FIELD OF THE INVENTION

[0001] The present invention relates to the field of films and the production thereof, in particular, the present Invention relates to the production of free-standing mineral or mineral-composite films. The present invention also relates to the production of mineral or mineral-composite films that are formed as coatings on substrates. Applications of the invention include, but are not limited to, medical devices, research, and tissue engineering. However, it will be appreciated that the invention is not limited to these particular fields of use.

BACKGROUND OF THE INVENTION

[0002] The following discussion of the background and prior art provides context to the invention. However, discussion of prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Mammalian bone mineral is carbonate-rich hydroxyapatite and calcium carbonate is regarded as a model for biomineralization research. Hence, the synthesis of mineral and mineral-composite films comprised of these minerals have widespread commercial applications spanning multiple fields, including, inter alia, medical devices, research, and tissue engineering. Such films could be formed as a coating on orthopaedic implants for enhanced integration with bone (osseointegration) or as a free-standing film that functions as a scaffold for biomineralization or in vitro research.

[0004] The incidence of joint replacement surgeries is increasing as longer lives and greater activity levels drive the need for repair and replacement. Orthopaedic implants are central to these procedures and recovery is directly influenced by the osseointegration and bioresorbability of these devices. Calcium-based minerals are ideal in this regard, owing to their chemical, structural, and morphological similarity to bone mineral. However, they are inappropriate as the bulk material for weight-bearing orthopaedic implants due to their brittleness. Generally, non-ceramic materials, such as polyether-ether ketone (PEEK) and titanium (Ti), are most used to fabricate orthopaedic implants because they meet the standards for mechanical properties.

[0005] The bulk material of the implant is commonly coated with materials conducive to bone growth, such as commercially pure Ti and hydroxyapatite (H-Ap). Ti has a higher Young’s modulus than bone, is chemically different from bone mineral, and causes interference with imaging. H-Ap has been used as a standard orthopaedic material for decades but limitations, such as low resorbability and contribution to the requirement of revision surgery, have been reported. Carbonate-rich hydroxyapatite (C-Ap) potentially addresses the issues with Ti and H-Ap. C-Ap exhibits chemical similarity to bone mineral, displays bioresorbability, and has higher osteoconductivity. However, the decomposition of C-Ap at typical sintering temperatures has been a key impediment to its clinical application in the past.

[0006] Common commercial strategies to coat implants with H-Ap, such as plasma spray and cold spray techniques, result in weakly adhered and thick coatings (>100 μm), multiple phases, and high temperatures at the coating-substrate interface that exceed the glass transition temperature of some bulk implant materials (e.g. PEEK, 143°C). Additionally, their unidirectional application may be inappropriate for the complex structural features characteristic of state-of-the-art implants made with additive manufacturing techniques.

[0007] Mineral or mineral-composite coatings also have applications in research and tissue engineering owing to their resemblance to biominerals. Sintered H-Ap blocks and uncoated glass cover slips are typically used as scaffolds for these purposes. Glass cover slips coated with nanocrystalline calcium-based minerals represents a cost-effective, scalable, and user-friendly alternative that more closely resembles biominerals. Mineral or mineral-composite coatings on glass could also be incorporated in microfluidic devices for bone-on-a-chip devices that mimic the bone microenvironment.

[0008] Free-standing mineral or mineral-composite films represent an alternative to coated substrates for applications in research and tissue engineering. Other functional applications have been reported in the literature for free-standing mineral films, including superoleophobicity, self- cleaning, and filtration. Despite their importance, free-standing mineral films are still rarely reported - partly owing to the technical challenges of forming a crystalline template/substrate-free film at low temperatures.

[0009] In the 2000s, however, free-standing mineral films were produced, but they required heat treatment to convert amorphous precursors to a crystalline phase. These methods also required the use of a polymer template to initiate deposition of the bulk film. For example, some workers have formed carbonate-rich dicalcium hydroxide phosphate (carbonate-rich hydroxyapatite) films (~1 μm) by a template-growth inhibition method that requires subsequent heat treatment at 900 °C to convert an amorphous precursor to a crystalline film. Other workers in the field have formed a crystalline carbonate-rich hydroxyapatite film (~1 μm) by using an octadecanoic acid (stearic acid) monolayer as a template to induce deposition from a solution of calcium phosphate, followed by subsequent heat treatment at 700 °C.

[0010] Other previous studies have reported the production of continuous crystalline films (~1 μm) at relatively low temperature. However, the films were comprised of vaterite, which is an unstable polymorph of CaCO3. Additionally, poly(l-carboxyethylene) (poly(acrylic acid)) was used as a template in a saturated solution of calcium hydrogen carbonate (Ca(HCO3)2 prepared by the Kitano method (viz., Y. Kitano, “A study of the polymorphic formation of calcium carbonate in thermal springs with an emphasis on the effect of temperature”, Bull. Chem. Soc. Jpn. 35 (1962) 1980-1985). Therefore, whilst free-standing mineral films have been produced in the past, they are characterised by the incorporation of an organic phase that enables the formation of crystalline films of a stable phase at low temperatures.

[0011] It is an object of the present invention to overcome or ameliorate one or more the disadvantages of the prior art, or at least to provide a useful alternative.

[0012] It is an object of the present invention to provide a process that enables the production of a mineral or mineral-composite film is free-standing or forms a coating on a substrate. Applications include, but are not limited to, research, tissue engineering, and medical devices.

