WO2022195280A1 - Bone repair kit - Google Patents

Bone repair kit Download PDF

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Publication number
WO2022195280A1
WO2022195280A1 PCT/GB2022/050670 GB2022050670W WO2022195280A1 WO 2022195280 A1 WO2022195280 A1 WO 2022195280A1 GB 2022050670 W GB2022050670 W GB 2022050670W WO 2022195280 A1 WO2022195280 A1 WO 2022195280A1
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WIPO (PCT)
Prior art keywords
composition
bone
component
iron
chitosan
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PCT/GB2022/050670
Other languages
French (fr)
Inventor
Animesh Jha
Panagiotis GIANNOUDIS
Elena Anatolyevna JONES
Neelam IQBAL
Antonios ANASTASIOU
Dina ABDULAZIZ
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University Of Leeds
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Publication of WO2022195280A1 publication Critical patent/WO2022195280A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/0047—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L24/0073—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix
    • A61L24/0084—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix containing fillers of phosphorus-containing inorganic compounds, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/0047—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L24/0073—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix
    • A61L24/0089—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix containing inorganic fillers not covered by groups A61L24/0078 or A61L24/0084
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • This invention relates to a composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron-doped calcium phosphate mineral component; and (ii) a collagen component.
  • This invention also relates to a freeze-dried composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron-doped calcium phosphate mineral component; and (ii) a collagen component that has been subjected to one or more processing steps to remove solvents.
  • This invention also relates to a bone scaffold in which a first layer comprises a porous titanium/TiO2-based composition and a second layer comprises the freeze dried composition of the invention.
  • Bone is a multiphasic tissue, in which hydroxyapatite (HAp) [Ca5(PO4)3(OH)] is the dominant inorganic phase, with collagen, blood vessels and bone marrow forming the soft material in the matrix [Toroian, D., J.E. Lim, and P.A. Price, The size exclusion characteristics of type I collagen implications for the role of noncollagenous bone constituents in mineralization. Journal of Biological Chemistry.
  • Hp hydroxyapatite
  • the hard HAp mineral is combined with collagen fibres to form the strong, lightweight structure that supports the body weight and ambulation. Collagen is essential for the bio- and electro-mechanical actuation which is necessary for regular osteoconduction.
  • the damaged bone When the damaged bone is treated surgically, it may not heal completely, and this leads to the development of delayed union or non-union. Infection at the site of injury can also delay or stop healing. In case of fragility related fracture (e.g. osteoporotic bone), the risk of non union and infection may increase due to ageing.
  • fragility related fracture e.g. osteoporotic bone
  • a load-bearing long bone (e.g. femur and tibia) following a fracture must be stabilised to be given the best chance for repair and healing.
  • Intramedullary nails (IMN) inserted within the medullary cavity of the bone and plates applied extramedullary (lateral aspect of the bone) are the most common metal devices used to stabilise the affected long bone. The surgical application of these devices induces some damage to the blood supply of the bone compromising the so call ‘biological component’ of fracture healing. Extensive periosteal stripping or vascular damage can lead to aseptic bone death and development of fracture non-union.
  • the implant used to stabilise the bone can withstand loading until the fracture unites; (on average approximately 1 ,000,000 loading cycles) and then its function of maintaining stability becomes obsolete)
  • the implant will start loosening and break/fail. This will lead to revision surgery.
  • revision surgery after the implant is extracted and the dead tissues are debrided, a larger void to fill may be present in the bone requiring some form of bone grafting.
  • infection may have been established where was the previous fracture, which can lead to failure of healing, revision surgery, bone debridement and subsequent need for bone grafting.
  • the bone tissue is intrinsically piezoelectric and it responds to material and charge transport when loaded. This also means that the osteoconduction in bone is dependent on the biophysical piezoelectric properties [Anderson JC and Eriksson, C., Piezoelectric Properties of Dry and Wet Bone, Nature volume 227, pages491-492(1970) [also see reference therein by E Fukada in J. Phys. Soc. Jap., 12, 1158 (1957)] of multiphasic material, in which the collagens, HAp and proteins all contribute to the overall biophysical piezoelectricity in a complex manner, which is not linearly dependent on weight or volume fractions of the constituents.
  • a load bearing bone for example, has a cortex which is denser than the inner trabecular or cancellous layer. Both the cortex and trabecular layers are made up of HAp. Since the cortex is more dense than cancellous bone, a smaller number of blood vessels permeate through the cortex than that through the trabecular bone. More soft matter (collagen and proteins) is also present in the trabeculae than in the cortex, which is essential for the bodily function of bone.
  • the centre of the bone is called the medullary canal, and is full of cellular elements (progenitor stem cells), inductive molecules and other autocoids, all contributing to the underlying repair mechanisms, which control daily bodily need of blood formation and bone mineralisation and resorption through complex signal processing, in which intrinsic piezoelectricity controlling the ion/mass transport play important role.
  • progenitor stem cells progenitor stem cells
  • inductive molecules inductive molecules
  • autocoids autocoids
  • a bone adhesive e.g. a bone glue
  • a filler or a scaffold e.g. a filler or a scaffold
  • biomaterials e.g. a bone adhesive (bone glue) or a filler or a scaffold
  • a biomaterial to promote bone regeneration must possess inductive attributes (which may be added by the patient’s own platelet rich plasma or exogenously using e.g.
  • Bone Morphogenic Protein -2 (BMP-2), or other commercially available growth factors), conductive and osteogenic properties and an element of ‘being contained’ where is placed amongst others so that it can act as a bridge to support neo-angiogenesis and mechano-transduction.
  • BMP-2 Bone Morphogenic Protein -2
  • a material having therefore some adhesive properties (acting as a glue) would address this important necessity.
  • the present invention provides a composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron- doped calcium phosphate mineral component; and (ii) a collagen component.
  • the present invention provides a freeze-dried composition comprising the composition of the first aspect that has been subjected to one or more processing steps to remove solvents.
  • the present invention provides a bone scaffold comprising: (i) a first layer comprising a porous titanium/TiO 2 -based composition; and (ii) a second layer comprising a composition of the second aspect.
  • the present invention provides the composition of the first aspect or the bone scaffold of the third aspect for use in surgical bone repair.
  • the present invention provides the composition of the first aspect or the bone scaffold of the third aspect for use as a prophylactic to prevent the risk of bone failure.
  • the present invention provides the composition of the first aspect or the bone scaffold of the third aspect for use in the treatment of a condition selected from: bone fractures (diaphyseal, metaphyseal and with intra-articular extension), craniofacial fracture, bone gaps, critical size bone defects, bone voids, comminuted fractures, segmental fractures, open fractures, fragility/osteoporotic fractures, fracture non- union, avascular necrosis, bone infection, diabetic foot, and bone failure.
  • a condition selected from: bone fractures (diaphyseal, metaphyseal and with intra-articular extension), craniofacial fracture, bone gaps, critical size bone defects, bone voids, comminuted fractures, segmental fractures, open fractures, fragility/osteoporotic fractures, fracture non- union, avascular necrosis, bone infection, diabetic foot, and bone failure.
  • the present invention provides a use of a graphene, graphene oxide, reduced graphen
  • Figure 1 shows two pieces of bone substitute (Orthoss) glued together with a glue of the present invention.
  • the arrow shows the area where the glue was applied.
  • Figure 2 shows the effect of sterilisation of the glue under UV for 20 minutes (left column images), under UV for 60 minutes (middle column images) and dipped in 70% ethanol (right column images). As can be seen in Figure 2, no bacterial cultures are observed over the time period tested.
  • Figure 3 shows microscope of pictures of a direct toxicity assay observing the effect of the glue of the invention on human mesenchymal stem cells (MSCs).
  • Figure 4 shows a plot of the absorbance measured four hours after the addition of XTT agent against the initial number of cells placed within a reaction well.
  • the conditions tested were a positive control, a negative control, Orthoss only, example Glue A with Orthoss, and example Glue B.
  • Figure 5 shows the average value of absorbance against time for each of the positive control, negative control, Orthoss only, example Glue A with Orthoss and example Glue B with Orthoss.
  • Figure 6 shows microscopic pictures, at 4x magnification, of the stained samples using methylene blue in the wells seeded with 1000 cells.
  • A Positive control.
  • B Extract from Orthoss only.
  • C Extract from Orthoss with example Glue A;
  • D Extract from Orthoss with Glue B.
  • Figure 7 shows a graphical representation of the scaffolds of the invention.
  • Figure 8 shows an image of an annular TiO 2 or Ti-alloy scaffold (diameter of 20 mm) and chitosan scaffold fabricated through freeze drying.
  • Figure 9 shows a freeze-dried cortical-cancellous scaffold used in human mesenchymal stem cell studies.
  • Figure 10 shows the visual appearance of the cortical-cancellous scaffolds.
  • Figure 11 shows XTT absorbance results of mesenchymal stem cells cultured in MACS (positive control), conditioned media/extract form titanium (TIT) scaffold and titanium with iron scaffold (TIT+Fe) compared to DMEM media (negative control).
  • Figure 12 shows samples of bone marrow collected (a) prior to loading the scaffolds, (b) after loading onto a titanium-Fe scaffold, and (c) after loading onto a titanium scaffold. The culture plates were maintained in a MACS medium to support MSC colony formation and were stained with methylene blue to visualise the colonies.
  • Figure 13 shows flow cytometry results for bone marrow MSCs released from a loaded titanium scaffold (left) and a loaded titanium-Fe scaffold.
  • Figure 14 shows Glue A of Example 1 was used to join pieces of bovine jaw bones together.
  • Figure 15 shows the glued surface having a line of bonding.
  • Figure 16(A) shows a comminuted femoral fracture with reds arrows showing the bone gaps.
  • Figure 16(B) shows femoral gaps have been filled with bone glue.
  • Figure 16(C) shows segmental femoral fracture where the bone glue may be used in red high-lighted areas where the gaps are shown even after inserting and stabilising the top and bottom halves of the femur with an intramedullary nail (rod). The gaps are left (see red arrows) which cannot be stabilized easily with soft putty.
  • a glue which hardens stabilizes the bone and provides the biomechanic continuum.
  • Figure 17 Left image shows a femoral shaft fracture after high energy trauma (side, 29 years old man); central image shows the gap in a simulation; and right image shows when a porous Ti-alloy scaffold will be used to match with the biaxial and torsional biomechanical loading of the femur, whilst the bone has been stabilized with an intramedullary nail.
  • Figure 18 Compares the FTIR spectroscopic data for 3 different glue samples from Example 11 with chitosan. Note that the chitosan structure dominates in all three compositions of glue. The order of the FTIR plots is the same as the order in the key in Figure 18 (top to bottom).
  • Figure 19 viscosity of each of Compositions 1 to 3 (glue) and Compositions 1 to 3 (putty) from Example 11.
  • Figure 20 (a) A comparison of synthesised cerium oxide nanoparticles calcined at 280 °C, 385 °C, and 815 °C, analysed using a Vertex 70 FTIR spectra from 400 to 4000 cm ⁇ 1 . The operating parameters consisted of a total of 32 scans at a resolution of 4 cm -1 and (b) UV–Vis absorbance spectra obtained from nanoparticle concentrations of 0.5 mg/ml. The inset Tauc Plot corresponds to the bandgap energies of synthesised cerium oxide nanoparticles.
  • UV-Vis plots The order of the UV-Vis plots is the same as the order in the key in Figure 20 (top to bottom).
  • Figure 21 UV-Vis absorbance spectra obtained from cerium oxide nanoparticles concentrations of 0.5 mg/ml and corresponding Tauc Plot.
  • Figure 22 a) Comparison of the viscosities of glues (Glue 1, Glue 2, Glue 3 of Example 15) in water; b) comparison of the viscosities of two different chitosan mixtures in water.
  • Figure 23 Osteoblast G292 cells at a cell density of 10 4 cells/well were tested against glue composition 1, 2 and 3. Giemsa staining was utilised to stain the cells for enhancing.
  • Figure 24 Extract cytotoxicity and proliferation testing using osteoblast cell line G292 cells on glue 1, 2 and 3 samples. Positive and negative controls consisted of McCoy's media and McCoy's media with 10% DMSO.
  • A optical density extract cytotoxicity results (left) and cell viability percentage live results (right) measured as the percentage of extracts collected after 1, 3 and 7 days.
  • Figure 25a and 25b data for the E. Coli and S.
  • FIG. 26a and 26b optical density measurements characterising the antibacterial properties of the cerium oxide (CeO 2 ) nanoparticles after direct incubation with Escherichia coli and Staphylococcus epidermidis for 48 hours. The reference point is the cultured bacteria (CB).
  • CB cultured bacteria
  • graphene oxide refers to chemically modified single-layer graphene having a high oxygen content, typically characterised as having a carbon:oxygen atomic ratio of from about 6:1 to about 1:1, depending on the method of synthesis.
  • Graphene oxide is typically prepared by oxidation and exfoliation of graphite, causing extensive oxidative modification of the basal plane.
  • reduced graphene oxide refers to a reduced oxygen content form of graphene oxide, typically characterised as having a carbon:oxygen atomic ratio of from about 20:1 to about 6:1.
  • Reduced graphene oxide is produced by chemical, thermal, microwave, photo-chemical, photo-thermal or microbial/bacterial reduction of graphene oxide or by exfoliating reduced graphite oxide.
  • nanocarbon refers to carbon-based materials having at least one external dimension in the nanoscale, i.e. from about 1 nm to about 100 nm.
  • the term ‘nanoparticle’ refers to particles having at least one external dimension in the nanoscale, i.e. from about 1 nm to about 500 nm.
  • Compositions [0033] According to a first aspect, the present invention provides a composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron- doped calcium phosphate mineral component; and (ii) a collagen component.
  • the iron-doped calcium phosphate mineral component is present in an amount of from about 1% w/w to about 30% w/w of the chitosan gel.
  • the iron-doped calcium phosphate mineral component is present in an amount of from about 5% w/w to about 30% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 10% w/w to about 30% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 15% w/w to about 30% w/w of the chitosan gel. [0035] In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 1% w/w to about 25% w/w of the chitosan gel.
  • the iron-doped calcium phosphate mineral component is present in an amount of from about 5% w/w to about 25% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 10% w/w to about 25% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 15% w/w to about 25% w/w of the chitosan gel. [0036] In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 20% w/w to about 25% w/w of the chitosan gel.
  • the iron-doped calcium phosphate mineral component is present in an amount of from about 15% w/w to about 20% w/w of the chitosan gel.
  • the iron-doped calcium phosphate mineral component may be present in an amount of about 20% w/w of the chitosan gel.
  • the iron-doped calcium phosphate mineral component may be present in an amount of about 25% w/w of the chitosan gel.
  • the iron-doped calcium phosphate mineral component is considered to facilitate oxygen regulation and/or neo-angiogenesis.
  • the iron-doped calcium phosphate mineral component is an iron- doped component selected from: hydroxyapatite (Ca10(PO4)6(OH)2), fluorapatite (Ca5(PO4)3F), brushite (CaHPO4.2H2O), ⁇ -pyrophosphate, monetite (CaHPO4) and tricalcium phosphate (Ca3(PO4)2).
  • the iron-doped calcium phosphate mineral component is fluoride free, e.g.
  • the iron-doped calcium phosphate mineral component may be selected from hydroxyapatite (Ca10(PO4)6(OH)2), brushite (CaHPO4.2H2O), monetite (CaHPO4) and tricalcium phosphate (Ca3(PO4)2).
  • the calcium phosphate mineral component is selected from the group consisting of: brushite (CaHPO 4 .2H 2 O), ⁇ -pyrophosphate, monetite (CaHPO 4 ) and tricalcium phosphate (Ca3(PO4)2). This is particularly advantageous when it is desirable to accelerate new bone formation (neo osteogenesis).
  • the calcium phosphate mineral component is fluorapatite (Ca 5 (PO 4 ) 3 F). This is particularly advantageous when the end use will be for jaw bone treatment and for dental implants.
  • the calcium phosphate mineral component is hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ).
  • an iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 30% w/w of the iron-doped calcium phosphate mineral component. In embodiments, the iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 25% w/w of the iron-doped calcium phosphate mineral component.
  • the iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 20% w/w of the iron-doped calcium phosphate mineral component. In embodiments, the iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 15% w/w of the iron-doped calcium phosphate mineral component. In embodiments, the iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 10% w/w of the iron-doped calcium phosphate mineral component. [0043] The amount of iron ions present in the calcium phosphate mineral component can be adjusted according to the application.
  • iron ions are incorporated into the calcium phosphate mineral component during the preparation of the calcium phosphate mineral component.
  • the calcium phosphate mineral component can be prepared by combining a calcium salt containing solution with a phosphate salt containing solution.
  • the calcium phosphate mineral component may be prepared from a calcium nitrate solution and an ammonium phosphate solution.
