor is present in an amount of 100 ng to 1 μg.

In one embodiment the active ingredient is a vascular endothelial growth factor (VEGF) and the VEGF is typically present in an amount of 1 pg to 10 mg. In one embodiment the VEGF is present in an amount of 1 ng to 10 μg. In one embodiment the VEGF is present in an amount of 100 ng to 1 μg.

In one embodiment the active ingredient is VEGF-A and the VEGF-A is typically present in an amount of 1 pg to 10 mg. In one embodiment the VEGF-A is present in an amount of 1 ng to 10 μg. In one embodiment the VEGF-A is present in an amount of 100 ng to 1 μg.

The active ingredient may be present in any concentration capable of exerting a biological effect. In one embodiment the active ingredient is present in a

concentration of from 1 ng to 1 g per g of the composite material, such as from 10 ng to 500 mg per g of the composite material, such as from 100 ng to 100 mg per g of the composite material, such as from 1 μg to 10 mg per g of the composite material, such as from 10 μg to 1 mg per g of the composite material.

When the active ingredient is a growth factor, the growth factor may be present in a concentration of from 1 ng to 1 g per g of the composite material, such as from 10 ng to 500 mg per g of the composite material, such as from 100 ng to 100 mg per g of the composite material, such as from 1 μg to 10 mg per g of the composite material, such as from 10 μg to 1 mg per g of the composite material.

When the active ingredient is an angiogenic growth factor, the angiogenic growth factor is typically present in a concentration of 100 ng to 50 mg per g of the composite material. In one embodiment the angiogenic growth factor is present in a

concentration of 10 g to 5 mg per g of the composite material. In one embodiment, the active ingredient is a BMP and the BMP is present in a concentration of 100 ng to 50 mg per g of the composite material. In one embodiment the BMP is present in a concentration of 10 μg to 5 mg per g of the composite material.

In one embodiment, the active ingredient is a BMP -4 and the BMP -4 is present in a concentration of 100 ng to 50 mg per g of the composite material. In one embodiment the BMP -4 is present in a concentration of 10 μg to 5 mg per g of the composite material.

When the active ingredient is an osteogenic growth factor, the osteogenic growth factor is typically present in a concentration of 10 ng to 1 mg per g of the composite material. In one embodiment the osteogenic growth factor is present in an amount of 1 μg to 100 μg per g of the composite material.

In one embodiment, the active ingredient is a VEGF, and the VEGF is present in a concentration of 10 ng to 1 mg per g of the composite material. In one embodiment the VEGF is present in a concentration of 1 μg to 100 μg per g of the composite material.

In one embodiment, the active ingredient is a VEGF-A, and the VEGF-A is present in a concentration of 10 ng to 1 mg per g of the composite material. In one embodiment the VEGF-A is present in a concentration of 1 g to 100 μg per g of the composite material.

In one embodiment, the active ingredient is a combination of an angiogenic growth factor and an osteogenic growth factor, the angiogenic growth factor is typically present in a concentration of 100 ng to 50 mg per g of the composite material, preferably 10 μg to 5 mg per g of the composite material, and the osteogenic growth factor is present in a concentration of 10 ng to 1 mg per g of the composite material, preferably 1 μg to 100 μg per g of the composite material.

In one embodiment, the active ingredient is a combination of an angiogenic growth factor and an osteogenic growth factor, the angiogenic growth factor is typically present in a concentration of 100 ng to 50 mg per g of the composite material, preferably 10 μg to 5 mg per g of the composite material, and the osteogenic growth factor is present in a concentration of 10 ng to 1 mg per g of the composite material, preferably 1 μg to 100 μg per g of the composite material.

In one embodiment, the active ingredient is a combination of a BMP and a VEGF, the BMP is typically present in a concentration of 100 ng to 50 mg per g of the composite material, preferably 10 μg to 5 mg per g of the composite material, and the VEGF is present in a concentration of 10 ng to 1 mg per g of the composite material, preferably 1 μg to 100 g per g of the composite material.

In one embodiment, the active ingredient is a combination of a BMP-4 and a VEGF- A, the BMP-4 is present in a concentration of 100 ng to 50 mg per g of the composite material, preferably 10 μg to 5 mg per g of the composite material, and the VEGF-A is present in a concentration of 10 ng to 1 mg per g of the composite material, preferably 1 μg to 100 μg per g of the composite material.