[0013] It is an object of at least one preferred form of the present invention to provide a process for forming free-standing mineral or mineral-composite films that exhibits at least one of the following advantages: ability to introduce functional grading or produce different phases of calcium-based minerals, additive-free, option for low temperatures that are below the denaturation temperature of biologies.

[0014] It is an object of at least one preferred form of the present invention to provide a process for forming a mineral or mineral-composite film as a coating on a substrate at least one of the following advantages: multidirectional application that is suitable for standard surfaces and/or complex structural features characteristic of additively manufactured surfaces, ability to form a contiguous or discontinuous layer on a substrate, rapid application, option for low temperatures that are below the denaturation temperature of biologies and glass transition temperatures of substrates, ability to introduce functional grading or produce different phases of calcium-based minerals, and is scalable. It is a further object of at least one preferred form of the invention to provide a mineral-or mineral- composite film as a coating on a substrate with at least one of the following advantages over prior art: reduced laws (irregularities and discontinuities), tuneable thickness, bonding with the substrate (chemical or mechanical interlocking), tribological/mechanical properties , osseointegration, bioresorbability, optional functional graded coatings or different phases of calcium-based minerals, incorporation of biologies.

SUMMARY OF THE INVENTION

[0015] According to a first aspect, the present invention provides a method for providing a mineral or mineral-composite film, the method comprising the steps of: a) providing a layer comprising a calcium-based mineral, wherein the layer is produced using convective self-assembly (CSA) of particles in a suspension of a calcium-based mineral and/or by precipitation of a calcium-based mineral, and b) conversion of at least some of the layer to apatite, with or without the incorporation of biologies, to thereby produce said mineral or mineral-composite film.

[0016] According to a second aspect, the present Invention provides a method for providing a mineral film, the method comprising the steps of: a) providing a Ca(HCO3)2 solution having an air-solution interface, b) forming a layer comprising a CaCO3 film at the air-solution interface, and c) conversion of at least some of the layer to apatite, with or without the incorporation of biologies, to thereby produce said mineral or mineral-composite film.

[0017] It will be appreciated that the CaCO3 forms by nucleation and intergrowth of crystals at the air-solution interface.

[0018] In some preferred embodiments, the Ca(HCO3)2 solution is prepared by dissolving particles of CaCO3 in a reactor at elevated pressure, for example at 6 MPa for 30 mins.

[0019] In some preferred embodiments, the apatite is selected from hydroxyapatite (HAp) or carbonate-rich hydroxyapatite (CAp).

[0020] According to a third aspect, the present invention provides a method for coating a substrate with a mineral or mineral-composite film, the method comprising the steps of: a) coating on said substrate a layer comprising a calcium-based mineral, wherein the layer is produced using CSA of particles in a suspension of a first calcium-based mineral and/or by precipitation of a second calcium-based mineral, and b) and/or conversion of at least some of the layer to apatite, with or without the incorporation of biologies, to thereby produce said mineral or mineral-composite film as a coating on said substrate.

[0021] It will be appreciated that in some embodiments, the first calcium-based mineral is the same as the second calcium-based mineral. In other embodiments, the first calcium-based mineral is different to the second calcium-based mineral.

[0022] It will be appreciated that in a preferred embodiment of the third aspect, the layer is produced using CSA of a CaCO3, followed by precipitation of CaCO3, the first calcium-based mineral is the same as the second calcium-based mineral. [0023] In another embodiment of the third aspect, the layer is produced using CSA of a calcium- based mineral (such as H-Ap), followed by precipitation of a CaCO3. In this embodiment, the first calcium-based mineral is different to the second calcium-based mineral. Other embodiments will be apparent to the skilled person.

[0024] According to fourth aspect, the present invention provides a method for coating a substrate with a mineral film, the method comprising the steps of: a) contacting said substrate with a calcium-based mineral suspension and using CSA to thereby produce a coated substrate, b) providing a Ca(HCO ₃ ) ₂ Solution, c) contacting the coated substrate with said Ca(HCO3)2 solution and precipitating CaCO3 on said coated substrate, and d) converting at least some of the layer to apatite, with or without the incorporation of biologies, to thereby produce said mineral film as a coating on said substrate.

[0025] In preferred embodiments, the Ca(HCO3)2 solution is prepared by dissolving particles of CaCO3 in a reactor at elevated pressure, for example at 6 MPa for 30 mins.

[0026] In preferred embodiments, the apatite is selected from hydroxyapatite (HAp) or carbonate-rich hydroxyapatite (CAp).

[0027] According to a fifth aspect, the present invention provides a method for coating a substrate with a layer of calcium-based mineral, the method comprising the steps of: a) contacting said substrate with a suspension of a calcium-based mineral and using CSA to produce said layer coated to said substrate.

[0028] According to a sixth aspect, the present invention provides a method for coating a substrate with a layer of calcium-based mineral, the method comprising the steps of: a) contacting said substrate with a suspension of a calcium-based mineral and using CSA to produce said layer of calcium-based mineral coated to said substrate, and b) providing a Ca(HCO3)2 solution, and c) contacting the coated substrate with a Ca(HCO3)2 solution and precipitating CaCO3 on said coated substrate to thereby produce said layer of CaCO3 coated to said substrate. [0029] According to a seventh aspect, the present invention provides a method for coating a substrate with a layer of calcium-based mineral, the method comprising the steps of: a) contacting said substrate with a suspension of a calcium-based mineral and using CSA to produce said layer of calcium-based mineral coated to said substrate, and b) converting at least some of the layer to apatite, with or without the incorporation of biologies, to produce said mineral film as a coating on said substrate.