  • An iron salt can, e.g. Fe(NO3)3.3H2O
  • Fe(NO3)3.3H2O may be added to the calcium salt containing solution (e.g. calcium nitrate solution) prior to combination with the phosphate salt containing solution (e.g. ammonium phosphate solution).
  • the calcium phosphate mineral component can be prepared by combining a calcium salt containing solution with a phosphate salt containing solution and a fluoride salt containing solution.
  • An iron salt can, (e.g.
  • Fe(NO 3 ) 3 .3H 2 O) may be added to the calcium salt containing solution (e.g. calcium nitrate solution) prior to combination with the phosphate salt containing solution (e.g. ammonium phosphate solution) and fluoride salt containing solution (e.g. NH 4 F).
  • the collagen component is present in an amount of from about 1% w/w to about 30% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 5% w/w to about 30% w/w of the chitosan gel.
  • the collagen component is present in an amount of from about 10% w/w to about 30% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 15% w/w to about 30% w/w of the chitosan gel. [0046] In an embodiment, the collagen component is present in an amount of from about 1% w/w to about 25% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 5% w/w to about 25% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 10% w/w to about 25% w/w of the chitosan gel.
  • the collagen component is present in an amount of from about 15% w/w to about 25% w/w of the chitosan gel. [0047] In an embodiment, the collagen component is present in an amount of from about 20% w/w to about 25% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 15% w/w to about 20% w/w of the chitosan gel. Preferably, the collagen component may be present in an amount of about 20% w/w of the chitosan gel. [0048] Without meaning to be bound by theory, collagen is made up chains of amino acids organised in the form of a triple helix of elongated fibril (also known as a collagen helix).
  • the collagenous fibrils are considered to be essential for bone formation. Together with the presence of the other components of the composition of the present invention, the collagen aids essential ion transport under load-bearing conditions in bone.
  • the collagen component is Type I or Type III collagen.
  • the collagen component is Type I collagen.
  • the collagen component is Type III collagen.
  • For bone repair Type III collagen helps in promoting angiogenesis.
  • Type I collagen is important for enthesis and connective tissue engineering.
  • Type I collagen is also important for osteogenesis.
  • the composition of the first aspect further comprises a graphene, graphene oxide, reduced graphene oxide or nanocarbon component.
  • the composition of the first aspect further comprises a graphene oxide, reduced graphene oxide or nanocarbon component.
  • Conductivity of the graphene, graphene oxide, reduced graphene oxide or nanocarbon component may vary between 100 and 350 S/m (J. Phys. Chem. C 2008, 112, 20264–20268).
  • the graphene oxide is in the form of a powder.
  • the graphene is in the form of a powder.
  • the graphene oxide has from 10 to 25 layers, and preferably from 15 to 20 layers.
  • the graphene has from 3 to 20 layers, and preferably from 3 to 15 layers.
  • the graphene has 9 ⁇ 6 layers.
  • the graphene has a resistivity of 40-50 ⁇ . In an embodiment, the graphene oxide is from 4 to 10% edge-oxidised. In an embodiment, the graphene oxide is in the form of a powder, the graphene oxide has 15 to 20 layers and the graphene oxide is from 4 to 10% edge-oxidised. In an embodiment, the graphene is in the form of a powder and the graphene has 3 to 20 layers. [0051] Without meaning to be bound by theory, the graphene, graphene oxide, reduced graphene oxide or nano carbon component is thought to enhance the osteo-conducting properties (e.g. by supporting charge transport of ions and electrons) of the resulting product.
  • compositions that do not include the graphene, graphene oxide, reduced graphene oxide or nano carbon component have been found to perform less effectively. Since graphene and GO contains sp 3 - bonded hybridized carbon atoms, graphene and GO can readily capture water vapor from the surrounding external environment, owing to its hydrophilicity. The effect of atmospheric relative humidity (RH) on the electrical and mechanical properties of graphene and GO are beneficial for the practical application of graphene and GO-based medical implants. However, in the presence OH- ions, the electrical conductivity persists, as the electrical property appears to be accentuated in the presence of collagen and chitosan, the latter two also facilitate ion transport.
  • RH relative humidity
  • the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 4% w/w of the chitosan gel.
  • the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 3% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 2.5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 2% w/w of the chitosan gel.
  • the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 1.5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 1% w/w of the chitosan gel. [0053] In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 5% w/w of the chitosan gel.
  • the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 4% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 3% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 2.5% w/w of the chitosan gel.
  • the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 2% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 1.5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 1% w/w of the chitosan gel.
  • the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.2% to 0.9% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.3% to 0.8% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.4% to 0.7% w/w of the chitosan gel.
  • the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of about 0.5% w/w of the chitosan gel.
  • the graphene oxide has a C:O ratio of from 9:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 8:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 7:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 6:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 5:1 to 1:1.
  • the graphene oxide has a C:O ratio of from 4:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 3:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 2:1 to 1:1. [0056] In embodiments, the reduced graphene oxide has a C:O ratio of from 50:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 20:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 15:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 10:1 to 6:1.
  • the reduced graphene oxide has a C:O ratio of from 9:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 8:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 50:1 to 10:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 20:1 to 10:1.
  • the composition of the first aspect further comprises a graphene component (as opposed to a graphene oxide, reduced graphene oxide or nanocarbon component). Graphene oxide or reduced GO are less consistent and are more resistive than graphene.
  • the composition of the invention also includes chitosan.
  • Chitosan Chitosan
  • the inclusion of chitosan (Chi) in the composition of the present invention is thought to increase the binding and adhesive properties of the composition (both adhesion to the surrounding bone and adhesion between the constituents to help the constituent components of the composition to form as a paste which may be then applied along the damaged bone surfaces for adhesion for tissue restoration).
  • Chitosan is a polysaccharide meaning that, during the regenerative process, it can support the cell growth by providing energy. Chitosan also has several biological properties selected from the group consisting of: biodegradability, lack of toxicity, anti-fungal effects, wound healing acceleration and immune system stimulation.
  • the chitosan gel may comprise chitosan in an amount of from 0.5 to 10% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 8% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 6% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 5% w/w of the chitosan gel.
  • the chitosan gel may comprise chitosan in an amount of from 0.5 to 4% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 3% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 3 to 5% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 4 to 5% w/w of the chitosan gel. [0060] In embodiments, the chitosan gel is an aqueous based gel.
  • the chitosan is a high molecular weight chitosan.
  • the composition of the first aspect further comprises a nanoparticle component.
  • the nanoparticle is an inorganic oxide nanoparticle, for example a metal oxide nanoparticle.
  • the nanoparticle component is in the form of a nanopowder, for example an inorganic oxide nanopowder.
  • the nanoparticle is selected from the group consisting of cerium oxide and strontium oxide.
  • the nanoparticle component has a particle size of 1-10,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-5,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-4,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-3,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-2,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-1,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-900 nm (BET).
  • the nanoparticle component has a particle size of 1-800 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-700 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-600 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-500 nm (BET). [0064] In an embodiment, the nanoparticle component has a particle size of 1-500 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-400 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-300 nm (BET).
  • the nanoparticle component has a particle size of 1-200 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-100 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-50 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-40 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-30 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-25 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-20 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-15 nm (BET).
  • the nanoparticle component has a particle size of 1-10 nm (BET).
  • BET nm
  • the nanoparticle component is thought to enhance the antimicrobial properties of the resulting product.
  • Compositions that include the nanoparticle component have been shown to have improved antimicrobial properties.
  • the nanoparticle component is present in the composition in amount of from 0.01% to 10% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 9% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 8% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 7% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 6% w/v of the chitosan gel.
  • the nanoparticle component is present in the composition in amount of from 0.01% to 5% w/v of the chitosan gel. [0067] In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 10% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 9% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 8% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 7% w/v of the chitosan gel.
  • the nanoparticle component is present in the composition in amount of from 0.1% to 6% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 5% w/v of the chitosan gel. [0068] In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 10% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 9% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 8% w/v of the chitosan gel.
  • the nanoparticle component is present in the composition in amount of from 1% to 7% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 6% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 5% w/v of the chitosan gel. [0069] In embodiments, the nanoparticle component is present in the composition in amount of from 1% w/v, 2% w/v, 3% w/v, 4% w/v or 5% w/v of the chitosan gel.
  • the chitosan has a molecular weight of from 10 kilo Daltons to 2220 kilo Daltons. The molecular weight of the chitosan impacts the viscosity and/or rheology of the composition.
  • the composition of the first aspect is in the form of a glue.
  • the glue may have a viscosity of from about 1 Pa-s to about 10 Pa-s.
  • the chitosan gel may comprise chitosan in an amount of from 0.5 to 3% w/w of the chitosan gel.
  • the composition of the first aspect is in the form of a putty.
  • the glue may have a viscosity of from about 10 Pa-s to about 10,000 Pa-s.
  • the chitosan gel may comprise chitosan in an amount of from 3 to 5% w/w of the chitosan gel, preferably from 4 to 5% w/w of the chitosan gel.
  • the rheological property of the composition can be adjusted by varying the overall surface charge (e.g. measured by the zeta potential).
  • the percentage of protonation may be controlled between 100% to less than 1%, depending on the need for the mineral mixture with chitosan for a particular bone repair.
  • the composition of the first aspect is useful in restoring an impaired bone that has failed to repair itself.
  • the composition of the first aspect may be useful in the augmentation of missing bone; and/or in reducing a bone fracture gap; and/or in treating non-union of bones and bone fragments; and/or in bone fracture reduction; and/or in treating open bone fractures; and/or in treating bone infection; and/or in the treatment of fragile/osteoporotic bone; and/or in the treatment of avascular necrosis.
  • the composition of the first aspect has rapid adhesive bonding properties with natural bone.
  • the composition of the first aspect provides a matrix material having one of more of the following properties: antimicrobial properties; and/or osteogenesis enhancing properties; and/or osteo-conduction enhancing properties; and/or osteo- integration enhancing properties; and/or ossification enhancing properties.
  • the composition can be bonded by a process that exposes the composition to heat source and/or a light source.
  • the composition of the first aspect may be formulated to have a viscosity that renders the composition suitable as a bone glue.
  • the bone glue can be used in the joining of multiple fragments together into a shape or form. Further stabilisation using nails or plates may be required.
  • the composition of the first aspect may be formulated to have a viscosity that renders the composition suitable as a putty.
  • the putty can be used in the treatment of bone voids (i.e. small areas where bone is missing, e.g. bone loss less than 1 cm, optionally less than 3 cm), such as bone gaps, non-unions, fracture reduction, diabetic foot and avascular necrosis.
  • bone voids i.e. small areas where bone is missing, e.g. bone loss less than 1 cm, optionally less than 3 cm
  • bone gaps i.e. small areas where bone is missing, e.g. bone loss less than 1 cm, optionally less than 3 cm
  • bone gaps i.e. small areas where bone is missing, e.g. bone loss less than 1 cm, optionally less than 3 cm
  • bone gaps i.e. small areas where bone is missing, e.g. bone loss less than 1 cm, optionally less than 3 cm
  • fracture reduction e.g. bone loss less
  • the first layer is a cortical layer.
  • the second layer is a cancellous layer.
  • the porous titanium-based composition comprises Ti alloy or a mixture of Ti-alloy with TiO2.
  • the porous titanium-based composition further comprises an iron-doped calcium phosphate mineral component.
  • the porous titanium-based composition further comprises Fe 2 O 3 nanoparticles.
  • the second layer comprises a composition of the first aspect that has been subjected to one or more processing steps to remove solvents.
  • the composition of the first aspect is first subjected to a freeze drying process to produce a freeze-dried composition, which is then contacted with the first layer of the bone scaffold.
  • the composition of the first aspect is contacted with the first layer of the bone scaffold to form a bone scaffold precursor material, which is then freeze dried to produce a bone scaffold of the third aspect.
  • the bone scaffold of the third aspect is useful in the treatment of bone defects (e.g. long bone defects, craniomaxilofacial defects, pelvis defects and spine defects).
  • a bone scaffold can be used in the treatment of extensive bone loss (bone defect; >1cm to 15cm, optionally >3cm to 10cm) for facilitating bone continuity and load distribution.
  • Method for preparing a composition comprising: (1) preparing a chitosan gel comprising mixing chitosan flakes with water and/or a mixture of water and acetic acid; (2) preparing an iron-doped calcium phosphate material comprising combining: (a) an iron salt and a calcium salt containing solution; and (b) adding a phosphate salt containing solution to the iron salt / calcium salt containing solution; (3) combining the chitosan gel of step (1), the iron-doped calcium phosphate material of step (2) and collagen.
  • the calcium phosphate mineral component may be prepared from a calcium nitrate solution and an ammonium phosphate solution.
  • the iron salt is Fe(NO 3 ) 3 .3H 2 O.
  • from 0.1 to 1 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution.
  • 0.1 to 0.5 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution.
  • More preferably 0.1 to 0.2 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution.
  • the calcium phosphate mineral component can be prepared by combining a calcium salt containing solution with a phosphate salt containing solution and a fluoride salt containing solution.
  • An iron salt can, (e.g. Fe(NO3)3.3H2O) may be added to the calcium salt containing solution (e.g. calcium nitrate solution) prior to combination with the phosphate salt containing solution (e.g. ammonium phosphate solution) and fluoride salt containing solution (e.g. NH4F).
  • the method further comprises additionally combining a graphene oxide, reduced graphene oxide or nanocarbon component in step (3).
  • the method further comprises additionally combining a nanoparticle component in step (3).
  • Method for preparing a bone scaffold [0099]
  • a method of preparing a bone scaffold of the third aspect comprising: (1) pressing a powder comprising Ti and/or TiO2 and a calcium phosphate mineral component and/or Ti and Fe2O3, and sintering the pressed powder to produce a sintered component; (2) applying to the sintered component of step (1) the composition of the first or third aspect to produce a scaffold precursor component; (3) subjecting the scaffold precursor component to a freeze drying process.
  • step (1) involves pressing the powder at a pressure of >5 ton, e.g.6 ton or 7 ton for a period of time of >15 minutes, e.g.30 minutes or 60 minutes.
  • step (1) involves sintering the pressed powder for > 1 hour, e.g. 2, 3, 4, 5 or 6 hours.
  • step (1) involves sintering the pressed powder at 800 – 1200°C, preferably 900 – 1100°C, more preferably 1000°C.
  • step (3) involves freezing the scaffold precursor component, optionally at ⁇ -50°C, e.g. -60°C, -70°C or -80°C.
  • step (3) involves freezing for >1 hour, e.g.6, 12, 18 or 24 hours. In an embodiment, step (3) involves freeze drying at ⁇ -80°C, e.g. -90°C or -100°C. In an embodiment, step (3) involves freeze drying at about 40 to about 50 milli torr pressure. In an embodiment, step (3) involves freeze drying for >1 hour, e.g.6, 12, 18 or 24 hours.
  • a method of preparing a bone scaffold of the third aspect comprising: (1) pressing a powder comprising Ti and/or TiO 2 and a calcium phosphate mineral component and/or Ti and Fe2O3, and sintering the pressed powder to produce a sintered component; (2) subjecting a composition of the first or third aspect to a freeze drying process to produce a freeze dried composition; (3) applying to the sintered component of step (1) the composition of the first or third aspect, and then contacting the applied composition of the first or third aspect with the freeze dried composition of step (2) to produce a scaffold precursor component; (4) subjecting the scaffold precursor component to a freeze drying process.
  • step (1) involves pressing the powder at a pressure of >5 ton, e.g.6 ton or 7 ton for a period of time of >15 minutes, e.g.30 minutes or 60 minutes.
  • step (1) involves sintering the pressed powder for > 1 hour, e.g. 2, 3, 4, 5 or 6 hours.
  • step (1) involves sintering the pressed powder at 800 – 1200°C, preferably 900 – 1100°C, more preferably 1000°C.
  • step (2) involves freezing the composition of the first or third aspect, optionally at ⁇ -50°C, e.g.
  • step (2) involves freezing for >1 hour, e.g.6, 12, 18 or 24 hours. In an embodiment, step (2) involves freeze drying at ⁇ -80°C, e.g. -90°C or -100°C. In an embodiment, step (2) involves freeze drying at about 40 to about 50 milli torr pressure. In an embodiment, step (2) involves freeze drying for >1 hour, e.g.6, 12, 18 or 24 hours. [00109] In an embodiment, step (4) involves freezing the scaffold precursor component, optionally at ⁇ -50°C, e.g. -60°C, -70°C or -80°C.
  • step (4) involves freezing for >1 hour, e.g.6, 12, 18 or 24 hours. In an embodiment, step (4) involves freeze drying at ⁇ -80°C, e.g. -90°C or -100°C. In an embodiment, step (4) involves freeze drying at about 40 to about 50 milli torr pressure. In an embodiment, step (4) involves freeze drying for >1 hour, e.g.6, 12, 18 or 24 hours.
  • a method of joining bone comprising: applying a glue composition of the first aspect to a first bone fragment; contacting the first bone fragment having the glue composition with a second bone fragment; allowing the glue to join the first and second bone fragments.