Method The present invention also comprises methods for preparing the composition of the invention.

In one aspect, the method comprises mixing the organic polymer, the active ingredient and, if required, the inorganic ceramic material or inorganic glass material, such that the active ingredient or combination thereof is covalently bonded to the composite material.

In one embodiment, the method is carried out such that the active ingredient or combination thereof is covalently bonded to the organic polymer.

The method for covalently linking of the active ingredient to the composite material, typically the organic polymer, may be carried out by a number of processes well known to those skilled in the polymer arts. Typically, any process which permits a hydrogen atom to be abstracted from an organic polymer may be used. For example, the covalent-linking can be initiated by heat, pressure, change in pH (acid or alkaline), or electromagnetic radiation (particularly visible or ultraviolet light).

Typically, the method is a reaction initiated by external excitation, such as using a photoinitiator and/or ultraviolet or visible light. Such methods typically follow a free- radical mechanism and involve the formation of carbon-carbon bonds. In contrast to the polar (typically amide bond or ester bond formation) processes found in the prior art, forming the linker using external excitation, such as UV crosslinking according to the present invention, takes both a shorter time (typically 40 minutes as opposed to 3 days) and enables improved control of the pore size of the composite material leading to more controlled diffusion of the active ingredient through the membrane and better control of the bioavailability of the active ingredient.

Preferably, the covalent linking is carried out (typically initiated) using ultraviolet light or other photoexcitation, especially UV excitation.

In one embodiment, the method of the present invention, comprising the covalent linking of the active ingredient to the composite material, typically the organic polymer, is carried out using a cross-linking agent, as defined and exemplified above. Examples of cross-linking agents are well known to those skilled in the art, and include diolefins (when referring to cross-linking agents, the term "diolefin" means any organic compound having more than one carbon-carbon double bond, preferably at the terminal carbon atoms). Examples of diolefins include diacrylates and dimethacrylates, such as polyalkylene glycol diacrylates and polyalkylene glycol dimethacrylates, especially polyethylene glycol dimethacrylate. The polyethylene glycol moiety of polyethylene glycol diacrylates and polyethylene glycol

dimethacrylates typically has a molecular weight ranging from 200 to 20,000, typically 200 to 1000, suitably from 200 to 800, more preferably 200 to 600. In one embodiment, the diolefin comprises polyethylene glycol 600 dimethacrylate (i.e. wherein the polyethylene glycol moiety of the polyethylene glycol dimethacrylate has a molecular weight of about 600, typically from 570 to 630).

In one embodiment, the process is carried out in the presence of an acrylate or methacrylate co-monomer. Examples of acrylate and methacrylate monomers include acrylic acid, methacrylic acid, and acrylic and methacrylic esters such as methyl acrylate, ethyl acrylate, 2-ethylhexyl acrylate, hydroxyethyl methacrylate, butyl acrylate, butyl methacrylate and trimethylolpropane triacrylate (TMPTA). It is preferred that acrylic acid or methacrylic acid, particularly acrylic acid is used as the co-monomer, as it is believed hydrogen bonding between the acrylic acid or methacrylic acid and the organic polymer increases the strength of the gel, as described in Peppas, N.A., et al.. Euro J Pharmace Biopharmace, 2000, 50, 27-46.

In the method of the present invention, comprising the covalent linking of the scaffold (typically the organic polymer) to the active ingredient is typically carried out using a photoinitiator, as defined and exemplified above. A photoinitiator can form radicals of the surface of the composite material, typically the organic polymer, such that it can form a covalent bond with the active ingredient and, if necessary, the cross- linking agent.

Type II photoinitiators are particularly preferred for use in the present invention. Type II photoinitiators abstract a hydrogen atom from the surface of the organic polymer to form a radical on the polymer surface, thereby enabling the polymer to react with the active ingredient to form a covalent linkage between the polymer and the active (either directly or via the cross-linking agent, if present). Type II photoinitiators are especially preferred when the organic polymer is chitosan, as they are capable of abstracting a hydrogen atom from the backbone chain of the chitosan phase of the composite material and allows the attachment of an unsaturated crosslinking agent.

In one embodiment, the photoinitiator is benzophenone. In another embodiment, the photoinitiator is a water soluble Type II photoinitiator. Examples of water soluble Type II photoinitiators include sulfonate and carboxylate salts of quinones. A particular example is anthraquinone 2-sulfonic acid sodium salt.