[0030] According to an eighth aspect, the present invention provides a method for coating a substrate with a layer of calcium-based mineral, the method comprising the steps of: a) providing a Ca(HCO3)2Solution, and b) contacting the substrate with a Ca(HCO3)2 solution and precipitating CaCO3 on said substrate to produce said layer of CaCO3 coated to said substrate.

[0031] According to a ninth aspect, the present invention provides a method for coating a substrate with a layer of calcium-based mineral, the method comprising the steps of: a) providing a Ca(HCO3)2 solution, and b) contacting the substrate with a Ca(HCO3)2 solution and precipitating CaCO3 on said substrate to thereby produce said layer of CaCO3 coated to said substrate. c) converting at least some of the layer to apatite, with or without the incorporation of biologies, to thereby produce said mineral film as a coating on said substrate.

[0032] According to a tenth aspect, the present invention provides a substrate coated with a layer of calcium-based mineral when produced by the method according to the fifth, sixth, seventh, eighth, or ninth aspects.

[0033] It will be appreciated that, for the purposes herein, reference to a suspension may also include a solution that forms a suspension.

Preparation of a Solution of Ca(HCO3)2

[0034] In a preferred embodiment of the present invention, the Ca(HCO3)2 solution is a supersaturated Ca(HCO3)2 solution. However, it will be appreciated that, in some embodiments, a solution that is not supersaturated can still be used with substantially similar effect as a supersaturated solution. The person skilled in the art will understand that supersaturation occurs with a chemical solution when the concentration of a solute exceeds the concentration specified by the equilibrium solubility under standard -state conditions. Most commonly, the term is applied to a solution of a solid in a liquid. A supersaturated solution is in a metastable state; it may be brought to equilibrium by forcing the excess of solute to separate from the solution.

[0035] As will be explained below, a Ca(HCO3)2 solution can be prepared under certain conditions of pressure, temperature, and composition. However, it will be appreciated by the person skilled In the art that other methods or combinations of methods to prepare an equivalent or substantially similar or identical solution are possible. In one preferred embodiment, a Ca(HCO3)2 solution is prepared by dissolving CaCO3 in water that is saturated with CO ₂ at elevated pressure (e.g., 8 MPa) in order to achieve a CaCO3 solubility of approximately 1 ,250 mg/L at approximately 25°C. However, it will be appreciated that other pressures will be possible, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, or 20 MPa, or any pressure in between. Additionally, it will be appreciated that other dissolution temperatures will be possible, such as 1 , 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280 or 300°C, or any temperature in between. It also will be appreciated that the temperature and pressure can be selected in appropriate combinations to achieve a CaCO3 solubility of approximately 1 ,250 mg/L. It will be appreciated that other predetermined or target solubilities will be appropriate for the invention, such as 1 , 5, 10, 15, 20, 25, 30 ,40, 50, 100, 200, 300, 400, 500, 750, 1000, 1250, 1500, 2000 or 2500, 3000, 4000 or 5000 mg/L, or any concentration in between. It will also be appreciated that while a range of solubilities is appropriate, more concentrated solutions are preferred. In preferred embodiments, preparation of the solution is conducted over a reaction time of approximately 30 minutes. However, it will be appreciated that other reaction times fall within the purview of the present invention, for example, 5, 10, 15, 20, 25, 35, 40, 45, 60, 90, 120 or 240 minutes, or any reaction time in between.

[0036] In a preferred embodiment of the present Invention the CaCO3 used to prepare the solution is in the form of nanoparticles that have an average particle size of approximately 90 nm. It will be appreciated by the person skilled in the art that while smaller particle sizes are preferred, a range of suitable particle sizes is suitable, for example, 1 , 5, 10, 25, 50, 75, 100, 200, 500 or 1000 nm or any particle size in between.

[0037] The solution prepared according to the method of the invention is an advance over prior art solutions. For example, CaCO3 solubility is very limited in -water under atmospheric conditions (approximately 10 mg/L) but methods such as those used by Kitano (bubbling CO2 through the solution) can be used for modest increase in this solubility. The method of the present invention is superior to the Kitano method, which achieves lower levels of Ca(HCO3)2 saturation.

[0038] In preferred embodiments of the present invention, a Ca(HGG3)2 solution is provided. However, it will be appreciated that at least some of the Ca can be substituted with other group two elements, such as beryllium, magnesium, strontium and/or barium. Substrates for Coating

[0039] In preferred embodiments, the substrate is a surface of an implant. The substrate may be a portion of the implant or the entire surface of the implant.

[0040] In preferred embodiments, the substrate is polyether ether ketone (PEEK) or a PEEK containing composite. However, the person skilled in the art will appreciate that the substrate also can be any biocompatible material known in the art, for example, titanium, tantalum, cobalt, chromium, and their respective alloys, polyurethane, stainless steel, PEEK, PEEK composites, ceramics, glass, polymeric materials such as biopolymers, bioceramics, and the composites or combinations thereof.

[0041] The implant may be formed from one material and coated with another, for example, the implant may be formed from a material selected from the group consisting of: titanium, tantalum, cobalt, chromium, and their respective alloys, polyurethane, stainless steel, PEEK, PEEK composites, ceramics, glass, polymeric materials and the composites or combinations thereof and coated with a material selected from the group consisting of: titanium, tantalum, cobalt, chromium, and their respective alloys as well as biopolymers, bioceramics, and composites or combinations thereof.

[0042] Some or all of the substrate surface may be treated to adjust its surface roughness reactivity, and/or wettability through techniques such as hand grinding, plasma treatment or corona discharge.