  • the allowing step involves irradiating the glue with a source of electromagnetic radiation.
  • the source of electromagnetic radiation is a near-IR radiation source.
  • the source of electromagnetic radiation is a near-IR laser (e.g. a pulsed laser).
  • the allowing step takes less than 10 minutes, optionally less than 5 minutes and further optionally 2 or 3 minutes.
  • a method of bridging a bone defect comprising: applying a glue composition of the first aspect to a first and second bone fragment; positioning a bone scaffold between the first and second bone fragments; allowing the glue to join the first and second bone fragments to the bone scaffold.
  • the allowing step involves irradiating the glue with a source of electromagnetic radiation.
  • the source of electromagnetic radiation is a near-IR radiation source.
  • the source of electromagnetic radiation is a near-IR laser (e.g. a pulsed laser).
  • the specific electromagnetic radiation source used in the examples of this application is generally applicable to the rest of the disclosure and is a near- IR 1040nm pulsed laser with 400 mW average incident power.
  • the allowing step takes less than 10 minutes, optionally less than 5 minutes and further optionally 2 or 3 minutes.
  • the abbreviations used here have their conventional meaning within the chemical and biological arts.
  • Example 1 Glue synthesis: [00123] Materials: Chitosan flakes - (Sigma-Aldrich, CAS: 9012-76-4; 3100000 – 3750000 Da, > 75 % deacetylated) Acetic acid - (Acros Organics, MFCD00036152) Calcium nitrate – (Fisher Chemicals, CAS: 13477-34-4 Ca(NO3)2.4H2O) Diammonium phosphate solution – (Acros Organics, CAS: 7783-28-0, (NH 4 ) 2 HPO 4 ) Sodium hydroxide Iron nitrate – Fe(NO 3 ) 3 .3H 2 O Collagen (Type I) Graphene Oxide [00124] Synthesis of Chito
  • Glue Synthesis For the response of bone marrow stem cells, the following two compositions of glue were formulated for processing: (a) GLUE A: 20% w/w Fe-HA and 20% w/w Col in Chi (3% w/w) Gel and (b) GLUE B: 0.5% w/w Gr, 20% w/w Fe-HA and 20% w/w Col in Chi (3% w/w) Gel.
  • the glue is synthesized by combining chitosan (Chi) gel, iron doped hydroxyapatite (Fe-HA) powder, collagen (Col) powder and graphene (Gr) oxide powder.
  • Cho chitosan
  • Fe-HA iron doped hydroxyapatite
  • Col collagen
  • Gr graphene oxide powder.
  • Glue A we added 0.1g of Fe-HA, 0.1 g of Col and 0.5 g of Chi Gel on a glass plate and mix them until one homogeneous gel is formed.
  • Glue B we added 0.0025g of Gr oxide, 0.1g of Fe-HA, 0.1g of Col and 0.5g of Chi Gel on a glass plate and mix them until one homogeneous gel is formed.
  • Example 2 – Cell Toxicity of Glue Composition [00139] Cell toxicity: The tests were divided into three parts – sterilization and stability, direct and indirect toxicity. [00140] Sterilization and Stability [00141] The glue was used to bond two fragments of Orthoss, mimicking the bonding of bone fragments (Error! Reference source not found.). [00142] Three techniques for sterilization were used; (a) UV for twenty minutes; (b) UV for sixty minutes; (c) Dipped in 70% Ethanol. [00143] All three techniques were successful (Error! Reference source not found.), as the materials remained sterile and stable for at least two months. The culture remained clear, showing no sign of infection from bacteria.
  • the conditions tested for this assay were (a) positive control (only containing StemMACS); (b) negative control (10%DMSO in StemMACS); (c) extract from two separate Orthoss pieces; (d) extract from two Orthoss pieces glued with Glue A; and (e) extract from two Orthoss pieces glued with Glue B. [00150] Once calculating the average values per cell density and time, data were plotted against the cell number placed initially in each well (Error! Reference source not found.), four hours after the addition of XTT agent). [00151] Error! Reference source not found. shows that as expected, the higher the cell density, the stronger the absorbance signal in all conditions, apart from the negative control.
  • Example 3 Demonstration of glue to join bone
  • Glue A of Example 1 was used to join pieces of bovine jaw bones together. A near- IR 1040nm pulsed laser with 400 mW average incident power was used. This is shown in Figure 14. These pieces 1, 2 and 3 can be further joined to augment and reconstruct bone. After putting the two surfaces together, the process takes less than few minutes.
  • Figure 16(A) shows a comminuted femoral fracture with reds arrows showing the bone gaps and 16(B) shows femoral gaps have been filled with bone glue.
  • the comminuted fracture in the femoral shaft consists of several pieces. After the femur is stabilized with an intramedullary nail, the fragments are scattered around the zone of fragmentation.
  • the glue can be implanted (activated with a heat source) in between the fragments to provide a bridge to connect the so ‘called fracture gaps’. This approach will connect all the fragments together allowing uniform distribution of the mechanical forces applied and of the mechanical stimuli generated supporting a timely fracture healing response.
  • FIG. 16(C) shows segmental femoral fracture where the bone glue may be used in red high-lighted areas where the gaps are shown even after inserting an intramedullary nail (rod) for stabilizing the bone between the top and bottom halves.
  • the gaps are left (see red arrows) which cannot be stabilized easily with soft putty.
  • a glue which hardens stabilizes the bone and provides the biomechanic continuum. As the pieces are large, they can be held together by the glue, restoring continuity of the whole bone.
  • Example 4 Putty synthesis: [00158] The same synthetic procedure as described above in Example 1 was followed, except that the chitosan gel was made by the following process: [00159] We added 2 mL of acetic acid in a glass beaker with 98 mL of distilled water and then placed the beaker on top of a hot plate with a magnetic stirrer. Then we added 4-5g of chitosan flakes slowly into the acetic acid solution and leave the solution mixing for 24 hours, for the polymerization to occur. [00160]
  • Example 5 Fabrication of scaffolds [00161] For the fabrication of TiO2 scaffolds, Ti powder (Goodfellas, CAS number: 7440- 32-6) was pressed in pellets.
  • Example 6 Fabrication of TiO 2 /CaP scaffolds: [00163] A similar procedure was followed to that described in Example 4 for the fabrication of TiO 2 /CaP scaffolds. Ti powder was mixed with brushite crystals at different concentrations (i.e. 10, 20 and 40% w/w).
  • Example 7 Fabrication of Oxygenated ⁇ -Ti scaffolds
  • Oxygenated ⁇ -Ti scaffolds were fabricated by sintering Ti powder mixed with Fe2O3 nanoparticles. Specifically, 0.50 g Ti powder was mixed with 0.1 g Fe2O3 (atomic ratio of Ti/O is 100/20) and pressed in pellet (20 mm diameter die and load of 7 ton for 30 min).
  • Example 8 Fabrication of annular scaffolds: [00167] Annular scaffolds were fabricated with TiO2 and oxygenated Ti ( Figures 7 and 8) which will be used for the future animal trials. To achieve this, we designed and constructed a die for pressing powders, in annular shape, under high loads (up to 9 ton).
  • the cortex (Ti-alloy with mineral) and cancellous scaffold (chitosan with mineral) structures were joined together during a freeze-drying process.
  • the Ti-alloy scaffold is first coated with glue/putty (formulation as per Example 1 and Example 3) and then the material placed inside a -80°C fridge for few hours to 24 hrs until a bond is formed. After that the conjoined scaffold was placed inside a freeze drier (e.g. VirTis 4KB ZL Benchtop K (SP Industries)).
  • Freeze drier time is shorter for smaller scaffolds ( ⁇ 10mm in diameter and ⁇ 10 mm in length), by comparison longer period is required for larger size (25mm diameter and 25 mm length) scaffolds.
  • the freeze drier chamber should be maintained at around 40-50 milli torr pressure.
  • the freeze dryer temperature was set at -100 oC, the pressure was set at 43 millitorrs and the freeze drier duration was 24 hours.
  • Example 9 Stem cell attachment and proliferation in novel scaffolds for cortical and cancellous bone applications using chitosan and titanium alloy powder
  • h-MSC human mesenchymal stem cell
  • h-MSC human mesenchymal stem cell
  • Figure 9 Experiments were performed with the combination of chitosan and titanium scaffolds ( Figure 9). These scaffolds were processed in sterile conditions and tested for their effects on human mesenchymal stem cells (MSCs): • Cell toxicity: by testing MSC proliferation in media extracts in which these scaffolds were incubated (indirect cytotoxicity measurements according to ISO10993-5:2009).
  • MSC cell adhesion I immediate attachment: by visualizing if the leftover of bone marrow used to load these scaffolds (for 2 hours) contain MSCs that failed to attach and therefore adhere to plastic culture wells and form colonies.
  • Cell adhesion II longer term colonization during culture: by phenotyping bone marrow cells released from digested scaffolds after being loaded and cultured for 2 weeks. [00175] In Figure 10, the visual appearance of the cortical-cancellous scaffolds are shown. The scaffold with whiter appearance has superficial layer of chitosan and calcium phosphate mineral whereas the darker scaffold has no mineral content.
  • the illustration in Figure 17 shows an AP Radiograph of right femur (A), followed by two simulated images based on the biomechanical simulation for using bone substitute for filling the missing femoral bone (femoral fracture stabilized with an intramedullary nail).
  • the scaffold for replacing the missing bone will have the following features: [00188] Properties of porous Ti-alloy with calcium phopshate minerals (CaP) as osteogenic scaffolds. [00189]
  • the composition of calcium phosphate mineral is 80 weight % CaHPO4 (dried brushite called monetite) and 20 wt%Fe2O3). Instead of CaHPO4, tri or di calcium phosphate and HAp can also be used with porous Ti structures.
  • the scaffold may be loaded also with autologous bone, platelet rich plasma harvested from the patient and/or exogenous growth factors, existing commercially such as the bone morphogenic proteins (e.g. BMP-2). Liposomes as carriers of growth factors may also be used in the scaffold to be implanted to restore the bone segment missing and in the bone glue.
  • BMP-2 bone morphogenic proteins
  • Example 11 Glue and Putty containing cerium oxide synthesis: [00193] Constituents of Materials used for Glue Synthesis ⁇ Graphene (Goodfellow Cambridge CAT: 4447-5224-38, part number: GR006096, bulk density: 0.030 gcm -3 , platelet thickness: 3 + 2nm (9 + 6 layers). ⁇ Cerium (IV) oxide nanopowder (Sigma-Aldrich, product number: 544841, CAS: 1306-38-3, molecular weight: 172.11 g/mol, ⁇ 25nm particle size (BET).
  • Type-1 collagen was mixed as described above.
  • Composition 1 ⁇ 1 (w/v)% graphene (0.0025g) ⁇ 2 (w/v)% cerium (IV) oxide nanoparticles (0.005g) ⁇ 10 mol% iron-doped brushite (0.2425g) ⁇ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen
  • Composition 2 Without graphene ⁇ 2 (w/v)% cerium oxide nanoparticles (0.005g) ⁇ 10 mol% iron-doped brushite (0.2450g) ⁇ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen
  • Composition 3 Without cerium oxide nanoparticles ⁇ 1 (w/v)% graphene (0.0025g) ⁇ 10 mol% iron-doped brushite (0.2475g) ⁇ 3 (w/v)% high molecular weight chitosan (1.0g
  • Type-1 collagen was mixed as described above.
  • Composition 4 ⁇ 5(w/v)% graphene (0.0125g) ⁇ 5(w/v)% cerium (IV) oxide nanoparticles (0.0125g) ⁇ 10 mol% iron-doped brushite (1.125g) ⁇ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen
  • Composition 5 Without graphene ⁇ 5 (w/v)% cerium oxide nanoparticles (0.0125g) ⁇ 10 mol% iron-doped brushite (1.1875g) ⁇ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen
  • Composition 6 Without cerium oxide nanoparticles ⁇ 5 (w/v)% graphene (0.0125g) ⁇ 10 mol% iron-doped brushite (0.2475g) ⁇ 3 (w/v)% high molecular weight chitosan (1.0
  • Putty Composition 1 ⁇ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ⁇ 1 (w/v)% graphene (0.0125g) ⁇ 2 (w/v)% cerium (IV) oxide nanoparticles (0.025g) ⁇ 10 mol% iron-doped brushite (1.2125g)
  • Putty Composition 2 Without graphene ⁇ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ⁇ 2 (w/v)% cerium (IV) oxide nanoparticles (0.025g) ⁇ 10 mol% iron-doped brushite (1.225g)
  • Putty Composition 3 Without cerium oxide nanoparticles ⁇ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ⁇ 1 (w/v)% graphene (0.0125g)
  • Putty Composition 4 ⁇ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ⁇ 5 (w/v)% graphene (0.0625g) ⁇ 5 (w/v)% cerium (IV) oxide nanoparticles (0.0625g) ⁇ 10 mol% iron-doped brushite (1.125g)
  • Putty Composition 5 Without graphene ⁇ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ⁇ 5 (w/v)% cerium (IV) oxide nanoparticles (0.0625g) ⁇ 10 mol% iron-doped brushite (1.1875g)
  • Putty Composition 6 Without cerium oxide nanoparticles ⁇ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ⁇ 5 (w/v)% graphene (0.0625g) ⁇ 5 (w/v)% graphene (0.0625g) ⁇ 10 mol
  • Each sample was scanned 200 times in the 400cm-1 to the 4000cm- 1 range at a spectral resolution was 4cm-1.
  • the beam splitter was KBr, and the light source used was a MIR lamp. See Figure 18.
  • the viscosity of each of Compositions 1 to 3 (glue) and Compositions 1 to 3 (putty) are shown in Figure 19.
  • the zeta potential testing solutions were prepared by diluting the glue/putty compositions to a concentration of 2.9g/dm 3 . The measurements were taken in cell DTS 1070 cuvettes, and the Malvern Zetasizer equipment was utilised to conduct the zeta potentials measurements.
  • the zeta potential data for glue compositions compared with the minerals and cerium oxide are as follows: Sample Zeta Potential (mv) Glue 1 (composition 1) 15.7 Glue 2 (composition 2) 6.3 Glue 3 (composition 3) 43.3 Cerium Oxide Nanoparticles -12.6 10% Fe DCPD -5.4 [00223] The addition of cerium oxide nanoparticles caused the zeta potential to decrease, as observed in glue composition 2 containing no graphene. The zeta potential measures average charge distribution in a dispersed medium, for the glue compositions discussed above. The zeta potential values little meaning when the medium is not dispersed, as in a glue or in a powder form.
  • FIG. 20 shows a comparison of synthesised cerium oxide nanoparticles calcined at 280 °C, 385 °C, and 815 °C, analysed using a Vertex 70 FTIR spectra from 400 to 4000 cm ⁇ 1 . The operating parameters consisted of a total of 32 scans at a resolution of 4 cm -1 .
  • Figure 20 (b) shows the UV–Vis absorbance spectra obtained from nanoparticle concentrations of 0.5 mg/ml.
  • the inset Tauc Plot corresponds to the bandgap energies of synthesised cerium oxide nanoparticles.
  • Example 13 Ultra-Violet Visible Spectroscopy of nanoparticles
  • the PerkinElmer®, LAMDA 950 UV/VIS/NIR Spectrometer was used to characterise clear suspensions of nanoparticles suspended in deionised water. The absorption spectrum data was collected between 190 and 600 nm. The maximum absorbance for cerium oxide nanoparticles is in the 210nm region, as shown in figure 21.
  • the electronic behaviour, oxygen defects, particle size, and the bivalence of the cerium oxide nanoparticles affect the nanoparticles' optical properties.
  • the band energy is related to the presence of different oxidation states (Ce 3+ and Ce 4+ ) on the outer nanoparticles' surfaces, where the Ce3+:Ce4+ ratio is known to be affected by oxygen vacancies and surface defects [00231]
  • the direct optical band gap energies for a range of nano particles synthesised under furnace dried and freeze dried conditions were found to be in the range of 3.4 eV for coarser particles to 5.6 eV, for the finest particles in our investigations.
  • the difference in the bandgap energies is also related to the presence of different oxidation states (Ce 3+ and Ce 4+ ) on the surface of nano particles which then controls the overall charge on surface, as explained in the zeta potential measurements.
  • Example 14 Physical properties of Glue and chitosan materials [00233] Glue samples were tested in a wet environment where the film thickness was 1mm, and the data are shown in the below Table. There was no resistance observed in the dry environment in putty or glue, which means that the graphene was solely responsible high electrical conductivity.
  • Example 15 Contact Cytotoxicity Assay by Giemsa Staining (qualitative)
  • Scaffolds in triplicate were attached to 6 well plates with the aid of steri-strips [Medisave, cat no. R1540C, the negative and positive controls, consisted of steri-stips and 40% dimethyl sulfoxide (DMSO), respectively.