In one embodiment, the photoinitiator is present in an amount of 10 pg to 1 mg, such as 100 pg to 100 μg, such as 1 ng to 10μg, such as 10 ng to 1 g, per g of the composite material. In one embodiment the method of the present invention is carried out using both a photo initiator (as defined and exemplified above) and a crosslinldng agent (such as those defined and exemplified above). In this embodiment, the method may cause the composite material, typically the organic polymer, to form a covalent bond with one reactive site on the cross-linking agent (typically by a radical formed on the surface of the polymer reacting with a carbon-carbon double bond on the cross-linking agent) and the active ingredient to forms another covalent bond with another reactive site on the cross-linking agent, such that the residue of the cross-linking agent forms a linker group.

In the method of the invention, the organic polymer is preferably chitosan. In this aspect, the chitosan is preferably introduced in the form of a solution in acidic environment. Any acid, organic or inorganic, may be used provided it is capable of acting as an acid and is capable of dissolving the chitosan at least to some extent. Preferably, the chitosan is introduced in the form of a solution in acetic acid. More preferably, the chitosan is introduced in the form of a 20% (weight/volume) solution in a 1 % (volume/volume) aqueous acetic acid.

In another aspect, the method comprises:

(a) contacting the inorganic ceramic material or inorganic glass material with the organic polymer to form the composite material; and

(b) contacting the composite material with the active ingredient such that the active ingredient is covalently bonded to the composite material, typically the organic polymer.

In this aspect, the photoinitiator is preferably introduced in the form of a solution in a solvent. The precise nature of the solvent is not critical, provided it is substantially inert to the reaction and is capable of mixing/interfacing the chitosan at least to some extent. Examples of suitable solvents include alcohols such as methanol, ethanol, propanol and t-butanol, and especially ethanol. Uses, Applications and Methods of Treatment

The compositions of the present invention have a number of applications, particularly in tissue replacement and/or regeneration.

In one aspect, the composition of the present invention is used to regenerate and/or repair musculoskeletal tissue. Typically, a method of treating a musculoskeletal disease or injury using the composition of the invention comprises applying the composition to a site affected by said musculoskeletal disease or injury. Examples of suitable methods are known to those skilled in the art.

In one aspect, the composition of the present invention is used to restore or regenerate muscle tissue. Typically, a method of treating a muscle condition using the composition of the invention comprises applying the composition to a site affected by said condition. Examples of suitable methods are known to those skilled in the art.

In one aspect, the composition of the present invention is used as a bone replacement and/or bone regeneration material. Typically, a method of treating a bone disease or injury using the composition of the invention comprises applying the composition to a site affected by said bone disease or injury. Examples of suitable methods are known to those skilled in the art.

In one aspect, the composition of the present invention is used as a cartilage replacement and/or cartilage regeneration material. Typically, a method of treating a cartilage disease or injury using the composition of the invention comprises applying the composition to a site affected by said cartilage disease or injury. Examples of suitable methods are known to those skilled in the art.

In one aspect, the composition of the present invention is used as a nerve replacement and/or nerve regeneration material. Typically, a method of treating a nerve disease or injury comprises applying the composition of the invention to a site affected by said nerve disease or injury. Examples of suitable methods are known to those skilled in the art. Examples

Example 1 - Formation of a composition of the invention

Composites of hydroxyapatite (HAp) and chitosan have been successfully prepared via a method described generally in R. Murugan and S. RaiBakrishna, Biomaterials 25 (2004) 3829-3835 through the mixing of chitosan and HAp at various concentrations (as shown in Figures 1 and 2) by firstly weighing various amounts of the base materials chitosan and HAp. These materials were subsequently dry mixed in a beaker. To this dry mixture was added a 1 % aqueous solution of acetic acid to form a paste. The quantity of acetic acid solution added equalled five times the weight of chitosan used. However, these composites were not crosslmked and therefore dissolved in slightly acidic solutions such as is present in the tissue surrounding a fracture repair. This is described in more detail in Devine et al., European cells and Materials, 2009, 18, 40-48.