In preferred embodiments, the substrate surface has a roughness profile (R _a ) of approximately 0.3-0.4 μm. It will be appreciated that a range of surface roughness profiles is appropriate, for example, 0.0- 0.1 , 0.1 -0.2, 0.2-0.3, 0.4-0.5, 0.6-0.7, 0.8-0.9 or 0.9-1 .0 μm, or any roughness profile in between.

[0043] Suitable implants that may be coated with the mineral film of the invention may be selected from the group consisting of: orthopaedic, dental, oral and/or craniofacial implants (including, but not limited to, intervertebral spacers, spinal implants), orthopaedic screws, nails, pins, plates, and prostheses. The person skilled in the art will appreciate that the method of the present invention is not limited to the examples provided, and may be applied to any implant in which osseointegration is advantageous. In a preferred embodiment, the coating method of the present invention is multidirectional in that it can coat a substrate having complex structural features, including open porosity. It will be appreciated that due to the multidirectional nature of the present method, it may be used on substrates of any shape or size.

[0044] The person skilled in the art will appreciate that the method of the present invention is not limited to biomedical materials and procedures. Any material or device that would be advantaged by the application of a surface coating, particularly when applied at room temperature and so avoiding interaction with or alteration of the substrate, can utilise the invention. Such applications include, but are not limited to, surface coatings on metals for corrosion and thermal protection, surface coatings on polymers for chemical and/or thermal protection, and surface coatings on ceramics chemical and/or thermal protection. In some embodiments, the surface coating may provide the functional aspect itself rather than this being provided by the substrate. Such applications include, but are not limited to, the present invention, which provides a bioactive surface coating, and optical coatings

Mineral Film Properties

[0045] in some embodiments, the mineral films that are produced by the methods of the present invention are free-standing. A free-standing film is defined as a film that is formed without contact with a support material such as a substrate or scaffold. In preferred embodiments, the free-standing mineral film can have a thickness of: 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7', 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9,

10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 μm, or any thickness in between. In preferred embodiments, the “air surface” (i.e., the surface of the film that is exposed to air during production) has a roughness of approximately Ra = 2.3 μm, and the “solution surface” (i.e., the surface of the film that is immersed in the reaction solution during production) has a roughness of about Ra = 7.4 μm. It also will be appreciated that the air surface roughness may vary according to the specific reaction conditions. For example, the Ra may be 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4,

2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4, 5, 6, 7, 8, 9 or 10 μm, or any Ra value in between. It will also be appreciated that the “solution surface” roughness may vary according to the specific reaction conditions, for example, the Ra may be 0.1 , 0.5, 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30 ,35, 40 or 50 μm, or any Ra value in between. In preferred embodiments of the present invention, the “solution surface” of the film has a Ca/P ratio of approximately 1 .63, however it will be appreciated that a range of Ca/P ratios can be produced, for example 1 ,0, 1 .10, 1 .20, 1 .30,

1 .40, 1 .50, 1.51 , 1 .52, 1 .53, 1 .54, 1 .55, 1 .56, 1 .57, 1 .58, 1 .59, 1 .60, 1 .61 , 1 .62, 1 .63, 1 .64, 1 .65, 1 .66,

1 .67, 1 .68, 1 .69, 1 .70, 1 .80, 1 .90, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0, or any Ca/P ratio in between.

[0046] In other embodiments, the mineral film is coated onto a substrate. In preferred embodiments, when coated onto a substrate, the mineral film can have a thickness of approximately: 0.1 , 0.2, 0.3,

0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, 2.5, 2.7, 3.0, 4.0, 5, 10, 20, 50, 100, 200, 400, 500, 600, 700, 800,

900, or 1000 μm or any thickness in between. It will be appreciated that the coated substrate may have an increased surface roughness compared to an uncoated substrate or vice versa. In preferred embodiments, the coated substrate has a surface roughness of approximately Ra = 0.6 μm (for example, when the substrate is PEEK OPTIMA™ HA) or about Ra = 1 .2 μm (for example when the substrate is PEEK OPTIMA™). It also will be appreciated that the surface roughness may vary, for example, the Ra may be 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30 ,35, 40 or 50 μm, or any Ra value in between. In preferred embodiments of the present invention, the mineral film that is coated onto the substrate has a Ca/P ratio of approximately 1 .62. However, it will be appreciated that a range of Ca/P ratios can be produced, for example, 1 .0, 1.10, 1 .20, 1 .30, 1 .40, 1 .50, 1 .51 , 1 .52, 1 .53, 1 .54,

1 .55, 1 .56, 1 .57, 1 .58, 1 .59, 1 .60, 1.61 , 1 .62, 1 .63, 1 .64, 1 .65, 1 .66, 1 .67, 1 .68, 1 .69, 1 .70, 1 .80, 1 .90, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0, or any Ca/P ratio in between. In a preferred embodiment, the contact pressure is about 327 MPa (when the substrate is PEEK OPTIMA™ HA) or about 284 MPa (when the substrate is PEEK OPTIMA™). It will be appreciated that the contact pressure may vary with factors such as substrate composition, coating thickness and coating composition, and as such a range of contact pressures can be produced, for example, 50, 100, 150, 200, 250, 275, 300, 325, 350, 375,

400 , 425, 450, 500, 600, 700 or 800 MPa, and any contact pressure in between. In a preferred embodiment, the coating has an adhesion strength of 0.67 MPa (when the substrate is PEEK OPTIMA™ HA) or about 0.73 MPa (when the substrate is PEEK OPTIMA™). It will be appreciated that the adhesion strength may vary with factors such as substrate composition, coating thickness and coating composition, and as a result the coating can comprise a range of adhesion strengths, for example, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.5, 0.51 , 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61 , 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71 , 0.72, 0.73, 0.74, 0.75 ,0.76, 0.77, 0.78, 0.79, 0.80, 0.85, 0.90, 1 .0, 1 .2, 1 .4, 1 .6, 1 .8, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 MPa or even higher, or any adhesion strength in between.