  • DMSO dimethyl sulfoxide
  • DPBS Dulbecco's Phosphate Buffered Saline
  • the culture plates were incubated at 37 ⁇ C for 48 hours in 5 (v/v)% CO 2 in an incubator. After 48 hours, the media was aspirated from the wells and washed twice with DPBS. 1ml of 10 (v/v)% neutral-buffered formalin (NBF) was added to each well and incubated for 15 minutes. The formalin was aspirated and, all wells were stained for 5 minutes using Giemsa solution, then subsequently washed using distilled water. The culture plates were air-dried for 24 hours and the cytotoxic effects were determined via qualitative evaluation.
  • the tissue plates were examined microscopically to record any changes in cell morphology, confluency, membrane integrity, attachment and detachment of the osteoblast cells using an Olympus IX 7 Inverted Microscope under bright field illumination (CellB software; Olympus). All images were collected digitally, as shown in Figure 23. [00237] All glue compositions tested presented no cytotoxic effect. The images show that the osteoblast cells proliferated up to (adjacent) the glue samples. Glue compositions are numbered per Example 11.
  • Example 16 Extract Cytotoxicity by XTT Assay
  • XTT assay is used to quantify the potential cytotoxicity of glue compositions, whereby a decrease in the number of living cells decreases the total activity of mitochondrial dehydrogenases in the glue samples tested. The decrease directly correlates to the amount of orange formazan formed, as monitored by the optical density at 450nm.
  • Glue extracts were prepared according to the ISO standard: ISO 10993-12:2007 Part 12, and the cytotoxicity of the samples was evaluated according to ISO: 10993-5:2009(E) Part 5: Tests for in vitro cytotoxicity.
  • a decrease in the total mitochondrial dehydrogenase activity of living cells was used to quantify the potential cytotoxicity of the synthesised freeze-dried scaffolds.
  • a decrease in mitochondrial dehydrogenase activity is correlated to the amount of orange formazan formed, and this is monitored by characterising the optical density at 450nm.
  • the glue eluates were prepared according to the ISO standard: ISO10993-12:2007 part 12. Briefly, glue samples in triplicate were placed into 24 well plates containing 2ml of supplemented McCoy's 5A media and incubated at 37°C in 5 (v/v)% CO2 for 1, 3, and 7 days.
  • Extract cytotoxicity evaluation of the synthesised freeze-dried scaffolds was assessed according to the ISO10993-5:2009(E) part 5: Tests for in vitro cytotoxicity. Briefly, cell line G292 osteoblast cells were seeded into 96 well plates at a cell density of 10,000 cells/well and incubated at 37°C in 5 (v/v)% CO 2 for 24 hours.
  • the media was aspirated from the wells and replaced with 100 ⁇ l of the thawed collected media containing scaffold eluates.
  • the positive and negative controls consisted of McCoy's 5A media and McCoy's 5A media with 10% DMSO, respectively.
  • the well plates were incubated at 37°C in 5(v/v)% CO 2 for 24 hours. After 24 hours, the media in the wells were removed and replaced with 100 ⁇ l of McCoy's 5A media, 10% FCS and 50 ⁇ l of the XTT assay solution, then incubated for 4 hours at 37°C in 5(v/v)% CO2.
  • Example 17 Antibacterial Testing
  • Bacterial stock cultures of Escherichia coli, Staphylococcus epidermis and Pseudomonas aeruginosa were procured from a stock of 30% glycerol solutions kept at - 80 ⁇ C. 10 ⁇ l sterile loops were used to streak Staphylococcus epidermis, Pseudomonas aeruginosa and Escherichia coli onto BHI agar plates.
  • Inoculated plates were all incubated at 37 ⁇ C for 24 hours, after which a single colony was picked from each bacterium type and grown in 25ml of BHI broth in an incubator at 37 ⁇ C 150rpm for 24 hours.
  • Optical density (OD) measurement is a widely used method to assess the number of growing bacteria in a culture; thus, the absorbance values of bacterial suspensions can be measured using a photometer.
  • the initial optical density of each bacterial type was measured using the Jenway 6305 UV/Visible Spectrophotometer at 600nm (OD600). Triplicate bacterial solutions for each bacterium were produced, and the growth rate with the addition of the glue samples was measured.
  • FRNP refers to freeze dried nano particles which are compared with commercial C385 and C815 powders, which were heat treated at 385 o and 815 o C, respectively.
  • the dose specified is considerably small, and if increased to higher concentrations the effectiveness of antibacterial control will increase.
  • Summary The antibacterial tests for the glue samples with low and high graphene, cerium oxide, chitosan and brushite mixtures have been tested.
  • the data for S Epidermis and E coli show the reduction in bacterial colonies at these modest concentrations of cerium oxide and graphene in a mixture of iron oxide doped brushite and chitosan. Larger ratios of cerium oxide and graphene with brushite seem more effective in antibacterial control in general.

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Abstract

This invention relates to: a composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron-doped calcium phosphate mineral component; and (ii) a collagen component; to a freeze-dried composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron-doped calcium phosphate mineral component; and (ii) a collagen component that has been subjected to one or more processing steps to remove solvents; a bone scaffold in which a first layer comprises a porous titanium/TiO2-based composition and a second layer comprises the freeze dried composition of the invention; and methods of manufacturing these compositions, freeze dried compositions and bone scaffolds.

Description

Bone Repair Kit [0001] This invention relates to a composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron-doped calcium phosphate mineral component; and (ii) a collagen component. [0002] This invention also relates to a freeze-dried composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron-doped calcium phosphate mineral component; and (ii) a collagen component that has been subjected to one or more processing steps to remove solvents. [0003] This invention also relates to a bone scaffold in which a first layer comprises a porous titanium/TiO2-based composition and a second layer comprises the freeze dried composition of the invention. [0004] This invention further relates to methods of manufacturing these compositions, freeze dried compositions and bone scaffolds. [0005] BACKGROUND [0006] Bone is a multiphasic tissue, in which hydroxyapatite (HAp) [Ca5(PO4)3(OH)] is the dominant inorganic phase, with collagen, blood vessels and bone marrow forming the soft material in the matrix [Toroian, D., J.E. Lim, and P.A. Price, The size exclusion characteristics of type I collagen implications for the role of noncollagenous bone constituents in mineralization. Journal of Biological Chemistry. 282 (2007) 22437-22447, https://doi.org/10.1074/jbc.M700591200; LeGeros, R.Z., Properties of osteoconductive biomaterials: calcium phosphates. Clinical Orthopaedics and Related Research; 395 (2002) 81-98, https://journals.lww.com/clinorthop/Fulltext/2002/02000/Properties_of_Osteoconductive_Bi omaterials_.9.aspx]. The hard HAp mineral is combined with collagen fibres to form the strong, lightweight structure that supports the body weight and ambulation. Collagen is essential for the bio- and electro-mechanical actuation which is necessary for regular osteoconduction. Without osteoconduction, the daily replenishment of bone tissue due to wear/tear is not feasible, as is the case when the bone is damaged. Thus, Hap and collagen with bone-morphogenic proteins are also essential for bio-mechanical and electromechanical actuation, necessary for supporting the osteoconductive pathways [Md Minary-Jolandan and Min-Feng Yu, ACS Nano, 2009, 3, 7, 1859–1863, 10.1021/nn900472n]. By comparison, blood vessels and bone marrow provide a physiological environment for maintaining continuous tissue regeneration and biological function. [0007] A bone can be damaged as a result of trauma, fragility, or pathological bone disease (tumors). The loss of bone tissue can seriously compromise the bodily function. When the damaged bone is treated surgically, it may not heal completely, and this leads to the development of delayed union or non-union. Infection at the site of injury can also delay or stop healing. In case of fragility related fracture (e.g. osteoporotic bone), the risk of non union and infection may increase due to ageing.
[0008] A load-bearing long bone (e.g. femur and tibia) following a fracture must be stabilised to be given the best chance for repair and healing. Intramedullary nails (IMN) inserted within the medullary cavity of the bone and plates applied extramedullary (lateral aspect of the bone) are the most common metal devices used to stabilise the affected long bone. The surgical application of these devices induces some damage to the blood supply of the bone compromising the so call ‘biological component’ of fracture healing. Extensive periosteal stripping or vascular damage can lead to aseptic bone death and development of fracture non-union. Moreover, as there is a race between fracture healing and implant failure (the implant used to stabilise the bone can withstand loading until the fracture unites; (on average approximately 1 ,000,000 loading cycles) and then its function of maintaining stability becomes obsolete), if the fracture fails to unite in a timely fashion, the implant will start loosening and break/fail. This will lead to revision surgery. During revision surgery, after the implant is extracted and the dead tissues are debrided, a larger void to fill may be present in the bone requiring some form of bone grafting. Similarly, one also will have to take into account that infection may have been established where was the previous fracture, which can lead to failure of healing, revision surgery, bone debridement and subsequent need for bone grafting.
[0009] The above case scenarios are few common examples of bone void or tissue gap to be filled for restoring and maintaining the bodily function. It should be noted that traumatic bone loss, fracture non-union, infection, and pathological bone conditions can occur in any type of bone, although the load-bearing bones are more prone than non-load bearing ones.
[0010] It is also important to note that the bone tissue is intrinsically piezoelectric and it responds to material and charge transport when loaded. This also means that the osteoconduction in bone is dependent on the biophysical piezoelectric properties [Anderson JC and Eriksson, C., Piezoelectric Properties of Dry and Wet Bone, Nature volume 227, pages491-492(1970) [also see reference therein by E Fukada in J. Phys. Soc. Jap., 12, 1158 (1957)] of multiphasic material, in which the collagens, HAp and proteins all contribute to the overall biophysical piezoelectricity in a complex manner, which is not linearly dependent on weight or volume fractions of the constituents. [0011] A load bearing bone, for example, has a cortex which is denser than the inner trabecular or cancellous layer. Both the cortex and trabecular layers are made up of HAp. Since the cortex is more dense than cancellous bone, a smaller number of blood vessels permeate through the cortex than that through the trabecular bone. More soft matter (collagen and proteins) is also present in the trabeculae than in the cortex, which is essential for the bodily function of bone. The centre of the bone is called the medullary canal, and is full of cellular elements (progenitor stem cells), inductive molecules and other autocoids, all contributing to the underlying repair mechanisms, which control daily bodily need of blood formation and bone mineralisation and resorption through complex signal processing, in which intrinsic piezoelectricity controlling the ion/mass transport play important role. For these reasons, the healing of damaged bone is a complex physiological process and certain conditions must be satisfied for a successful outcome after surgery. [0012] Several aetiological factors have been identified over the years to affect a successful fracture healing response. These can be broadly divided into: injury related (severity of injury matters), patient related (co-morbidities, medication intake, smoking, etc) and surgeon related (type and quality of surgery carried out) [Santolini E, West R, Giannoudis PV. Risk factors for long bone fracture non-union: a stratification approach based on the level of the existing scientific evidence. Injury.2015 Dec;46 Suppl 8: S8-S19]. [0013] However, in general terms the following four factors must be present in order to achieve the restoration of damaged and fractured bone: a) osteogenic and osteo-conducting environment for cells to grow and colonise the matrix (scaffold), b) angiogenesis, c) growth factors for controlling and stimulating bone growth, and d) bio-mechanical stabilization for maintaining local stability, alignment and transduction of mechanical stimuli promoting healing. These four factors are known as the “Diamond Concept” and underpin the standard approach for achieving healing and bone remodelling [Giannoudis, P.V., T.A. Einhorn, and D. Marsh, Fracture healing: the diamond concept. Injury. 38 (2007) S3-S6, https://doi.org/10.1016/S0020-1383(08)70003-2]. [0014] Based on the aforementioned underpinning factors for restoring the damaged bone tissue, the most important challenge in the development of biomaterials, e.g. a bone adhesive (bone glue) or a filler or a scaffold is the lack of combination of characteristic functions for supporting osteo-induction, osteogenesis, osteo-conduction, and biomechanical support for bone fragment stabilisation. Ideally, a biomaterial to promote bone regeneration must possess inductive attributes (which may be added by the patient’s own platelet rich plasma or exogenously using e.g. Bone Morphogenic Protein -2 (BMP-2), or other commercially available growth factors), conductive and osteogenic properties and an element of ‘being contained’ where is placed amongst others so that it can act as a bridge to support neo-angiogenesis and mechano-transduction. A material having therefore some adhesive properties (acting as a glue) would address this important necessity. The lack of optimal biomechanical stress may disrupt the necessary for neo-osteogenesis bridge across the damaged area by exploiting the underpinning property of bone’s piezoelectric induced ion transport, chemotaxis signalling and onset of blood vessel formation [Bosch, G., et al., The effect of platelet-rich plasma on the neovascularization of surgically created equine superficial digital flexor tendon lesions. Scandinavian J Medicine & Science of Sports. 21 (2011) 554-561, https://doi.org/10.1111/j.1600-0838.2009.01070.x]. A mechanically unstable bone leads to compromised healing and non-union [Stewart, S.J.M.o.j., Fracture Non-Union: A Review of Clinical Challenges and Future Research Needs. Malaysian Orthopaedics Journal. 13 (2019) 1, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702984/], demanding further surgical intervention and risk of procrastinated healing [Stewart, S.J.M.o.j., Fracture Non-Union: A Review of Clinical Challenges and Future Research Needs. Malaysian Orthopaedics Journal. 13 (2019) 1, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702984/] due to other co-morbid factors [Aghaloo, T., et al., The Effects of Systemic Diseases and Medications on Implant Osseointegration: A Systematic Review. International Journal of Oral & Maxillofacial Implants. 34 (2019), https://www.ncbi.nlm.nih.gov/pubmed/31116832]. [0015] Infections associated with bacterial colonisation around implants and scaffolds is a significant clinical problem both in orthopaedics and regenerative dentistry. Epidemiological works suggest that between 12% and 43% of the dental implants will develop at some point symptoms of peri-implantitis while in orthopaedics, 2-5% of all the implant related procedures will be complicated by bacterial infections. Although so far, the use of antibiotics is common practice for preventing or treating these conditions, the potential risk of antibiotic resistance is a concern, and the effectiveness of their long-term use is disputable. In order to meet this critical clinical need and overcome the implications of the current treatment strategies, there is increased interest for the development of novel biomaterials with both antimicrobial properties and the potential to trigger bone regeneration. [0016] BRIEF SUMMARY OF THE DISCLOSURE [0017] According to a first aspect, the present invention provides a composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron- doped calcium phosphate mineral component; and (ii) a collagen component. [0018] According to a second aspect, the present invention provides a freeze-dried composition comprising the composition of the first aspect that has been subjected to one or more processing steps to remove solvents. [0019] According to a third aspect, the present invention provides a bone scaffold comprising: (i) a first layer comprising a porous titanium/TiO2-based composition; and (ii) a second layer comprising a composition of the second aspect. [0020] According to a fourth aspect, the present invention provides the composition of the first aspect or the bone scaffold of the third aspect for use in surgical bone repair. [0021] According to a fifth aspect, the present invention provides the composition of the first aspect or the bone scaffold of the third aspect for use as a prophylactic to prevent the risk of bone failure. [0022] According to a sixth aspect, the present invention provides the composition of the first aspect or the bone scaffold of the third aspect for use in the treatment of a condition selected from: bone fractures (diaphyseal, metaphyseal and with intra-articular extension), craniofacial fracture, bone gaps, critical size bone defects, bone voids, comminuted fractures, segmental fractures, open fractures, fragility/osteoporotic fractures, fracture non- union, avascular necrosis, bone infection, diabetic foot, and bone failure. [0023] According to a seventh aspect, the present invention provides a use of a graphene, graphene oxide, reduced graphene oxide or nanocarbon component for enhancing osteo- conduction of a bone scaffold. [0024] BRIEF DESCRIPTION OF THE DRAWINGS [0025] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows two pieces of bone substitute (Orthoss) glued together with a glue of the present invention. The arrow shows the area where the glue was applied. Figure 2 shows the effect of sterilisation of the glue under UV for 20 minutes (left column images), under UV for 60 minutes (middle column images) and dipped in 70% ethanol (right column images). As can be seen in Figure 2, no bacterial cultures are observed over the time period tested. Figure 3 shows microscope of pictures of a direct toxicity assay observing the effect of the glue of the invention on human mesenchymal stem cells (MSCs). Four conditions were tested: StemMACS only, bone substitute (Orthoss) without glue, Orthoss with example Glue A, and Orthoss with example Glue B. Figure 4 shows a plot of the absorbance measured four hours after the addition of XTT agent against the initial number of cells placed within a reaction well. The conditions tested were a positive control, a negative control, Orthoss only, example Glue