Crosslinking the structure was achieved through the use of a type II photoinitiator. It was found that if benzophenone was dissolved in ethanol at a lwt/vol % concentration and then added to the chitosan/HAp mixture at a concentration of 0.1vol/wt% based on the weight of chitosan in the sample, its availability to generate free radicals on the chitosan backbone chain was sufficient to crosslink the reaction. Subsequently, polyethylene glycol dimethacrylate (PEGDMA) was added to crosslink the structure at 0.1 vol/vol%. As chitosan dissolves in an acidic solution, when composite samples were prepared without crosslinking and without neutralising the acetic acid solution used to prepare the samples, the composite readily dissolved. Therefore, dissolution tests were carried out to determine if the crosslinking had occurred. Once procedures were developed to ensure this occurred, the success of the crosslinking reaction was confirmed using Fourier transform infrared spectrometry (described in more detail in Example 10). As shown in Figure 9, it was found that the amide I group on chitosan and the carbonyl group on PEGDMA reduced in intensity following UV curing indicating that this was the site where crosslinking occurred. Example 2: Mechanical testing

Compression testing was performed on a Lloyd LrlOK, screw driven testing machine fitted with a 2.5kN load cell with a bespoke 30mm diameter testing head.

Compression tests were performed on ~27mm diameter circular samples with a height of 3mm in order to calculate Young's modulus and stress at limit parameters of the samples. All samples were submerged in PBS pH 7.4 for 1 hour prior to testing.

Samples were subsequently compressed to 60% of their original height at a compression rate of 0.5mm/min. The results are shown in Figure 1.

Example 3: Surface wettability

Contact angle measurements were performed using a goniometer, (FTA 1000

Analyzer Systems) to assess the relative hydrophobicity and/or hydrophilicity of selected samples. In this test a lmL droplet of water was placed onto the surface of the scaffold (sessile drop method), a camera then captured ten separate images of the droplet over a 15 second time period. The contact angle Θ (degrees) is measured as the tangent drawn to the surface of the droplet. The value indicated is an average angle for all ten measurements (or smaller number if an error occurred during measurement) for both the left and right extremities of the droplet. Tests were carried out on all five batches of samples in duplicate with three separate droplets of water analysed on each sample type. The results are shown in Figure 2.

Example 4: Bovine serum albumin ELISA - release rate measurement

400 g of Bovine serum albumin (BSA) was added to 200 mg of chitosan/HAp composite scaffold. These samples were placed into a mould and UV cured for 40 minutes according to the process described in more detail in Example 5 below.

The samples were placed into a sterile 24 well plate and immersed in 1mm of sterile phosphate buffered solution (PBS) pll 7.4 and incubated at 37°C. At predetermined time intervals the PBS was removed and replaced with fresh PBS. The collected aliquots were centrifuged at lOOOg for 20 minutes and then frozen until required for analysis. The concentration of BSA in each sample was determined using a commercially available ELISA kit (USCN Life Sciences Inc.) using manufacturer's instructions available via the following link. http://v\ waiscm^ om/uscn/ELISA-Kit-for-Bovme-Serum- Albumin-Rudimental- BSA-294.htm

The results are shown in Figure 3. Example 5: BMP-4 and VEGF-A ELISA - release rate measurement

200 mg composite samples were prepared as in Example 1 above. The samples were weighed and placed into a mould with a suitable shape for UV irradiation for 40 minutes. Midway through the reaction the samples were removed from the mould and turned so that both sides were exposed to the same level of irradiation.

The samples were combined with 2μg of BMP-4 or 500ng of VEGF-A or a combination of both. As previously described the samples were placed into a sterile 24 well plate and immersed in 1mm of sterile phosphate buffered solution (PBS) pH 7.4 and incubated at 37°C. At predetermined time intervals the PBS was removed and replaced with fresh PBS. The collected aliquots were centrifuged at lOOOg for 20 minutes and then frozen until required for analysis. The concentration of BSA in each sample was determined using a commercially available ELISA ldt (USCN Life Sciences Inc.) using manufacturer's instructions linked below:

http://www.uscnk.corn/uscn/ELISA-Kit-for-Human-Bone-Morph ogenetic-Protein-4- BMP4-24.htm

http://v w■uscrlk■com uscrl ELISA-Kit-for-Human-Vascular-Endothe

Growth-Factor- A- VEGF-A-2010.htm The results are shown in Tables 1 to 3 below and in Figures 4 to 6. In Tables 1 to 3, the term "pore 1" means that polyethylene glycol (200) dimethacrylate was added to crosslink the structure, and the term "pore 2" means that polyethylene glycol (600) dimethacrylate was added to crosslink the structure. Therefore the pore size in the scaffold is larger in the pore 2 samples.