Biologies and/or Bioactive Compounds

[0047] In preferred embodiments, the methods disclosed herein for producing a mineral film also may include biologies to elicit responses in cells. The biologies may be incorporated into the mineral film as it is being produced, adsorbed on the surface, and/or absorbed into the pores of the final film once produced. It will be appreciated that incorporating the biologies into the mineral film as it is being produced is preferred as this may result in a more uniform distribution of biologies throughout the film. It also will be appreciated that the biologies may be any biocompatible elements or compounds that produces a desirable biological response, such as osteoinduction, antimicrobial activity, antibacterial activity, antiviral activity or antifungal activity. Biologies related to orthopaedic applications for patients with or without comorbidities include bone morphogenic proteins, recombinant bone morphogenic proteins, p15 peptide, and sclerostin. Other biologies can be copper, zinc, silver, gold or other metals and metal-based composites with antibacterial effects, or other compounds/materials such as cephalosporin, polypeptide antibiotics, fluoroquinolone, acyclovir, ganciclovir, idoxuridine, amantadine, interferon, azidothymidine, nystatin, amphotericin B, liposomal amphotericin B, flucytosine, aminoglycosides such as gentamicin, kanamycin, neomycin, paromomycin, streptomycin, or tobramycin; rifamycin, or ansamycin such as rifampin, cephaloporins such as cephalexin, cephaloridin, cephalosin, defazoline, cephapirin, cefradine or cephaloglysin; chloramphenicol; macrolides such as erythromycin, tylosin, oleandomycin or spiramycin; penicillin G & V, pheneticillin, methicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin, ampicillin, amoxicillin, or penicillins such as carbenicillin; tetracycline; trimethoprim-sulfamet xoxazoles; polypeptides such as bacitracin, polymyxin, thyrosurisin or vancomycin: as well as various antibiotics such as lincomycin, clindamycin, or spectinomycin, fungicidal agents such as phenol; cresol; resorcinol; substituted phenol; aldehyde; benzoic acid; salicylic acid; iodine; iodophors such as betadine; chlorophores such as hypochlorite, peroxides such as hydrogen peroxide and zinc peroxide; heavy metals such as merbromine, silver nitrate, zinc sulfate and salts thereof; surfactants such as benzalkonium chloride; furan derivatives such as nitrofurazone; thiosulfates; salicylanilides; and carbanilides. [0048] As described above, the present invention produces mineral films at relatively low temperatures. This enables the more uniform incorporation of biologies into the mineral films of the invention, and additionally enables the incorporation of thermally sensitive biologies that would not be otherwise possible using prior art processes. Accordingly, it is an advantage of the invention to be able to utilise a broader range of biologies and therefore achieve significant improvements in the functionality of the resulting mineral film that would not be possible, or would be extremely difficult to do, with prior art mineral films that are produced at relatively high temperatures.

Convective Self-Assembly (CSA) step

[0049] When utilising the methods of the invention to coat a substrate (or prepare free standing films), one of the first typical steps is to coat the substrate with a layer of CaCO3 to form a coated substrate. A preferred method is through a convective self-assembly (CSA) step, which is a process whereby the substrate is immersed in a solution containing CaCO3 nanoparticles and heated at a temperature sufficient to cause evaporation of the solution. As the solution evaporates, the CaCO3 grains self-assemble on the surface of the substrate through capillary action at the meniscus, thus forming a coating. It will be appreciated that there may be other methods to form an equivalent or substantially similar or identical coating on the substrate surface.

[0050] In some embodiments, the convective self-assembly (CSA) step comprises the steps of: a) immersing the substrate in a suspension or solution of Ca(HCO3)2 and CaCO3, and b) heating the suspension and the substrate at approximately 130°C for approximately 45 minutes, and c) removing the substrate from the prior suspension or solution and immersing it in a suspension or solution of CaCO3 nanoparticles without Ca(HCO3)2for about 10 minutes, and d) heating the suspension or solution of CaCO3 and the substrate at approximately 130°C for approximately 2-3 hours, so as to promote evaporation of some or all of the suspension and until the substrate is coated.

[0051 ] In some embodiments, the convective self-assembly step comprises the steps of: a) immersing the substrate in a suspension of CaCO3 nanoparticles and Ca(HCO3)2, and b) heating the suspension and the substrate at about 130°C for approximately 45 minutes, and c) removing the substrate from the prior suspension and immersing the substrate in a suspension of CaCO3 nanoparticles without Ca(HCO3)2, and d) heating the suspension and the substrate at about 130°C to promote evaporation until the substrate is coated.

[0052] In some embodiments, the convective self-assembly step comprises the steps of: a) immersing the substrate in a solution of CaCO3 for about 10 minutes, and b) heating the CaCO3 solution and the substrate at about 130°C for approximately 2-3 hours, so as to promote evaporation of some or all of the suspension.