A with Orthoss, and example Glue B. Figure 5 shows the average value of absorbance against time for each of the positive control, negative control, Orthoss only, example Glue A with Orthoss and example Glue B with Orthoss. Figure 6 shows microscopic pictures, at 4x magnification, of the stained samples using methylene blue in the wells seeded with 1000 cells. (A) Positive control. (B) Extract from Orthoss only. (C) Extract from Orthoss with example Glue A; (D) Extract from Orthoss with Glue B. Figure 7 shows a graphical representation of the scaffolds of the invention. Figure 8 shows an image of an annular TiO2 or Ti-alloy scaffold (diameter of 20 mm) and chitosan scaffold fabricated through freeze drying. Figure 9 shows a freeze-dried cortical-cancellous scaffold used in human mesenchymal stem cell studies. Figure 10 shows the visual appearance of the cortical-cancellous scaffolds. Figure 11 shows XTT absorbance results of mesenchymal stem cells cultured in MACS (positive control), conditioned media/extract form titanium (TIT) scaffold and titanium with iron scaffold (TIT+Fe) compared to DMEM media (negative control). Figure 12 shows samples of bone marrow collected (a) prior to loading the scaffolds, (b) after loading onto a titanium-Fe scaffold, and (c) after loading onto a titanium scaffold. The culture plates were maintained in a MACS medium to support MSC colony formation and were stained with methylene blue to visualise the colonies. Figure 13 shows flow cytometry results for bone marrow MSCs released from a loaded titanium scaffold (left) and a loaded titanium-Fe scaffold. Figure 14 shows Glue A of Example 1 was used to join pieces of bovine jaw bones together. Figure 15 shows the glued surface having a line of bonding. Figure 16(A) shows a comminuted femoral fracture with reds arrows showing the bone gaps. Figure 16(B) shows femoral gaps have been filled with bone glue. Figure 16(C) shows segmental femoral fracture where the bone glue may be used in red high-lighted areas where the gaps are shown even after inserting and stabilising the top and bottom halves of the femur with an intramedullary nail (rod). The gaps are left (see red arrows) which cannot be stabilized easily with soft putty. A glue which hardens stabilizes the bone and provides the biomechanic continuum. Figure 17 – Left image shows a femoral shaft fracture after high energy trauma (side, 29 years old man); central image shows the gap in a simulation; and right image shows when a porous Ti-alloy scaffold will be used to match with the biaxial and torsional biomechanical loading of the femur, whilst the bone has been stabilized with an intramedullary nail. Figure 18: Compares the FTIR spectroscopic data for 3 different glue samples from Example 11 with chitosan. Note that the chitosan structure dominates in all three compositions of glue. The order of the FTIR plots is the same as the order in the key in Figure 18 (top to bottom). Figure 19: viscosity of each of Compositions 1 to 3 (glue) and Compositions 1 to 3 (putty) from Example 11. Figure 20: (a) A comparison of synthesised cerium oxide nanoparticles calcined at 280 °C, 385 °C, and 815 °C, analysed using a Vertex 70 FTIR spectra from 400 to 4000 cm−1. The operating parameters consisted of a total of 32 scans at a resolution of 4 cm-1 and (b) UV–Vis absorbance spectra obtained from nanoparticle concentrations of 0.5 mg/ml. The inset Tauc Plot corresponds to the bandgap energies of synthesised cerium oxide nanoparticles. The order of the UV-Vis plots is the same as the order in the key in Figure 20 (top to bottom). Figure 21: UV-Vis absorbance spectra obtained from cerium oxide nanoparticles concentrations of 0.5 mg/ml and corresponding Tauc Plot. Figure 22: a) Comparison of the viscosities of glues (Glue 1, Glue 2, Glue 3 of Example 15) in water; b) comparison of the viscosities of two different chitosan mixtures in water. Figure 23: Osteoblast G292 cells at a cell density of 104 cells/well were tested against glue composition 1, 2 and 3. Giemsa staining was utilised to stain the cells for enhancing. Figure 24: Extract cytotoxicity and proliferation testing using osteoblast cell line G292 cells on glue 1, 2 and 3 samples. Positive and negative controls consisted of McCoy's media and McCoy's media with 10% DMSO. (A) optical density extract cytotoxicity results (left) and cell viability percentage live results (right) measured as the percentage of extracts collected after 1, 3 and 7 days. (B) 5-day percentage live extract proliferation cytotoxicity results where testing consisted of 500 cells/well osteoblast G292 cells. Data are shown with mean and standard error of mean and n = 3. In each group of three bars, the left bar is “Glue 1”, the middle bar is “Glue 2” and the right bar is “Glue 3”. Figure 25a and 25b: data for the E. Coli and S. epidermidis strain of bacteria for the two different types of glues (Compositions 1-3). Figure 26a and 26b: optical density measurements characterising the antibacterial properties of the cerium oxide (CeO2) nanoparticles after direct incubation with Escherichia coli and Staphylococcus epidermidis for 48 hours. The reference point is the cultured bacteria (CB). [0026] DETAILED DESCRIPTION [0027] Definitions [0028] The term ‘graphene oxide’ refers to chemically modified single-layer graphene having a high oxygen content, typically characterised as having a carbon:oxygen atomic ratio of from about 6:1 to about 1:1, depending on the method of synthesis. Graphene oxide is typically prepared by oxidation and exfoliation of graphite, causing extensive oxidative modification of the basal plane. [0029] The term ‘reduced graphene oxide’ refers to a reduced oxygen content form of graphene oxide, typically characterised as having a carbon:oxygen atomic ratio of from about 20:1 to about 6:1. Reduced graphene oxide is produced by chemical, thermal, microwave, photo-chemical, photo-thermal or microbial/bacterial reduction of graphene oxide or by exfoliating reduced graphite oxide. [0030] The term ‘nanocarbon’ refers to carbon-based materials having at least one external dimension in the nanoscale, i.e. from about 1 nm to about 100 nm. [0031] The term ‘nanoparticle’ refers to particles having at least one external dimension in the nanoscale, i.e. from about 1 nm to about 500 nm. [0032] Compositions [0033] According to a first aspect, the present invention provides a composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron- doped calcium phosphate mineral component; and (ii) a collagen component. [0034] In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 1% w/w to about 30% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 5% w/w to about 30% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 10% w/w to about 30% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 15% w/w to about 30% w/w of the chitosan gel. [0035] In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 1% w/w to about 25% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 5% w/w to about 25% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 10% w/w to about 25% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 15% w/w to about 25% w/w of the chitosan gel. [0036] In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 20% w/w to about 25% w/w of the chitosan gel. In an embodiment, the iron-doped calcium phosphate mineral component is present in an amount of from about 15% w/w to about 20% w/w of the chitosan gel. Preferably, the iron-doped calcium phosphate mineral component may be present in an amount of about 20% w/w of the chitosan gel. Preferably, the iron-doped calcium phosphate mineral component may be present in an amount of about 25% w/w of the chitosan gel. [0037] Without meaning to be bound by theory, the iron-doped calcium phosphate mineral component is considered to facilitate oxygen regulation and/or neo-angiogenesis. Compositions that do not include iron-doped calcium phosphate mineral component (instead containing ‘bare’ calcium phosphate mineral component) have been found to perform less effectively. [0038] In embodiments, the iron-doped calcium phosphate mineral component is an iron- doped component selected from: hydroxyapatite (Ca10(PO4)6(OH)2), fluorapatite (Ca5(PO4)3F), brushite (CaHPO4.2H2O), β-pyrophosphate, monetite (CaHPO4) and tricalcium phosphate (Ca3(PO4)2). In embodiments, the iron-doped calcium phosphate mineral component is fluoride free, e.g. the iron-doped calcium phosphate mineral component may be selected from hydroxyapatite (Ca10(PO4)6(OH)2), brushite (CaHPO4.2H2O), monetite (CaHPO4) and tricalcium phosphate (Ca3(PO4)2). [0039] In an embodiment, the calcium phosphate mineral component is selected from the group consisting of: brushite (CaHPO4.2H2O), β-pyrophosphate, monetite (CaHPO4) and tricalcium phosphate (Ca3(PO4)2). This is particularly advantageous when it is desirable to accelerate new bone formation (neo osteogenesis). Recent studies have suggested that enzymatic action accelerates dissolution of the inorganic pyrophosphate ions causing a simultaneous loss of mineralisation and a localised rise in ion saturation and as a result the presence of pyrophosphate can stimulate bone mineralisation and healing. [0040] In an embodiment, the calcium phosphate mineral component is fluorapatite (Ca5(PO4)3F). This is particularly advantageous when the end use will be for jaw bone treatment and for dental implants. [0041] In an embodiment, the calcium phosphate mineral component is hydroxyapatite (Ca10(PO4)6(OH)2). This is particularly advantageous when the end use involves an application where it is desired to slow new bone formation, such as when seeking to avoid hyper calcification or uncontrolled cancerous growth in patients with compromised hormone control. [0042] In embodiments, an iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 30% w/w of the iron-doped calcium phosphate mineral component. In embodiments, the iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 25% w/w of the iron-doped calcium phosphate mineral component. In embodiments, the iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 20% w/w of the iron-doped calcium phosphate mineral component. In embodiments, the iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 15% w/w of the iron-doped calcium phosphate mineral component. In embodiments, the iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 10% w/w of the iron-doped calcium phosphate mineral component. [0043] The amount of iron ions present in the calcium phosphate mineral component can be adjusted according to the application. In an embodiment, iron ions are incorporated into the calcium phosphate mineral component during the preparation of the calcium phosphate mineral component. In an embodiment, the calcium phosphate mineral component can be prepared by combining a calcium salt containing solution with a phosphate salt containing solution. For example, the calcium phosphate mineral component may be prepared from a calcium nitrate solution and an ammonium phosphate solution. An iron salt can, (e.g. Fe(NO3)3.3H2O) may be added to the calcium salt containing solution (e.g. calcium nitrate solution) prior to combination with the phosphate salt containing solution (e.g. ammonium phosphate solution). In an embodiment, from 0.1 to 1 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution. Preferably, 0.1 to 0.5 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution. More preferably 0.1 to 0.2 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution. [0044] In embodiments where the iron-doped calcium phosphate mineral component includes fluoride, the calcium phosphate mineral component can be prepared by combining a calcium salt containing solution with a phosphate salt containing solution and a fluoride salt containing solution. An iron salt can, (e.g. Fe(NO3)3.3H2O) may be added to the calcium salt containing solution (e.g. calcium nitrate solution) prior to combination with the phosphate salt containing solution (e.g. ammonium phosphate solution) and fluoride salt containing solution (e.g. NH4F). [0045] In an embodiment, the collagen component is present in an amount of from about 1% w/w to about 30% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 5% w/w to about 30% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 10% w/w to about 30% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 15% w/w to about 30% w/w of the chitosan gel. [0046] In an embodiment, the collagen component is present in an amount of from about 1% w/w to about 25% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 5% w/w to about 25% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 10% w/w to about 25% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 15% w/w to about 25% w/w of the chitosan gel. [0047] In an embodiment, the collagen component is present in an amount of from about 20% w/w to about 25% w/w of the chitosan gel. In an embodiment, the collagen component is present in an amount of from about 15% w/w to about 20% w/w of the chitosan gel. Preferably, the collagen component may be present in an amount of about 20% w/w of the chitosan gel. [0048] Without meaning to be bound by theory, collagen is made up chains of amino acids organised in the form of a triple helix of elongated fibril (also known as a collagen helix). The collagenous fibrils are considered to be essential for bone formation. Together with the presence of the other components of the composition of the present invention, the collagen aids essential ion transport under load-bearing conditions in bone. [0049] In an embodiment, the collagen component is Type I or Type III collagen. In an embodiment, the collagen component is Type I collagen. In an embodiment, the collagen component is Type III collagen. For bone repair Type III collagen helps in promoting angiogenesis. Type I collagen is important for enthesis and connective tissue engineering. Type I collagen is also important for osteogenesis. [0050] In an embodiment, the composition of the first aspect further comprises a graphene, graphene oxide, reduced graphene oxide or nanocarbon component. In an embodiment, the composition of the first aspect further comprises a graphene oxide, reduced graphene oxide or nanocarbon component. Conductivity of the graphene, graphene oxide, reduced graphene oxide or nanocarbon component may vary between 100 and 350 S/m (J. Phys. Chem. C 2008, 112, 20264–20268). In an embodiment, the graphene oxide is in the form of a powder. In an embodiment, the graphene is in the form of a powder. In an embodiment, the graphene oxide has from 10 to 25 layers, and preferably from 15 to 20 layers. In an embodiment, the graphene has from 3 to 20 layers, and preferably from 3 to 15 layers. In an embodiment, the graphene has 9 ± 6 layers. In an embodiment, the graphene has a resistivity of 40-50Ω. In an embodiment, the graphene oxide is from 4 to 10% edge-oxidised. In an embodiment, the graphene oxide is in the form of a powder, the graphene oxide has 15 to 20 layers and the graphene oxide is from 4 to 10% edge-oxidised. In an embodiment, the graphene is in the form of a powder and the graphene has 3 to 20 layers. [0051] Without meaning to be bound by theory, the graphene, graphene oxide, reduced graphene oxide or nano carbon component is thought to enhance the osteo-conducting properties (e.g. by supporting charge transport of ions and electrons) of the resulting product. Compositions that do not include the graphene, graphene oxide, reduced graphene oxide or nano carbon component have been found to perform less effectively. Since graphene and GO contains sp3 - bonded hybridized carbon atoms, graphene and GO can readily capture water vapor from the surrounding external environment, owing to its hydrophilicity. The effect of atmospheric relative humidity (RH) on the electrical and mechanical properties of graphene and GO are beneficial for the practical application of graphene and GO-based medical implants. However, in the presence OH- ions, the electrical conductivity persists, as the electrical property appears to be accentuated in the presence of collagen and chitosan, the latter two also facilitate ion transport. The OH- ion permeation is expected in the r-GO medium via the following reaction. The transport of OH- , H+ and H2O are expected alongside electrons. GO + 2H+ + 2e = r-GO + H2O [0052] In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 4% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 3% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 2.5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 2% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 1.5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 1% w/w of the chitosan gel. [0053] In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 4% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 3% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 2.5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 2% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 1.5% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.1% to 1% w/w of the chitosan gel. [0054] In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.2% to 0.9% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.3% to 0.8% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.4% to 0.7% w/w of the chitosan gel. In embodiments, the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of about 0.5% w/w of the chitosan gel. [0055] In embodiments, the graphene oxide has a C:O ratio of from 9:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 8:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 7:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 6:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 5:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 4:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 3:1 to 1:1. In embodiments, the graphene oxide has a C:O ratio of from 2:1 to 1:1. [0056] In embodiments, the reduced graphene oxide has a C:O ratio of from 50:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 20:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 15:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 10:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 9:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 8:1 to 6:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 50:1 to 10:1. In embodiments, the reduced graphene oxide has a C:O ratio of from 20:1 to 10:1. [0057] In a preferred embodiment, the composition of the first aspect further comprises a graphene component (as opposed to a graphene oxide, reduced graphene oxide or nanocarbon component). Graphene oxide or reduced GO are less consistent and are more resistive than graphene. [0058] As explained above, the composition of the invention also includes chitosan. Without meaning to be bound by theory, the inclusion of chitosan (Chi) in the composition of the present invention is thought to increase the binding and adhesive properties of the composition (both adhesion to the surrounding bone and adhesion between the constituents to help the constituent components of the composition to form as a paste which may be then applied along the damaged bone surfaces for adhesion for tissue restoration). Chitosan is a polysaccharide meaning that, during the regenerative process, it can support the cell growth by providing energy. Chitosan also has several biological properties selected from the group consisting of: biodegradability, lack of toxicity, anti-fungal effects, wound