Table 1 states the release rate of BMP4 (in ng) as a function of time, these results also being shown in Figure 4. The total BMP-4 content of the samples was 2μg. Time (hours) 2 4 24 72 168 240

Chitosan and BMP4 0.30 0.43 0.68 0.78 1.02 1.15

Chitosan, HAp and BMP4 (pore 1) 0.00 0.00 0.07 0.17 0.19 0.38

Chitosan, HAp and BMP4 (pore 2) 0.00 0.03 0.06 0.06 0.27 0.43

Table 1

Table 2 states the release rate of BMP4 (in ng) as a function of time; these results also being shown in Figure 5. The total BMP-4 content of the samples was 2μg and the total VEGF-A content 500 ng.

Table 3 states the release rate of VEGF-A (in pg) as a function of time; these results also being shown in Figure 6. The total BMP-4 content of the samples was 2μg and the total VEGF-A content 500 ng.

Table 3 Example 6 - Cell Viability Test

Cytocompatibility testing was conducted using an elution assays with Mc3t3-El cells. Prior to testing the constructs were placed in cell culture media for 24 hours at which timepoint the supernatant was collected and placed directly onto cells which had been precultured for 24 hours. Following 24 hours incubation with the scaffold supernatant the media was removed and cell titre blue was added to the media and the colorimetric response of the media was assessed using a plate reader in comparison to cells which had been cultured in fresh media. The results presented in Figure 7 as a percentage viability in comparison to cells cultured on cell culture plastic as a control The ratios shown in Figure 7 are HAp to chitosan.

Example 8: zone of inhibition

Agar zone of inhibition tests were conducted to confirm the bioactivity of the antibiotic, ciprofloxacin incorporated into the scaffolds. The in vitro tests were performed on growing cultures of Staphylococcus aureus ATCC 25923 (AIT micro- bank) distributed on nutrient agar plates (Sigma Aldrich - Steinheim, Germany). Tests were conducted in accordance with The American Society for Microbiology (ASM) guidelines, for the Kirby-Bauer disk diffusion susceptibility test method for antimicrobial susceptibility testing was used and adapted where required for this experiment (ASM 2009a). This test was conducted aseptically as follows; molten nutrient agar which had been previously sterilised by autoclaving at 121°C for 15 minutes was poured into sterile petri dishes. The plates were left to set before inverting and were left to dry overnight to prevent condensation arising which may alter the result of the test. Once dry, each plate was streaked with Staphylococcus aureus in a zig-zag motion. This procedure was repeated an additional two times turning the plate by 90° each time to ensure an even lawn of bacteria inoculated the agar plate. Each of the drug-loaded discs and control discs were placed dry onto the middle of the agar surface; specimens were gently pressed so they were in intimate contact with the agar. Plates were incubated at 37°C for approximately 18hrs. After incubation, the inhibition zones on the plates were assessed as efficacy of the released antibacterial drugs and compared with that of the blank samples without ciprofloxacin. The diameter (mm) of the inhibition zone was determined by measuring the zone present on the agar with a ruler and the agar plates inverted. Tests were carried out in triplicate and mean values were recorded. Pictures of the agar samples were taken on completion of the test to illustrate findings, and are shown in Figure 8. Example 9: In vivo animal experiment

These tests were performed in accordance with the procedure based on that described in Betz et al,. J Bone Joint Surg Am. 2006;88:355-365.

The results are described in Example 15 below. Example 10: Analysis of cross-linking by FTIR

Attenuated total reflectance Fourier transform infrared spectroscopy was carried out on a Perkin Elmer Spectrum One fitted with a universal ATR sampling accessory. Tests were ran in the spectral range of 4000 - 650cm ^"1 , utilising a 4 scan per sample cycle and a fixed universal compression force of 80 N at ambient temperature. FTIR testing was conducted to help to determine if the composite samples analysed were chemically cross-linked after exposure to UV light.