[0053] During step a) of the convective self-assembly step, the concentration of CaCO3 is preferably 0.375 wt%. However, it will be appreciated that a range of concentrations is suitable. For example, 0.050, 0.0750, 0.100, 0.125, 0.150, 0.200, 0.250, 0.300, 0.350, 0.400, 0.450, 0.500, 0.550, 0.600, 0.700, 0.800, 0.900, 1 .000 wt% or any concentration in between. In a preferred embodiment, during step a) of the convective self-assembly step, the concentration of Ca(HCO3)2 is about 0.125 g/L. In some embodiments, the reaction time of steps b) and d) of the convective self-assembly step may be selected independently from the group consisting of: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes or 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 8.0, 12.0, 16.0, 20.0, 24.0, 48.0 or 72.0 hours, or any reaction time in between. It will be appreciated that the temperature of steps b) and d) of the convective self-assembly step are tuneable and may be selected independently from the group consisting of: 1 , 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,

115, 120, 125, 130, 135, 140, 145, 150, 175, 200, 250, 300 , 400, 500, 600, 700, 800 or 850°C or any temperature in between.

‘Cold Sintering’ step

[0054] When utilising the methods of the invention to coat a substrate (or a free standing film), one of the steps is to immerse the substrate in a solution of Ca(HCO3)2 and subsequently apply a relatively low amount of heat to the substrate. This process is defined as the “cold sintering” step, due to the relatively low temperatures required compared to traditional sintering techniques. In preferred embodiments, the “cold sintering” step is performed at a temperature of approximately 130°C. It will be appreciated that the “cold sintering” step may be performed at a range of different temperatures, for example: 1 , 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 175, 200, 250, 300 , 400, 500, 600, 700, 800 or 850°C or any temperature in between. In preferred embodiments, the “cold sintering” step has a reaction time of approximately 10 minutes. However, it will be appreciated that this step can have a range of reaction times, for example: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes or 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5,

4.0, 4.5, 5.0, 5.5, 6.0, 8.0, 12.0, 16.0, 20.0, 24.0, 48.0 or 72.0 hours, or any reaction time in between. Conversion Step

[0055] A characteristic of the method of the present invention is the use of phosphate buffered saline (PBS) to convert at least some of a primary CaCO3 layer into apatite. In embodiments where the mineral film is free-standing, a sufficient amount of the primary CaCO3 layer may be converted to a secondary apatite layer to produce a layered biphasic film, for example, 50 wt%. It will be appreciated that, irrespective of whether the film is free-standing or coated onto a substrate, the method of the present invention may be used to convert any amount of the primary CaCO3 layer to apatite, for example, 1 , 5, 10, 20, 30, 40 ,50, 60, 70, 80 ,90 or 100 wt%, or any amount in between. In preferred embodiments, the conversion step has a reaction time 24 hours. However, it will be understood that the reaction time is scalable and related to the amount of the primary layer that is converted to apatite, for example, the reaction time may be 0.5, 1 .0, 3.0, 6.0, 12.0, 18.0, 24.0, 30.0, 36.0, 42.0, 48.0, 54.0, 60.0, 66.0, 72.0, 84.0, 96.0 or 108.0 hours, or any conversion time in between, in preferred embodiments, the conversion step uses PBS to convert the primary CaCO3 layer to apatite, it will be understood that other phosphate ([PCu] ^3' ) containing reagents, or combinations of reagents that produce phosphate in situ may be used, for example simulated body fluid (SBF).

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0057] Figure 1 A shows formation of the CaCO3 primary layer at the air-solution Interface of a supersaturated Ca(HCO ₃ ) ₂ solution showing an Increasing concentration gradient.

[0058] Figure 1 B shows conversion of the CaCO3 primary layer to a bone-like film by “incubation” in PBS at 80°C,

[0059] Figure 2 shows the caicite “raft” formation mechanism and the air-water interface, which has the highest saturation state. CaCO3 crystals heterogeneously nucleate and grow at the surface, eventually coalescing to form a CaCO3 “raft”, which is suspended at the air-water interface by surface tension effects.

[0060] Figure 3 shows a schematic of the CaCO3 primary layer formation mechanism: a) nucieation and formation of clusters at the surface of the calcium bicarbonate solution (similar to Fig. 1 A); b) crystal formation/growth; and c) film formation.

[0061] Figure 4 shows 3D laser confocal microscopy, Raman microspectroscopy (Raman), and scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS) micrographs of cross sections and surfaces of the mineral films: (a-d) CaCO3 primary layer and an apatite (i.e., bone-like) mineral film; (e,f) laser Raman spectral map of cross section of a bone-like mineral film, where white areas correspond to caicite at the air surface and red areas correspond to carbonate-rich hydroxyapatite at the solution surface; (g,h) SEM/EDS micrographs of a cross section of a bone-like mineral film (areas without P are calcite crystals and P-confaining areas are carbonate-rich hydroxyapatite (the circle highlights a large crystal that extends through the film with partial conversion to carbonate-rich hydroxyapatite around the perimeter); (h-j) SEM/EDS micrographs of the air surface of a bone-like mineral film.