healing acceleration and immune system stimulation. These properties make it an attractive material for use in medical applications. [0059] In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 10% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 8% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 6% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 5% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 4% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 0.5 to 3% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 3 to 5% w/w of the chitosan gel. In embodiments, the chitosan gel may comprise chitosan in an amount of from 4 to 5% w/w of the chitosan gel. [0060] In embodiments, the chitosan gel is an aqueous based gel. [0061] In embodiments, the chitosan is a high molecular weight chitosan. For example, the chitosan can have a viscosity of 800-2000cps (c=1%; 1% acetic acid), deacetylation: > 75%. [0062] In an embodiment, the composition of the first aspect further comprises a nanoparticle component. In an embodiment, the nanoparticle is an inorganic oxide nanoparticle, for example a metal oxide nanoparticle. Preferably, the nanoparticle component is in the form of a nanopowder, for example an inorganic oxide nanopowder. In an embodiment, the nanoparticle is selected from the group consisting of cerium oxide and strontium oxide. Cerium oxide is particularly preferred as the nanoparticle component. [0063] In an embodiment, the nanoparticle component has a particle size of 1-10,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-5,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-4,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-3,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-2,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-1,000 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-900 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-800 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-700 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-600 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-500 nm (BET). [0064] In an embodiment, the nanoparticle component has a particle size of 1-500 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-400 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-300 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-200 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-100 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-50 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-40 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-30 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-25 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-20 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-15 nm (BET). In an embodiment, the nanoparticle component has a particle size of 1-10 nm (BET). [0065] Without meaning to be bound by theory, the nanoparticle component is thought to enhance the antimicrobial properties of the resulting product. Compositions that include the nanoparticle component have been shown to have improved antimicrobial properties. The antimicrobial properties in an osteoconducting environment requires a bivalent state of an ion to work together with simultaneous osteoconduction. Cerium oxide exists predominantly in the 4+ state and if the abundance of 3+ is not maintained, the antibacterial properties becomes less effective. Ce4+ to Ce3+ transition is supported via electron exchange: Ce4+ = Ce3+ + e-. [0066] In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 10% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 9% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 8% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 7% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 6% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.01% to 5% w/v of the chitosan gel. [0067] In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 10% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 9% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 8% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 7% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 6% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 0.1% to 5% w/v of the chitosan gel. [0068] In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 10% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 9% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 8% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 7% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 6% w/v of the chitosan gel. In embodiments, the nanoparticle component is present in the composition in amount of from 1% to 5% w/v of the chitosan gel. [0069] In embodiments, the nanoparticle component is present in the composition in amount of from 1% w/v, 2% w/v, 3% w/v, 4% w/v or 5% w/v of the chitosan gel. [0070] In an embodiment, where the composition is in the form of a glue, the chitosan has a molecular weight of from 10 kilo Daltons to 2220 kilo Daltons. The molecular weight of the chitosan impacts the viscosity and/or rheology of the composition. [0071] In an embodiment, the composition of the first aspect is in the form of a glue. The glue may have a viscosity of from about 1 Pa-s to about 10 Pa-s. In embodiments when the composition is in the form of a glue, the chitosan gel may comprise chitosan in an amount of from 0.5 to 3% w/w of the chitosan gel. [0072] In an embodiment, the composition of the first aspect is in the form of a putty. The glue may have a viscosity of from about 10 Pa-s to about 10,000 Pa-s. In embodiments when the composition is in the form of a putty, the chitosan gel may comprise chitosan in an amount of from 3 to 5% w/w of the chitosan gel, preferably from 4 to 5% w/w of the chitosan gel. [0073] The rheological property of the composition can be adjusted by varying the overall surface charge (e.g. measured by the zeta potential). The percentage of protonation may be controlled between 100% to less than 1%, depending on the need for the mineral mixture with chitosan for a particular bone repair. [0074] The composition of the first aspect is useful in restoring an impaired bone that has failed to repair itself. For example, the composition of the first aspect may be useful in the augmentation of missing bone; and/or in reducing a bone fracture gap; and/or in treating non-union of bones and bone fragments; and/or in bone fracture reduction; and/or in treating open bone fractures; and/or in treating bone infection; and/or in the treatment of fragile/osteoporotic bone; and/or in the treatment of avascular necrosis. [0075] In an embodiment, the composition of the first aspect has rapid adhesive bonding properties with natural bone. [0076] In an embodiment, the composition of the first aspect provides a matrix material having one of more of the following properties: antimicrobial properties; and/or osteogenesis enhancing properties; and/or osteo-conduction enhancing properties; and/or osteo- integration enhancing properties; and/or ossification enhancing properties. [0077] In an embodiment, the composition can be bonded by a process that exposes the composition to heat source and/or a light source. [0078] The composition of the first aspect may be formulated to have a viscosity that renders the composition suitable as a bone glue. The bone glue can be used in the joining of multiple fragments together into a shape or form. Further stabilisation using nails or plates may be required. [0079] The composition of the first aspect may be formulated to have a viscosity that renders the composition suitable as a putty. The putty can be used in the treatment of bone voids (i.e. small areas where bone is missing, e.g. bone loss less than 1 cm, optionally less than 3 cm), such as bone gaps, non-unions, fracture reduction, diabetic foot and avascular necrosis. [0080] Bone Scaffold [0081] In an embodiment, the first layer is an outer layer mimicking the structure and physiological/anatomical properties of cortical bone and the second layer is an inner layer, which emulates the structural and anatomical/physiological features of cancellous bone such that the bone scaffold is a concentric bone scaffold structure. [0082] In an embodiment, the first layer is a cortical layer. [0083] In an embodiment, the second layer is a cancellous layer. [0084] In an embodiment, the porous titanium-based composition comprises Ti alloy or a mixture of Ti-alloy with TiO2. [0085] In an embodiment, the porous titanium-based composition further comprises an iron-doped calcium phosphate mineral component. [0086] In an embodiment, the porous titanium-based composition further comprises Fe2O3 nanoparticles. [0087] In an embodiment, the second layer comprises a composition of the first aspect that has been subjected to one or more processing steps to remove solvents. In an embodiment, the composition of the first aspect is first subjected to a freeze drying process to produce a freeze-dried composition, which is then contacted with the first layer of the bone scaffold. In an alternative embodiment, the composition of the first aspect is contacted with the first layer of the bone scaffold to form a bone scaffold precursor material, which is then freeze dried to produce a bone scaffold of the third aspect. [0088] The bone scaffold of the third aspect, is useful in the treatment of bone defects (e.g. long bone defects, craniomaxilofacial defects, pelvis defects and spine defects). [0089] A bone scaffold can be used in the treatment of extensive bone loss (bone defect; >1cm to 15cm, optionally >3cm to 10cm) for facilitating bone continuity and load distribution. [0090] Method for preparing a composition [0091] In an aspect of the invention, there is provided a method of preparing the composition of the first aspect, the method comprising: (1) preparing a chitosan gel comprising mixing chitosan flakes with water and/or a mixture of water and acetic acid; (2) preparing an iron-doped calcium phosphate material comprising combining: (a) an iron salt and a calcium salt containing solution; and (b) adding a phosphate salt containing solution to the iron salt / calcium salt containing solution; (3) combining the chitosan gel of step (1), the iron-doped calcium phosphate material of step (2) and collagen. [0092] In an embodiment, the calcium phosphate mineral component may be prepared from a calcium nitrate solution and an ammonium phosphate solution. [0093] In an embodiment, the iron salt is Fe(NO3)3.3H2O. [0094] In an embodiment, from 0.1 to 1 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution. Preferably, 0.1 to 0.5 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution. More preferably 0.1 to 0.2 molar equivalents of Fe ions are added to the Ca ions in the calcium salt solution. [0095] In embodiments where the iron-doped calcium phosphate mineral component includes fluoride, the calcium phosphate mineral component can be prepared by combining a calcium salt containing solution with a phosphate salt containing solution and a fluoride salt containing solution. An iron salt can, (e.g. Fe(NO3)3.3H2O) may be added to the calcium salt containing solution (e.g. calcium nitrate solution) prior to combination with the phosphate salt containing solution (e.g. ammonium phosphate solution) and fluoride salt containing solution (e.g. NH4F). [0096] In an embodiment, the method further comprises additionally combining a graphene oxide, reduced graphene oxide or nanocarbon component in step (3). [0097] In an embodiment, the method further comprises additionally combining a nanoparticle component in step (3). [0098] Method for preparing a bone scaffold [0099] In an aspect of the invention, there is provided a method of preparing a bone scaffold of the third aspect, the method comprising: (1) pressing a powder comprising Ti and/or TiO2 and a calcium phosphate mineral component and/or Ti and Fe2O3, and sintering the pressed powder to produce a sintered component; (2) applying to the sintered component of step (1) the composition of the first or third aspect to produce a scaffold precursor component; (3) subjecting the scaffold precursor component to a freeze drying process. [00100] In an embodiment, step (1) involves pressing the powder at a pressure of >5 ton, e.g.6 ton or 7 ton for a period of time of >15 minutes, e.g.30 minutes or 60 minutes. [00101] In an embodiment, step (1) involves sintering the pressed powder for > 1 hour, e.g. 2, 3, 4, 5 or 6 hours. [00102] In an embodiment, step (1) involves sintering the pressed powder at 800 – 1200°C, preferably 900 – 1100°C, more preferably 1000°C. [00103] In an embodiment, step (3) involves freezing the scaffold precursor component, optionally at < -50°C, e.g. -60°C, -70°C or -80°C. In an embodiment, step (3) involves freezing for >1 hour, e.g.6, 12, 18 or 24 hours. In an embodiment, step (3) involves freeze drying at < -80°C, e.g. -90°C or -100°C. In an embodiment, step (3) involves freeze drying at about 40 to about 50 milli torr pressure. In an embodiment, step (3) involves freeze drying for >1 hour, e.g.6, 12, 18 or 24 hours. [00104] In an aspect of the invention, there is provided a method of preparing a bone scaffold of the third aspect, the method comprising: (1) pressing a powder comprising Ti and/or TiO2 and a calcium phosphate mineral component and/or Ti and Fe2O3, and sintering the pressed powder to produce a sintered component; (2) subjecting a composition of the first or third aspect to a freeze drying process to produce a freeze dried composition; (3) applying to the sintered component of step (1) the composition of the first or third aspect, and then contacting the applied composition of the first or third aspect with the freeze dried composition of step (2) to produce a scaffold precursor component; (4) subjecting the scaffold precursor component to a freeze drying process. [00105] In an embodiment, step (1) involves pressing the powder at a pressure of >5 ton, e.g.6 ton or 7 ton for a period of time of >15 minutes, e.g.30 minutes or 60 minutes. [00106] In an embodiment, step (1) involves sintering the pressed powder for > 1 hour, e.g. 2, 3, 4, 5 or 6 hours. [00107] In an embodiment, step (1) involves sintering the pressed powder at 800 – 1200°C, preferably 900 – 1100°C, more preferably 1000°C. [00108] In an embodiment, step (2) involves freezing the composition of the first or third aspect, optionally at < -50°C, e.g. -60°C, -70°C or -80°C. In an embodiment, step (2) involves freezing for >1 hour, e.g.6, 12, 18 or 24 hours. In an embodiment, step (2) involves freeze drying at < -80°C, e.g. -90°C or -100°C. In an embodiment, step (2) involves freeze drying at about 40 to about 50 milli torr pressure. In an embodiment, step (2) involves freeze drying for >1 hour, e.g.6, 12, 18 or 24 hours. [00109] In an embodiment, step (4) involves freezing the scaffold precursor component, optionally at < -50°C, e.g. -60°C, -70°C or -80°C. In an embodiment, step (4) involves freezing for >1 hour, e.g.6, 12, 18 or 24 hours. In an embodiment, step (4) involves freeze drying at < -80°C, e.g. -90°C or -100°C. In an embodiment, step (4) involves freeze drying at about 40 to about 50 milli torr pressure. In an embodiment, step (4) involves freeze drying for >1 hour, e.g.6, 12, 18 or 24 hours. [00110] Uses of the composition of the invention and scaffold of the invention: [00111] In an aspect of the invention, there is provided a method of joining bone, the method comprising: applying a glue composition of the first aspect to a first bone fragment; contacting the first bone fragment having the glue composition with a second bone fragment; allowing the glue to join the first and second bone fragments. [00112] In an embodiment, the allowing step involves irradiating the glue with a source of electromagnetic radiation. Optionally, the source of electromagnetic radiation is a near-IR radiation source. Further optionally, the source of electromagnetic radiation is a near-IR laser (e.g. a pulsed laser). The specific electromagnetic radiation source used in the examples of this application is generally applicable to the rest of the disclosure and is a near- IR 1040nm pulsed laser with 400 mW average incident power. [00113] In an embodiment, the allowing step takes less than 10 minutes, optionally less than 5 minutes and further optionally 2 or 3 minutes. [00114] In an aspect of the invention, there is provided a method of bridging a bone defect, the method comprising: applying a glue composition of the first aspect to a first and second bone fragment; positioning a bone scaffold between the first and second bone fragments; allowing the glue to join the first and second bone fragments to the bone scaffold. [00115] In an embodiment, the allowing step involves irradiating the glue with a source of electromagnetic radiation. Optionally, the source of electromagnetic radiation is a near-IR radiation source. Further optionally, the source of electromagnetic radiation is a near-IR laser (e.g. a pulsed laser). The specific electromagnetic radiation source used in the examples of this application is generally applicable to the rest of the disclosure and is a near- IR 1040nm pulsed laser with 400 mW average incident power. [00116] In an embodiment, the allowing step takes less than 10 minutes, optionally less than 5 minutes and further optionally 2 or 3 minutes. [00117] The abbreviations used here have their conventional meaning within the chemical and biological arts. [00118] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. [00119] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. [00120] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. [00121] For the avoidance of doubt, it is hereby stated that the information disclosed earlier in this specification under the heading “Background” is relevant to the invention and is to be read as part of the disclosure of the invention. EXAMPLES [00122] Example 1: Glue synthesis: [00123] Materials: Chitosan flakes - (Sigma-Aldrich, CAS: 9012-76-4; 3100000 – 3750000 Da, > 75 % deacetylated) Acetic acid - (Acros Organics, MFCD00036152) Calcium nitrate – (Fisher Chemicals, CAS: 13477-34-4 Ca(NO3)2.4H2O) Diammonium phosphate solution – (Acros Organics, CAS: 7783-28-0, (NH4)2HPO4) Sodium hydroxide Iron nitrate – Fe(NO3)3.3H2O Collagen (Type I) Graphene Oxide [00124] Synthesis of Chitosan Gel [00125] We added 2 mL of acetic acid in a glass beaker with 98 mL of distilled water and then placed the beaker on top of a hot plate with a magnetic stirrer. Then we added 3 g of chitosan flakes slowly into the acetic acid solution and left the solution mixing for 24 hours, for the polymerization to occur. [00126] Synthesis of Iron-Doped Hydroxyapatite [00127] Prior to Fe-HA synthesis, we prepared three solutions: a) Solution A: Calcium Nitrate solution, by adding 47.230 g of Ca(NO3)2.4H2O in 200 mL of distilled water. b) Solution B: Ammonium phosphate solution, by adding 26.411 g of (NH4)2HPO4 in 200 mL of distilled water. c) Solution C: 1M of NaOH solution, by adding 40 g of sodium hydroxide in 1L of distilled water. [00128] We diluted 20mL of solution A in 180mL of distilled water, creating a 0.1M Calcium (Ca) solution. [00129] Similarly, we diluted 20 mL of solution B in 180mL of distilled water, creating a 0.1M Phosphate (P) solution. [00130] We then added 0.83g of Fe(NO3)3.3H2O to the Ca solution, and put onto a magnetic stirrer, with heat indication at 150°C and stirring level of 2. This solution was acidic, but the reaction for creating hydroxyapatite