According to Oliveira et al. Biomaterials 27 (2006) 6123-6137, amide (NH ₂ ) groups appear in the region of 1586 cm ^"1 . This is a typical band present in chitosan which can be seen on the non-UV cured spectrum at 1556 cm ^"1 , (see Figure 9). As can be seen from the lower line, this peak is not visible once the sample has been UV cured, the removal of this band from the IR spectra of chitosan indicates the breaking of the amide bond. The peak at 1416 cm ^"1 on the non- UV cured sample can be attributed to the carboxylic group found in PEGDMA (as described in Zhang et al. International Journal of Biological Macromolecules, 47 (2010) 546-550). The reduction in this peak indicates that the C=0 bond has broken during UV curing. The reduction or removal of these peaks during the UV crosshnliing procedure is evidence that these peaks were involved in the crosslinking reaction.

Example 11: UV crosslinking of polvvinylpyrrolidinone-based scaffolds with acrylic acid

Polyvinylpyrrolidinone (PVP; Sigma- Aldrich PVP k30 molecular weight circa 55,000, catalogue number 85656-8) was crosslinked via a UV initiated free radical polymerisation technique. PVP (200mg) was placed into a beakers to which varying volumes of distilled water was added (range ΙΟΟμΙ to 600μ1). To this solution ΙΟΟμΙ of benzophenone stock solution (BP sol; stock solution in ethanol] consisted of lOmg benzophenone per ml ethanol i.e. a 1% w/v solution) and 25μ1 acrylic acid as a crosslinking agent. Solutions were stirred using a magnetic stirrer until a homogenous solution was obtained. Subsequently, 200μ1 or 200mg samples were placed into silicone moulds and the solutions/mixtures were cured for 40 minutes under a UV curing system (Dr. GroObel UV-Electronik GmbH), turning the samples halfway through curing. The irradiation chamber utilised was a controlled radiation source with 20 UV tubes that provide a spectral range between 315 and 400nm at an average intensity of 10-13.5niW/cm ² . Following curing samples were dried overnight in a fumehood. Once dry samples were placed into a petri dish containing distilled water to determine if crosslinking had occurred rendering the sample insoluble.

It was observed that samples prepared with 100 and 600μ1 of distilled water readily dissolved whereas samples containing 200 and 400 μΐ of water retained their shape indicating that they had been successfully crosslinked. Example 12 - Optimisation of PVP crosslinking

PVP crosslinking was further optimised by varying the volume and concentration of the crosslinking agents polyethylene glycol dimethacrylate where the PEG moiety has a molecular weight of 600 (PEG600DMA) ], acrylic acid and the photo initiator (benzophenone solution in ethanol) as outlined in Tables 4 and 5. Samples were cured as outlined in Example 11 above for 40 minutes.

Table 4

Table 5 The following observations (n=3 per sample) were noted:

Samples 1 and 4 showed an increase in viscosity but was still liquid after curing. Samples 3 and 5 were partially solid and sample 2 had the best mechanical properties as assessed by handling. During swelling experiments in distilled water samples 1 and 4 dissolved within 1 hour. At 2 hours, sample 3 was mostly dissolved and sample 5 had broken into numerous small pieces. Sample 2 continued to swell up to 4 hours where it had reached equilibrium. Although these samples had started to break they still had good shape retention. For sample 2 it was found that the percentage swelling was 354%, the equilibrium water content (EWC) was 72% and the gel fraction was 35%.

When acrylic acid was used in place of PEG600DMA, samples 6 and 8 dissolved within 24 hours. However, samples 7, 9 and 10 retained their shape indicating successful crosslinking had taken place. However, the swelling percentage for these samples was between 80 and 98% indicating that the hydrogels had lost weight.

Nevertheless the gel fraction of these samples ranged from 38.5% to 42.7%. Due to the recorded weight loss the EWC resulted in negative values.

Example 13 - comparison of hydroxyapatite and beta-tricalcium phosphate composite scaffolds

Samples were UV crosslinked using various combinations of Hydroxyapatite (HAp) (synthesised using methods outlined by Hoctor et al, Australian Journal of Basic and Applied Sciences, 2013, 7(5): 140-149), beta-tricalcium phosphate (β-TCP; supplied by the Institute of Ceramics and Glass, Madrid), chitosan (Argos organics molecular weight 100,000-300,000, CAS 349055000) and polyvinylpyrrolidone as outlined in Example 11 and 12 above. These materials were combined with polyethylene glycol 600 dimethacrylate (PEG600DMA) and 0.1% (w/v) benzophenone solution in ethanol. The ceramics and polymers were firstly dry mixed, next the acetic acid (AA) solution was added and the samples were thoroughly mixed with a spatula.