[0062] Figure 5 shows SEM micrographs of film surfaces: (i) Air surface of CaCO3 films (digital photograph of the air surface in box): (a) Fiat air surface, with some voids and large crystal faces; (b) Arrows pointing to clusters of small crystallites (<5 μm) and larger crystals (<40 μm), with void areas also present; (c) Cluster of small crystallites showing orientations of crystals at air-solution interface; (ii) Solution surface of CaCO3 films (digital photograph of the air surface in box): (d) Solution surface comprised of rhombohedral crystals (approximately 10-60 μm); (e) Sharp rhombohedrai terminations (see arrow), with some not fully formed; (f) Arrows pointing to sharp rhombohedrai terminations and crystal growth terraces yielding foliated surfaces; (iii) Air surface of bone-like films: (g) Air surface similar to that of CaCO3 films; (h) Arrows pointing to large crystal faces (dark) and sheets (light), with growth terminations at air-solution interface; (i) Arrows pointing to large crystal faces, sheets, and small rosette clusters of sheets (rounded) (upper right: Dissolution of large crystal faces and recrystallisation as sheets, with surface-initiated conversion); (Iv) Solution surface of bone-like mineral films: (j) Solution surface comprised of rosette dusters of sheets (approx. 15-30 μm); (k) Box highlights dusters of rosette sheets arranged in the general rhombohedrai outline (retained from precursor calcite crystals); (i) Arrows pointing to a rosette duster of sheets, with irregular array to right.

[0063] Figure 6 shows a schematic of the bone-like mineral film formation mechanism: (a) Experimental set-up at time (t) = 0; (b) Conversion mechanism; (c) Dissolution-recrystallisation reactions at t = 5 hours, (d) Dissolution-recrystallisation reactions at t = 24 hours,

[0064] Figure 7 shows the method of coating PEEK with nanocrystailine carbonate apatite (C-Ap). Precursor CaCO3 layer: Deposition to form a contiguous template that encapsulates the PEEK substrate - nucleation followed by convective self-assembly (CSA) of CaCO3 nanoparticles (CaCO3- NPs). Rapid cold-sintering treatment - rapid heterogeneous nucleation and precipitation of CaCO3 from a solution of Ca(HCO3)2. Conversion to C-Ap: Phase change driven by dissolution- recrystallisation - conversion to C-Ap by Immersion in phosphate buffered saline (PBS).

[0065] Figure 8 shows SEM/EDS micrographs of uncoated and coated PEEK-OPTIMA™ HA and PEEK-OPTIMA™: (a) (i-iv) Uncoated PEEK-OPTIMA™ HA at various magnifications - (i) Surface pattern of microscale ridges-and-vaileys topography, (ii) EDS detection of Ca on surface, (iii) HA observed in crevices, (iv) Crevices with embedded HA and nanotopographicai features visible at high magnification; (b) (i-iv) Coated PEEK-OPTIMA™ HA at various magnifications - (i) General undulations of ridges-and-vaileys surface topography and large granular protrusions (<20 μm), (ii) increased magnification highlighting undulations, (iii) Coating comprised of rosette clusters (approximately 1 μm), (iv) High magnification showing rosette dusters comprised of nanoplatelets and adjacent voids; (c) (i-iv) Uncoated PEEK-OPTIMA™ at various magnifications - (i) Surface pattern of microscale ridges-and-valleys topography, (ii,iii) Swarf from roughening, (iv) Nanotopographical features visible at high magnification; (d) (i-iv) Coated PEEK-OPTIMA™ at various magnifications, with observations being the same as for coated PEEK-OPTIMA™ HA (b)(i-iv).

[0066] Figure 9 shows backscattered SEM/EDS micrographs of cross sections of coated PEEK- OPTIMA™ HA and PEEK- OPTIMA™: (a) Coated PEEK-OPTIMA™ HA at various magnifications - (i) Low magnification showing contiguous coating with H-Ap particles embedded in substrate, (ii) Higher magnification showing embedded H-Ap particles apposite to coating (circled and brighter than coating) and voids in coating (dark spots), (iii) EDS detection of Ca in coating and substrate; (b) Coated PEEK OPTIMA™ at various magnifications - (i) Low magnification showing contiguous coating, (ii) Dark spots visible in the coating at higher magnification, (iii) EDS detection of Ca in coating but not in substrate.

[0067] Figure 10 shows tribological properties of coatings on PEEK-Optima-HA and PEEK-Opiima (b) Wear path of PEEK-Optima-HA (progressive load of 1-5 N, sliding speed of 5 mm/min) - (i) Beginning of scratch, distance to point of failure of 56 μm, arrow for tensile cracks, (ii) Middle of scratch, arrows for loose particles of coating and residual coating, (iii) End of scratch, dense packing of coating into substrate still present at end of scratch, arrow for residual coating; (c) Wear path of PEEK-Optima (progressive load of 1 -5 N, sliding speed of 5 mm/min) - (i-iii) Same observations as (a)(l-ili)

[0068] Figure 11 shows a comparison of analogues of adhesion strength / coating performance for biomimetic coatings and the coating of the present invention, on polymeric substrates.

[0069] Figure 12 shows the cross section of a coating of the present invention on a 3D-printed porous titanium substrate. SEM/EDS showed that the coating was contiguous across the surface of the substrate (coating indicated by the presence of calcium, shown in white).

[0070] Figure 13 shows a time of flight secondary ion mass spectrometry (TOF-SIMS) 3D-rendering of the coating of the present invention with biologies incorporated throughout the structure, as indicated by the presence of nitrogen.

DEFINITIONS

[0071] In describing and claiming the present invention, the ioliowing terminology will be used in accordance with the definitions sot out below. It also is to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. [0072] Unless defined otherwise, ail technical and scientific terms used heroin have the same meaning as commonly understood by one having ordinary skill in the art to 'which the invention pertains.

[0073] Unless the context clearly requires otherwise, throughout the description and the claims, the terms “comprise”, “'comprising”, and the like are to be construed In an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “Including, but not limited to”. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.

[0074] The transitional phrase "consisting of” excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0075] The transitional phrase "consisting essentially of" is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term "consisting essentially of" occupies a middle ground between "comprising" and "consisting of".