should take place in a pH of 8 (ranging from 7.5 to 9). Additionally, the temperature of the reaction should be around 50°C (ranging from 47°C to 51°C). Therefore, we added solution C, drop by drop, until the pH reached 8. [00131] When both pH and temperature reached their values, then we poured the P solution into the dropwise pipette and started the drop-by-drop addition of P solution to the Ca solution. We continued adding solution C, drop by drop, until pH dropped below 8. [00132] When all P solution was added, we left the solution stirring for another two hours with the heat off. Then the solution is left for precipitation for another hour. We then poured the CaP solution in a filter paper/funnel and washed the solution three times with distilled water during filtration and left the material overnight. We collected the materials in the filter paper and put it in the freezer for 24 hours. On the next day, we freeze dried the material for 24 hours. Once the 24 hours passed, we collected the material and using mortar and pestle we ground the powder into finer particles. [00133] Glue Synthesis [00134] For the response of bone marrow stem cells, the following two compositions of glue were formulated for processing: (a) GLUE A: 20% w/w Fe-HA and 20% w/w Col in Chi (3% w/w) Gel and (b) GLUE B: 0.5% w/w Gr, 20% w/w Fe-HA and 20% w/w Col in Chi (3% w/w) Gel. [00135] The glue is synthesized by combining chitosan (Chi) gel, iron doped hydroxyapatite (Fe-HA) powder, collagen (Col) powder and graphene (Gr) oxide powder. [00136] For Glue A, we added 0.1g of Fe-HA, 0.1 g of Col and 0.5 g of Chi Gel on a glass plate and mix them until one homogeneous gel is formed. [00137] For Glue B, we added 0.0025g of Gr oxide, 0.1g of Fe-HA, 0.1g of Col and 0.5g of Chi Gel on a glass plate and mix them until one homogeneous gel is formed. [00138] Example 2 – Cell Toxicity of Glue Composition: [00139] Cell toxicity: The tests were divided into three parts – sterilization and stability, direct and indirect toxicity. [00140] Sterilization and Stability [00141] The glue was used to bond two fragments of Orthoss, mimicking the bonding of bone fragments (Error! Reference source not found.). [00142] Three techniques for sterilization were used; (a) UV for twenty minutes; (b) UV for sixty minutes; (c) Dipped in 70% Ethanol. [00143] All three techniques were successful (Error! Reference source not found.), as the materials remained sterile and stable for at least two months. The culture remained clear, showing no sign of infection from bacteria. [00144] Direct Toxicity [00145] The aim of the direct toxicity study was to observe the effect of the glue on human mesenchymal stem cells (MSCs), by adding glued Orthoss samples to a 50%-confluent cell monolayer in a 6-well plate, followed by a seven-day protocol according to ISO10993- 5:2009. After sterilization of the materials, four conditions were tested: (a) positive control (StemMACS only), (b) Orthoss without glue; (c) Orthoss with Glue A and (d) Orthoss with Glue B, with taking microscopic pictures in specific timepoints. [00146] In all conditions, cells continued to multiple and more or less cover the entirety of the well by the 6th day, showing that materials under investigation did not affect the cells. Note that the circled area points to Orthoss debris in Figure 3. [00147] Indirect Toxicity [00148] The aim of the indirect toxicity test was to identify any cytotoxic effects of the extracts of the glue materials on human MSCs. The experiments were performed in three different cell densities: 250, 500 and 1000 cells per well in triplicate, in a 96-well plate. MSC proliferation was quantified in the presence of different extracts by measuring the absorbance of the fluorogenic substrate XTT and by taking microscope pictures, following the ISO 10993:5-2009. [00149] The conditions tested for this assay were (a) positive control (only containing StemMACS); (b) negative control (10%DMSO in StemMACS); (c) extract from two separate Orthoss pieces; (d) extract from two Orthoss pieces glued with Glue A; and (e) extract from two Orthoss pieces glued with Glue B. [00150] Once calculating the average values per cell density and time, data were plotted against the cell number placed initially in each well (Error! Reference source not found.), four hours after the addition of XTT agent). [00151] Error! Reference source not found. shows that as expected, the higher the cell density, the stronger the absorbance signal in all conditions, apart from the negative control. Addition of Orthoss was non-toxic to cells, and interestingly extract from Orthoss alone was more beneficial to cells than Orthoss with Glue A. However, extract from Orthoss with Glue B produced results similar to positive control. Average ratios were calculated per condition (regardless cell seeding density), and ratios of absorbance at each time point against time zero were plotted (Error! Reference source not found.), showing that extract from Orthoss with Glue B had similar absorbance patterns as positive control. [00152] Microscopic pictures (4x magnification, Error! Reference source not found.) taken after the XTT assay and staining with methylene blue, supported the data, showing that wells from condition A and condition E had similar cell densities, with wells of conditions C and D being less populated with cells (with same initial cell density). [00153] Example 3: Demonstration of glue to join bone [00154] Glue A of Example 1 was used to join pieces of bovine jaw bones together. A near- IR 1040nm pulsed laser with 400 mW average incident power was used. This is shown in Figure 14. These pieces 1, 2 and 3 can be further joined to augment and reconstruct bone. After putting the two surfaces together, the process takes less than few minutes. [00155] Figure 16(A) shows a comminuted femoral fracture with reds arrows showing the bone gaps and 16(B) shows femoral gaps have been filled with bone glue. The comminuted fracture in the femoral shaft consists of several pieces. After the femur is stabilized with an intramedullary nail, the fragments are scattered around the zone of fragmentation. In this case the glue can be implanted (activated with a heat source) in between the fragments to provide a bridge to connect the so ‘called fracture gaps’. This approach will connect all the fragments together allowing uniform distribution of the mechanical forces applied and of the mechanical stimuli generated supporting a timely fracture healing response. Presence of fracture gaps post fracture fixation are well known to be associated with an increased risk of non-union. [00156] Figure 16(C) shows segmental femoral fracture where the bone glue may be used in red high-lighted areas where the gaps are shown even after inserting an intramedullary nail (rod) for stabilizing the bone between the top and bottom halves. The gaps are left (see red arrows) which cannot be stabilized easily with soft putty. A glue which hardens stabilizes the bone and provides the biomechanic continuum. As the pieces are large, they can be held together by the glue, restoring continuity of the whole bone. [00157] Example 4: Putty synthesis: [00158] The same synthetic procedure as described above in Example 1 was followed, except that the chitosan gel was made by the following process: [00159] We added 2 mL of acetic acid in a glass beaker with 98 mL of distilled water and then placed the beaker on top of a hot plate with a magnetic stirrer. Then we added 4-5g of chitosan flakes slowly into the acetic acid solution and leave the solution mixing for 24 hours, for the polymerization to occur. [00160] Example 5: Fabrication of scaffolds [00161] For the fabrication of TiO2 scaffolds, Ti powder (Goodfellas, CAS number: 7440- 32-6) was pressed in pellets. For each pellet approximately 0.50 g of Ti powder was filled inside a 20 mm diameter die before pressing with a load of 7 ton for 30 min. Afterwards the Ti pellets were placed in silica crucibles and heated at 1000 oC for 5 h. At this temperature and with the presence of atmospheric air, Ti transforms to TiO2. Sintering and crystal growth are taking place resulting the improved mechanical properties of the scaffolds. [00162] Example 6: Fabrication of TiO2/CaP scaffolds: [00163] A similar procedure was followed to that described in Example 4 for the fabrication of TiO2/CaP scaffolds. Ti powder was mixed with brushite crystals at different concentrations (i.e. 10, 20 and 40% w/w). After pressing pellets of 20 mm in diameter (0.50 g of mixture with a load of 7 ton) sintering took place at 1000oC for 5 h. As mentioned before, this process results the transformation of Ti into TiO2 while, brushite is transformed into b-pyrophosphate (transformation at 890 C). [00164] Example 7: Fabrication of Oxygenated α-Ti scaffolds [00165] Oxygenated α-Ti scaffolds were fabricated by sintering Ti powder mixed with Fe2O3 nanoparticles. Specifically, 0.50 g Ti powder was mixed with 0.1 g Fe2O3 (atomic ratio of Ti/O is 100/20) and pressed in pellet (20 mm diameter die and load of 7 ton for 30 min). The pellet was placed in a silica crucible and heated at 1000oC for 5 h. The sintering took place in the presence of Argon (for example: flow rate 0.8 l/min) for maintaining inert atmosphere. [00166] Example 8: Fabrication of annular scaffolds: [00167] Annular scaffolds were fabricated with TiO2 and oxygenated Ti (Figures 7 and 8) which will be used for the future animal trials. To achieve this, we designed and constructed a die for pressing powders, in annular shape, under high loads (up to 9 ton). [00168] For bone scaffold engineering to fill a bone defect, the cortex (Ti-alloy with mineral) and cancellous scaffold (chitosan with mineral) structures were joined together during a freeze-drying process. [00169] The Ti-alloy scaffold is first coated with glue/putty (formulation as per Example 1 and Example 3) and then the material placed inside a -80°C fridge for few hours to 24 hrs until a bond is formed. After that the conjoined scaffold was placed inside a freeze drier (e.g. VirTis 4KB ZL Benchtop K (SP Industries)). [00170] Freeze drier time is shorter for smaller scaffolds (< 10mm in diameter and <10 mm in length), by comparison longer period is required for larger size (25mm diameter and 25 mm length) scaffolds. The freeze drier chamber should be maintained at around 40-50 milli torr pressure. [00171] For the material shown in Figure 9, the freeze dryer temperature was set at -100 ºC, the pressure was set at 43 millitorrs and the freeze drier duration was 24 hours. [00172] Example 9: Stem cell attachment and proliferation in novel scaffolds for cortical and cancellous bone applications using chitosan and titanium alloy powder [00173] The examples of human mesenchymal stem cell (h-MSC) expression and proliferation are given where the hMSC on the conjoined scaffolds were tested. [00174] Experiments were performed with the combination of chitosan and titanium scaffolds (Figure 9). These scaffolds were processed in sterile conditions and tested for their effects on human mesenchymal stem cells (MSCs): • Cell toxicity: by testing MSC proliferation in media extracts in which these scaffolds were incubated (indirect cytotoxicity measurements according to ISO10993-5:2009). • MSC cell adhesion I (immediate attachment): by visualizing if the leftover of bone marrow used to load these scaffolds (for 2 hours) contain MSCs that failed to attach and therefore adhere to plastic culture wells and form colonies. • Cell adhesion II (longer term colonization during culture): by phenotyping bone marrow cells released from digested scaffolds after being loaded and cultured for 2 weeks. [00175] In Figure 10, the visual appearance of the cortical-cancellous scaffolds are shown. The scaffold with whiter appearance has superficial layer of chitosan and calcium phosphate mineral whereas the darker scaffold has no mineral content. [00176] Cell toxicity [00177] Culture-expanded MSCs (n=6 donors) were maintained in MACS media (StemMACS) or scaffolds’ extracts (indicated as conditioned media) for 1 week before performing the XTT assay (Figure 11) to measure MSC proliferation. [00178] Comparable levels of proliferation for MSCs in media in which scaffolds were maintained relative to specialized media for MSC growth (MACS media) ruled out the cytotoxicity of the scaffolds. The DMEM medium is not supportive of MSC growth was used as negative control. [00179] Cell adhesion I (Immediate attachment) [00180] Scaffolds were loaded with freshly collected bone marrow samples from two hours. The leftover bone marrow as well as pre-loading bone marrow (BM) samples were seeded in culture plates and maintained in MACS medium that support MSC colony formation. At end of the culture, colonies/plates were formalin-fixed then stained with methylene blue to visualise colonies. [00181] Having less colonies in B and C compared to pre-loading BM (A) indicated that both scaffolds provided support for cell attachment, but more leftover colonies in titanium alone scaffold compared to titanium-Fe suggested better cell attachment for the later (Figure 12). [00182] Cell adhesion II (Long term scaffold colonization during culture) [00183] Scaffolds were loaded with freshly collected bone marrow samples then the loaded scaffolds were cultured for 2 weeks. To release cells, scaffolds were digested using collagenase. Cells were then stained with antibodies for MSC markers (CD45negative, CD73positive). [00184] Detecting MSCs (green gated cells, Figure 13) among released cells confirmed that both scaffolds provided support for bone marrow MSC attachment. As these data were shown for one set of test samples, no conclusions can be made on the superiority of either scaffold. [00185] Example 10: Demonstration of scaffold for use in femoral shaft fracture [00186] Clinical Indication showing large bone defect (few cm to several cm) resulting from trauma or disease. The illustration in Figure 17 shows an AP Radiograph of right femur (A), followed by two simulated images based on the biomechanical simulation for using bone substitute for filling the missing femoral bone (femoral fracture stabilized with an intramedullary nail). [00187] The scaffold for replacing the missing bone will have the following features: [00188] Properties of porous Ti-alloy with calcium phopshate minerals (CaP) as osteogenic scaffolds.
Figure imgf000030_0001
[00189] The composition of calcium phosphate mineral is 80 weight % CaHPO4 (dried brushite called monetite) and 20 wt%Fe2O3). Instead of CaHPO4, tri or di calcium phosphate and HAp can also be used with porous Ti structures. [00190] In long bone defects, for maintaining a continuum of scaffolds, it is essential to bridge the gaps with a glue and/or a putty. This approach provides a stable mechanical environment which will maintain a ‘bridge’ for bone guided regeneration supported by angiogenesis and osteogenesis. [00191] The scaffold may be loaded also with autologous bone, platelet rich plasma harvested from the patient and/or exogenous growth factors, existing commercially such as the bone morphogenic proteins (e.g. BMP-2). Liposomes as carriers of growth factors may also be used in the scaffold to be implanted to restore the bone segment missing and in the bone glue. [00192] Example 11: Glue and Putty containing cerium oxide synthesis: [00193] Constituents of Materials used for Glue Synthesis ^ Graphene (Goodfellow Cambridge CAT: 4447-5224-38, part number: GR006096, bulk density: 0.030 gcm-3, platelet thickness: 3 + 2nm (9 + 6 layers). ^ Cerium (IV) oxide nanopowder (Sigma-Aldrich, product number: 544841, CAS: 1306-38-3, molecular weight: 172.11 g/mol, < 25nm particle size (BET). ^ High molecular weight chitosan (CAS: 9012-76-4, product number: 419419, viscosity: 800-2000cps (c=1%; 1% acetic acid), deacetylation: > 75%. ^ Collagen: (Sigma-Aldrich, product number: 08-115, Collagen Type I, Rat Tail). [00194] The ratio of mineral to chitosan must be 1:4, as established from prior experiments. The ratio is essential to ensure laser sintering by successfully drying the glue without damaging surrounding areas. [00195] Chitosan Stock Solutions [00196] The 3(wt)% and 5(wt)% chitosan stock solutions was prepared by dissolving high molecular weight chitosan flakes (3100000 – 3750000Da, >75% deacetylated) in a 2(v/v)% acetic acid solution under continuous mixing for 24 hours.20 v/v% (solution) collagen was added during the synthesis of the chitosan solutions before the addition of the minerals (see below). This implies that all glue and putty mixtures have collagen-type I, which was obtained from the commercial source. The resulting transparent CS stock solutions contained numerous air bubbles; therefore, the CS solutions was placed into an ultrasonic water bath for 4 hours. [00197] 10 mol% Iron doped Brushite Minerals [00198] 200 mL of a 0.1 M Ca(NO3)2∙4H2O (Fisher Chemicals, CAS:13477-34-4) aqueous solution (solution A), was heated to 37°C. At this temperature, 0.1M (NH4)3PO4 (Acros Organics, CAS:7783-28-0) solution (200 mL) mixed with 10 mol% iron nitrate powder (Fe(NO3)3·9H2O) (VWR Chemicals, CAS:7782-61-8) was added dropwise to solution A. The mixture was continuously stirred at 37°C for 2 hours, and then left to settle for 1 hour for allowing the precipitation of DCPD (CaHPO4·2H2O). The precipitated crystals were collected on a filter paper (Whatman grade 44 with pores of 3 μm), washed several times with distilled water and then dried for 24 hours at 80°C. [00199] Unloaded and Mineral Loaded Glue/Putty Compositions [00200] Each composition number refers to Glue number (Glue-1,.. etc), and so are the putties, described below. [00201] Glue Compositions [00202] Glue Compositions – 0.25g of total mineral mixed with 1.0g Chitosan to obtain an overall 1:4 ratio. Type-1 collagen was mixed as described above. [00203] Composition 1: ^ 1 (w/v)% graphene (0.0025g) ^ 2 (w/v)% cerium (IV) oxide nanoparticles (0.005g) ^ 10 mol% iron-doped brushite (0.2425g) ^ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen [00204] Composition 2: Without graphene ^ 2 (w/v)% cerium oxide nanoparticles (0.005g) ^ 10 mol% iron-doped brushite (0.2450g) ^ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen [00205] Composition 3: Without cerium oxide nanoparticles ^ 1 (w/v)% graphene (0.0025g) ^ 10 mol% iron-doped brushite (0.2475g) ^ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen [00206] Further Glue Compositions [00207] Glue Compositions – 1.25g of total mineral mixed with 5.0g Chitosan to obtain an overall 1:4 ratio. Type-1 collagen was mixed as described above. [00208] Composition 4: ^ 5(w/v)% graphene (0.0125g) ^ 5(w/v)% cerium (IV) oxide nanoparticles (0.0125g) ^ 10 mol% iron-doped brushite (1.125g) ^ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen [00209] Composition 5: Without graphene ^ 5 (w/v)% cerium oxide nanoparticles (0.0125g) ^ 10 mol% iron-doped brushite (1.1875g) ^ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen [00210] Composition 6: Without cerium oxide nanoparticles ^ 5 (w/v)% graphene (0.0125g) ^ 10 mol% iron-doped brushite (0.2475g) ^ 3 (w/v)% high molecular weight chitosan (1.0g) with collagen [00211] "Putty" Compositions – 1.25g of total mineral mixed with 5.0g Chitosan to obtain an overall 1:4 ratio. Type-I collagen was used as described in the case of glues above. [00212] Putty Composition 1: ^ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ^ 1 (w/v)% graphene (0.0125g) ^ 2 (w/v)% cerium (IV) oxide nanoparticles (0.025g) ^ 10 mol% iron-doped brushite (1.2125g) [00213] Putty Composition 2: Without graphene ^ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ^ 2 (w/v)% cerium (IV) oxide nanoparticles (0.025g) ^ 10 mol% iron-doped brushite (1.225g) [00214] Putty Composition 3: Without cerium oxide nanoparticles ^ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ^ 1 (w/v)% graphene (0.0125g) ^ 10 mol% iron-doped brushite (1.2375g) [00215] Further "Putty" Compositions – 1.25g of total mineral mixed with 5.0g Chitosan to obtain an overall 1:4 ratio. Type-I collagen was used as described in the case of glues above. [00216] Putty Composition 4: ^ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ^ 5 (w/v)% graphene (0.0625g) ^ 5 (w/v)% cerium (IV) oxide nanoparticles (0.0625g) ^ 10 mol% iron-doped brushite (1.125g) [00217] Putty Composition 5: Without graphene ^ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ^ 5 (w/v)% cerium (IV) oxide nanoparticles (0.0625g) ^ 10 mol% iron-doped brushite (1.1875g) [00218] Putty Composition 6: Without cerium oxide nanoparticles ^ 5 (w/v)% high molecular weight chitosan (5.0g) with collagen ^ 5 (w/v)% graphene (0.0625g) ^ 10 mol% iron-doped brushite (1.1875g) [00219] The molecular vibration spectroscopic analysis of the gluey samples 1 to 3 were analysed and characterised using the attenuated total reflection (ATR) mode in the Vertex 70 FTIR spectrometer. Each sample was scanned 200 times in the 400cm-1 to the 4000cm- 1 range at a spectral resolution was 4cm-1. The beam splitter was KBr, and the light source used was a MIR lamp. See Figure 18. [00220] The viscosity of each of Compositions 1 to 3 (glue) and Compositions 1 to 3 (putty) are shown in Figure 19. [00221] The zeta potential testing solutions were prepared by diluting the glue/putty compositions to a concentration of 2.9g/dm3. The measurements were taken in cell DTS 1070 cuvettes, and the Malvern Zetasizer equipment was utilised to conduct the zeta potentials measurements. [00222] The zeta potential data for glue compositions compared with the minerals and cerium oxide are as follows: Sample Zeta Potential (mv) Glue 1 (composition 1) 15.7 Glue 2 (composition 2) 6.3 Glue 3 (composition 3) 43.3 Cerium Oxide Nanoparticles -12.6 10% Fe DCPD -5.4 [00223] The addition of cerium oxide nanoparticles caused the zeta potential to decrease, as observed in glue composition 2 containing no graphene. The zeta potential measures average charge distribution in a dispersed medium, for the glue compositions discussed above. The zeta potential values little meaning when the medium is not dispersed, as in a glue or in a powder form. [00224] The refractive indices of chitosan, cerium oxide nanoparticles, and the 10% Fe- DCPD minerals were measured using the prism coupling technique rand found to be 1.52, 2.10 and 1.65, respectively at 633nm. These data are used for ascertaining the laser power absorption and scattering when joining bone with putties and glues using a known laser source. [00225] Example 12 - Fourier Transform Infrared Spectroscopy (FTIR) and UV–Vis absorbance spectra of synthesised cerium oxide nanoparticles [00226] Figure 20 (a) shows a comparison of synthesised cerium oxide nanoparticles calcined at 280 °C, 385 °C, and 815 °C, analysed using a Vertex 70 FTIR spectra from 400 to 4000 cm−1. The operating parameters consisted of a total of 32 scans at a resolution of 4 cm-1. [00227] Figure 20 (b) shows the UV–Vis absorbance spectra obtained from nanoparticle concentrations of 0.5 mg/ml. The inset Tauc Plot corresponds to the bandgap energies of synthesised cerium oxide nanoparticles. [00228] Example 13 - Ultra-Violet Visible Spectroscopy of nanoparticles [00229] The PerkinElmer®, LAMDA 950 UV/VIS/NIR Spectrometer was used to characterise clear suspensions of nanoparticles suspended in deionised water. The absorption spectrum data was collected between 190 and 600 nm. The maximum absorbance for cerium oxide nanoparticles is in the 210nm region, as shown in figure 21. The electronic behaviour, oxygen defects, particle size, and the bivalence of the cerium oxide nanoparticles affect the nanoparticles' optical properties. Bandgap energy Eg of the nanoparticles was investigated from the absorption spectra and calculated via the Tauc equation (equation 1), which determined the bandgap energy between valence and conduction bands of the nanoparticles spectrophotometrically: α h ʋ = A (h ʋ - Eg ) 0.5 eq (1) [00230] With A being the absorption, α refers to the absorption coefficient, hʋ is the photon energy (1240/λ), and Eg relates to the bandgap. Therefore, the bandgap energy was determined from the x-axis intersections of (αhʋ)2 vs hʋ plot (figure 21). The band energy is related to the presence of different oxidation states (Ce3+ and Ce4+) on the outer nanoparticles' surfaces, where the Ce3+:Ce4+ ratio is known to be affected by oxygen vacancies and surface defects [00231] The direct optical band gap energies for a range of nano particles synthesised under furnace dried and freeze dried conditions were found to be in the range of 3.4 eV for coarser particles to 5.6 eV, for the finest particles in our investigations. The difference in the bandgap energies is also related to the presence of different oxidation states (Ce3+ and Ce4+) on the surface of nano particles which then controls the overall charge on surface, as explained in the zeta potential measurements. The Ce3+ :Ce4+ ratio is known to be affected by oxygen vacancies and surface defects. The UV-visible absorption measurements confirmed two important properties that the absorption edge of particles are dependent on particle size, and that the particles have Ce3+/Ce4+ states present which are important for antibacterial control in glue and putty media. [00232] Example 14: Physical properties of Glue and chitosan materials [00233] Glue samples were tested in a wet environment where the film thickness was 1mm, and the data are shown in the below Table. There was no resistance observed in the dry environment in putty or glue, which means that the graphene was solely responsible high electrical conductivity. This is important to control for osteoconductance in the glue and putty materials, since the cytotoxicity data shows that the graphene containing materials are not cytotoxic. The electrical resistivity for higher graphene concentrations than 1 wt% in the glue and putty mixtures were not characterised because these materials showed high electrical conductivity at room temperature. Comparison of electrical resistivity data for glue compositions with commercial graphene
Figure imgf000036_0001
[00234] Rheometric measurements of chitosan with brushite mixtures and cerium oxide for 5, 10, and 20 wt% iron oxide doped brushite and 2 wt% cerium oxide, which were mixed with 3 wt% Chitosan dissolved in water are shown in Figure 22(a). The composition of chitosan used for making mixtures is discussed above. The viscosity of two different compositions of chitosan mixed with water are also compared Figure 22(b). [00235] Example 15: Contact Cytotoxicity Assay by Giemsa Staining (qualitative) [00236] Scaffolds in triplicate were attached to 6 well plates with the aid of steri-strips [Medisave, cat no. R1540C, the negative and positive controls, consisted of steri-stips and 40% dimethyl sulfoxide (DMSO), respectively. Dulbecco's Phosphate Buffered Saline (DPBS) was used to wash the wells twice, aspirated, and 2 ml of osteoblast cell suspension containing 1x104 cells were added to each well. The culture plates were incubated at 37˚C for 48 hours in 5 (v/v)% CO2 in an incubator. After 48 hours, the media was aspirated from the wells and washed twice with DPBS. 1ml of 10 (v/v)% neutral-buffered formalin (NBF) was added to each well and incubated for 15 minutes. The formalin was aspirated and, all wells were stained for 5 minutes using Giemsa solution, then subsequently washed using distilled water. The culture plates were air-dried for 24 hours and the cytotoxic effects were determined via qualitative evaluation. The tissue plates were examined microscopically to record any changes in cell morphology, confluency, membrane integrity, attachment and detachment of the osteoblast cells using an Olympus IX 7 Inverted Microscope under bright field illumination (CellB software; Olympus). All images were collected digitally, as shown in Figure 23. [00237] All glue compositions tested presented no cytotoxic effect. The images show that the osteoblast cells proliferated up to (adjacent) the glue samples. Glue compositions are numbered per Example 11. [00238] Example 16: Extract Cytotoxicity by XTT Assay [00239] XTT assay is used to quantify the potential cytotoxicity of glue compositions, whereby a decrease in the number of living cells decreases the total activity of mitochondrial dehydrogenases in the glue samples tested. The decrease directly correlates to the amount of orange formazan formed, as monitored by the optical density at 450nm. Glue extracts were prepared according to the ISO standard: ISO 10993-12:2007 Part 12, and the cytotoxicity of the samples was evaluated according to ISO: 10993-5:2009(E) Part 5: Tests for in vitro cytotoxicity. [00240] A decrease in the total mitochondrial dehydrogenase activity of living cells was used to quantify the potential cytotoxicity of the synthesised freeze-dried scaffolds. For the XTT assay, a decrease in mitochondrial dehydrogenase activity is correlated to the amount of orange formazan formed, and this is monitored by characterising the optical density at 450nm. The glue eluates were prepared according to the ISO standard: ISO10993-12:2007 part 12. Briefly, glue samples in triplicate were placed into 24 well plates containing 2ml of supplemented McCoy's 5A media and incubated at 37℃ in 5 (v/v)% CO2 for 1, 3, and 7 days. At each time point, the samples were imaged to access any change in size/change or appearance of each glue sample. Then, 330µl aliquots (n=6) of the media were collected and frozen in cryovials at -80℃ until required. [00241] Extract cytotoxicity evaluation of the synthesised freeze-dried scaffolds was assessed according to the ISO10993-5:2009(E) part 5: Tests for in vitro cytotoxicity. Briefly, cell line G292 osteoblast cells were seeded into 96 well plates at a cell density of 10,000 cells/well and incubated at 37℃ in 5 (v/v)% CO2 for 24 hours. After the stipulated time, the media was aspirated from the wells and replaced with 100µl of the thawed collected media containing scaffold eluates. The positive and negative controls consisted of McCoy's 5A media and McCoy's 5A media with 10% DMSO, respectively. The well plates were incubated at 37℃ in 5(v/v)% CO2 for 24 hours. After 24 hours, the media in the wells were removed and replaced with 100µl of McCoy's 5A media, 10% FCS and 50µl of the XTT assay solution, then incubated for 4 hours at 37℃ in 5(v/v)% CO2. After the stipulated time, 100µl were aliquoted into new 96 well plates and read on a microplate reader at 450, 570nm and 630, 670nm (reference wavelengths). The values at 650nm were deducted from 450nm to obtain the final optical density (OD). The test well ODs were normalised to the positive control ODs to measure cell viability. The data are compared in Figure 24. [00242] Statistical analysis of data across three-time points (1 day, 3 days and 7 days) was carried out using two-way ANOVA. Statistical analysis and graphical representations of the data were performed using GraphPad Prism (version 9.2.0). The normal distribution of the data was checked using the Shapiro–Wilk and Kolmogorov–Smirnov tests. The results were considered significant at a p-value of <0.05. [00243] Summary: [00244] The results are promising; all glue compositions presented minimal cytotoxic effects against the osteoblast G292 cells at all time points. In many cases, the % live seems to be above 100%, which is likely due to the addition of 10% Fe-DCPD mineral. As seen from previous experiments, the addition of iron-doped brushite can increase cell proliferation. Glue compositions are numbered per Example 11. [00245] Example 17: Antibacterial Testing [00246] Bacterial stock cultures of Escherichia coli, Staphylococcus epidermis and Pseudomonas aeruginosa were procured from a stock of 30% glycerol solutions kept at - 80 ̊C. 10μl sterile loops were used to streak Staphylococcus epidermis, Pseudomonas aeruginosa and Escherichia coli onto BHI agar plates. Inoculated plates were all incubated at 37 ̊C for 24 hours, after which a single colony was picked from each bacterium type and grown in 25ml of BHI broth in an incubator at 37 ̊C 150rpm for 24 hours. Optical density (OD) measurement is a widely used method to assess the number of growing bacteria in a culture; thus, the absorbance values of bacterial suspensions can be measured using a photometer. The initial optical density of each bacterial type was measured using the Jenway 6305 UV/Visible Spectrophotometer at 600nm (OD600). Triplicate bacterial solutions for each bacterium were produced, and the growth rate with the addition of the glue samples was measured. [00247] In Figures 25a and 25b, the effect of glue compositions Glue 1, Glue 2 and Glue 3 as numbered per Example 11 on the abundances of E Coli and S Epidermis is compared. [00248] In Figure 26a and 26b, the reference point is the cultured bacteria (CB) and the E- coli and S epidermis data are also compared the nano-scale cerium oxide. [00249] In the table below the effectiveness of nano-scale size cerium oxide on antibacterial activities is compared for three different strains. The antibacterial effectiveness of the synthesized CeO2 nano particles at a concentration of 200 µg/ml (0.2g/litre) is compared for three different strains of bacteria. FRNP refers to freeze dried nano particles which are compared with commercial C385 and C815 powders, which were heat treated at 385o and 815oC, respectively. The dose specified is considerably small, and if increased to higher concentrations the effectiveness of antibacterial control will increase.
Figure imgf000039_0001
[00250] Summary: The antibacterial tests for the glue samples with low and high graphene, cerium oxide, chitosan and brushite mixtures have been tested. The data for S Epidermis and E coli show the reduction in bacterial colonies at these modest concentrations of cerium oxide and graphene in a mixture of iron oxide doped brushite and chitosan. Larger ratios of cerium oxide and graphene with brushite seem more effective in antibacterial control in general.

Claims

CLAIMS 1. A composition comprising a chitosan gel as the primary component, wherein the composition comprises: (i) an iron-doped calcium phosphate mineral component; and (ii) a collagen component.
2. The composition of claim 1, wherein the iron-doped calcium phosphate mineral component is present at an amount of from about 1% w/w to about 30% w/w of the chitosan gel.
3. The composition of claim 2, wherein the iron-doped calcium phosphate mineral component is present at an amount of from about 10% w/w to about 25% w/w of the chitosan gel.
4. The composition of any preceding claim, wherein the collagen component is present at an amount of from about 1% w/w to about 30% w/w of the chitosan gel.
5. The composition of claim 4, wherein the collagen component is present at an amount of from about 10% w/w to about 25% w/w of the chitosan gel.
6. The composition of any preceding claim, wherein the iron-doped calcium phosphate mineral component is an iron-doped component selected from: hydroxyapatite (Ca10(PO4)6(OH)2), fluorapatite (Ca5(PO4)3F), brushite (CaHPO4.2H2O), β-pyrophosphate, monetite (CaHPO4) and tricalcium phosphate (Ca3(PO4)2).
7. The composition of any preceding claim, wherein the iron-doped calcium phosphate mineral component is fluoride free.
8. The composition of any preceding claim, wherein the iron-doped calcium phosphate mineral component is doped with Fe2O3 or a mixture of FeO and Fe2O3.
9. The composition of any preceding claim, wherein an iron-containing component is present in the iron-doped calcium phosphate mineral component in an amount of up to 30% w/w of the iron-doped calcium phosphate mineral component.
10. The composition of any preceding claim, further comprising a graphene, graphene oxide, reduced graphene oxide or nanocarbon component.
11. The composition of claim 10, wherein the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in the composition in amount of from 0.01% to 5% w/w of the chitosan gel.
12. The composition of claim 11, wherein the graphene, graphene oxide, reduced graphene oxide or nanocarbon component is present in amount of from 0.1% to 1% w/w of the chitosan gel.
13. The composition of any of claims 10 to 12, wherein the graphene oxide has a C:O ratio of from 6:1 to 1:1.
14. The composition of any of claims 10 to 12, wherein the reduced graphene oxide has a C:O ratio of from 20:1 to 6:1.
15. The composition of any preceding claim, further comprising a metal oxide nanoparticle.
16. The composition of claim 15, wherein the nanoparticle is selected from the group consisting of cerium oxide and strontium oxide.
17. The composition of any preceding claim, wherein the composition is in the form of a glue.
18. The composition of claim 17, wherein the glue has a viscosity of from 1 to 10 Pa-s.
19. The composition of any of claims 1 to 15, wherein the composition is in the form of a putty.
20. The composition of claim 19, wherein the putty has a viscosity of from 10Pa-s to 10,000 Pa-s.
21. The composition of any of claims 17 to 20, wherein the chitosan gel comprises chitosan in an amount of from 1 to 10% w/w of the chitosan gel, wherein the chitosan gel is an aqueous based gel.
22. The composition of any of claims 17 to 21, wherein the chitosan has a molecular weight of from less than 10 kilo Dalton to 2220 kilo Dalton.
23. A freeze-dried composition comprising the composition of any preceding claim that has been subjected to one or more processing steps to remove solvents.
24. A bone scaffold comprising: (i) a first layer comprising a porous titanium/TiO2-based composition; (ii) a second layer comprising a composition of claim 23.
25. The bone scaffold of claim 24, wherein the porous titanium-based composition comprises Ti alloy or a mixture of Ti-alloy with TiO2.
26. The bone scaffold of claim 24 or 25, wherein the porous titanium-based composition further comprises an iron-doped calcium phosphate mineral component.
27. The bone scaffold of any of claims 24 to 26 wherein the porous titanium-based composition further comprises Fe2O3 nanoparticles.
28. The composition of any of claims 1 to 22 or the bone scaffold of any of claims 24 to 27 for use in surgical bone repair.
29. The composition of any of claims 1 to 22 or the bone scaffold of any of claims 24 to 27 for use as a prophylactic to prevent the risk of bone failure.
30. The composition of any of claims 1 to 22 or the bone scaffold of any of claims 24 to 27 for use in the treatment of a condition selected from: bone fractures (diaphyseal, metaphyseal and with intra-articular extension), craniofacial fracture, bone gaps, critical size bone defects, bone voids, comminuted fractures, segmental fractures, open fractures, fragility/osteoporotic fractures, fracture non-union, avascular necrosis, bone infection, diabetic foot, and bone failure.
31. A use of a graphene, graphene oxide, reduced graphene oxide or nanocarbon component for enhancing osteo-conduction of a bone scaffold.
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