Subsequently, PEG600DMA and benzophenone (1% w/v solution in ethanol) were added separately with thorough mixing following the addition of each new component. 200mg samples were then placed into a silicone mould and curing was performed for 40 minutes as described previously. Samples were neutralised in 0.1 mol solution of sodium bicarbonate for 10 minutes to neutralise the acetic acid and prevent dissolution. Samples were subsequently placed into pH 7.4 phosphate buffered solution and allowed to swell. Following swelling, the samples were dried and placed into a 1% aqueous acetic acid solution to dissolve uncrosslinked portions of the scaffold and determine the gel fraction of the scaffold.

Table 6 summarises the various combinations of HAp, β-TCP chitosan and PVP which were combined to produce scaffolds. Table 7 summarises the swelling properties of these scaffolds.

Table 6

The following observations were noted: Samples (n=3 for each group) appeared to reach equilibrium at 48 hours. Higher EWC's were observed for composites with lower concentrations of ceramic. This is expected as it is the polymer phase that absorbs water. There was little difference for the EWC for equivalent samples containing HAp and β-TCP. However, the addition of PVP increased the EWC of the scaffolds.

When the samples were placed into the acetic acid solution, samples containing HAp did not completely dissolve and had gel fractions ranging between 62 and 90%.

However, samples containing higher concentrations of β-TCP did dissolve completely. However, the sample which contained chitosan and β-TCP at a ratio of 4:1 had a gel fraction of 48%. Similarly, the addition of PVP reduced the gel fraction of scaffolds containing HAp to 11.5% and again the sample containing β-TCP completely dissolved.

Example 14 - Alternative photoinitiators

As an alternative to benzophenone dissolved in ethanol (as in the previous examples), a water soluble Type II photoinitiator may be used. In this example, anthraquinone-2- sulfonic acid sodium salt monohydrate (Sigma Aldrich) was used in place of benzophenone solution.

A stock solution of anthraquinone-2-sulfonic acid sodium salt monohydrate was prepared by placing 67mg of anthraquinone-2-sulfonic acid sodium salt monohydrate into 10ml of distilled water at 30°C under stirring until the initiator had dissolved. Once samples containing anthraquinone-2-sulfonic acid sodium salt monohydrate were UV cured they did not dissolve in a 1% acetic acid solution indicating that they had crosslinked.

Based on the above findings, the stability of the scaffold can be tailored by varying the combination of components in the scaffold. This will allow better water uptake, better in vivo degradation and enable the release of higher levels of growth factor.

Example 15 - In vivo results

A rodent critical sized femoral defect was selected to assess the in vivo efficacy of the scaffold outlined in Table 8. In this model a custom made high density polyethylene (HDPE) plate is fixed in position using 4 threaded k-wires and a 5mm defect is created using a 0.22mm Gligi saw. For extra support and to prevent pin loosening, a resorbable suture was placed around the bone plate construct. Additionally, a 5-0 suture was placed through the scaffold to retain it in the defect.

In vivo characterisation of healing was performed using radiographical analysis and after 8 weeks in vivo, animals were euthanized and ex vivo analysis in the form of uCT was performed.

Table 8

* 30 samples weighing 50mg were prepared from the above combination of materials. The approximate dimensions of the scaffolds were 3x3x5mm. Each sample contained ~ 0^g BMP4 and ~ 0.25μ _§ VEGFa. The following observations were noted:

Radiographical analysis of healing indicated that the scaffold remained in the defect after 8 weeks. Therefore, the ability to alter the dissolution of the scaffold as outlined above is very important to future developments.

Radiographical analysis of bone healing indicate that the scaffold alone, as predicted, does not induce bone formation and minimal bone formation was observed after 8 weeks in vivo. However, the incorporation of growth factors appeared to enhance bone formation, albeit mainly outside the defect as the scaffolds failure to resorb prevented bone formation in the defect. Ex vivo micro computed tomography ^CT) data for bone volume is presented in Figure 10. From statistical analysis of the data using a one way ANOVA (SPSS for windows, IBM) it was found that a significant difference occurred between groups (p — 0.019). Using a LSD post hoc test it was found that the scaffolds containing VEGF- a and BMP-4 & VEGF-a had significantly more bone in the defect than the scaffold alone (p < 0.019 for both comparisons). It was also found that the BMP4 & VEGFa had significantly more bone formation that the scaffold containing BMP4 (p = 0.044). No further significant differences were detected (p > 0.128).

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.