[0076] Where applicants have defined an invention or a portion thereof with an open-ended term such as "comprising", it should be understood readily that (unless otherwise stated) the description should be interpreted to describe such an invention using the terms "consisting essentially of" or "consisting of." In other words, with respect to the terms “comprising”, “consisting of, and “consisting essentially of, where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of or, alternatively, by “consisting essentially of.

[0077] Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0078] Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0079] Other than In the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about” or “approximately”. The examples are not intended to limit the scope of the invention, in what follows, or where otherwise indicated, “%” will mean “weight %”, “ratio” will mean “weight ratio” and “parts” will mean “weight parts”.

[0080] The terms “predominantly” and “substantially” as used herein shall mean comprising more than 50% by weight, unless otherwise indicated.

[0081] As used herein, with reference to numbers In a range of numerals, the terms "about," "approximately" and "substantially" are understood to refer to the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to + 1 % of the referenced number, most preferably -0 .1 % to +0 .1 % of the referenced number. Moreover, with reference to numerical ranges, those terms should bo construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from 8 to 10, and so forth.

[0082] As used herein, “wt.%” refers to the weight of a particular component relative to total weight of the referenced composition,

[0083] The term "and/or" used in the context of "X and/or Y" should be interpreted as "X," or "Y," or "X and Y." Similarly, "at least one of X or Y" should be interpreted as "X," or "Y," or "both X and Y."

[0084] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments also may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and Is not intended to exclude other embodiments from the scope of the invention.

[0085] The complete disclosures of the patents, patent documents and publications cited herein are incorporated by reference in their entirety as If each were individually incorporated.

[0086] As used herein, the term "free-standing" is understood to refer to structures that are formed without contact with a support material such as a substrate or scaffold.

[0087] The term convective self-assembly (CSA) has been defined above as a process whereby the substrate is immersed in a solution containing mineral nanoparticles and heated at a temperature sufficient to cause evaporation of the solution. As the solution evaporates, the mineral grains self- assemble on the surface of the substrate through capillary action at the meniscus, thus forming a coating. It will be appreciated by the skilled person that the term convective self-assembly could also be described as colloidal self-assembly, evaporative self-assembly, evaporating meniscus assembly.

[0088] As used herein, the term "multidirectional" Is understood to refer to techniques that affect multiple faces of a substrate in 3D.

[0089] As used heroin, the terms "mineral” and calcium-based mineral Is understood to refer to calcium carbonate (including the polymorphs of calcite, aragonite, and vaterite) and minerals within the calcium phosphate group.

[0090] As used herein, the term "apatite” is understood to refer to minerals within the apatite mineral group, which includes hydroxylapatite, carbonate-rich hydroxylapatite, fluorapatite, carbonate apatite, and other variants. It will be appreciated that where the generic terms “bone-like apatite” and “bone- like” have been used herein, these are intended to refer to at least one of these specific minerals.

DETAILED DESCRIPTION

[0091] The skilled addressee will understand that the invention comprises the embodiments and features disclosed herein as well as all combinations and/or permutations of the disclosed embodiments and features.

EXAMPLES

[0092] The present invention now will he described with reference to the following examples which should be considered in all respects as illustrative and non-restrictive.

Raw Materials CaCO3 Nanoparticies and Preparation of Calcium Bicarbonate Solution

[0093] Precipitated CaCO3 nanoparticies ( CaCO3-NPs) of average particle size approximately 90 ± 15 nm) were used as the raw material to prepare precursor CaCO3 nanoparticulate suspensions (CaCO3 suspensions) and the subsequent calcium bicarbonate (Ca(HCO3)2) solutions. The Ca(HCO3)2 solution was prepared using a stainless steel batch reactor. A CaCO3 suspension (0.125 g/L, 40 ml water or ultrapure water) was transferred to the reactor, pressurised to the CO2 saturation pressure (varies by temperature, e.g., approximately 6 MPa at 22-25 °C), and held for approximately 30 minutes to term the solution of Ca(HCO3)2. The solution was magnetically stirred at 600 rprn throughout the entire pressurlsation process. The magnetic stirrer was stopped at the beginning of the depressurisation process and the solution of Ca(HCO3)2 was removed when the reactor reached atmospheric pressure. Bone-Like Film Synthesis

[0094] The following experimental information relates particularly to the first and second aspects of the invention as defined above.

Primary layer comprising a CaCO3 film

[0095] A solution of Ca(HCG3)2 (35 mL) was transferred to a Pyrex® measuring cylinder (17.5 cm H x 1 .4 cm ID) and evaporated/degassed in a drying oven at 40 °C for 24 hours. A primary layer comprising a CaCO3 film was formed at the air-solution interface during this stage.

Conversion to a “Bone-like” film

[0096] At least some of the primary layer then was converted to apatite to produce a mineral film, which is herein referred to as a “bone-like film”.

[0097] The primary layer comprising a CaCO3 film was converted to a bone-like film by incubation in PBS (Sigma Aldrich, 10 niM, pH = 7.6, 37.5 mL) at 80 °C for 24 hours. The precursor films wore placed at the air-solution interface with the flat/smooth air surface facing up and the rough solution surface down. The films were held at the air-solution interface by surface tension throughout the entire process.

Characterisation

[0098] 3D laser confocai microscopy (3D Microscopy, VK-X200; Keyence Corporation, Japan) was used to determine the topographical roughness (arithmetical mean height, Ra) of the samples (n = 3, 20X objective lens). The thicknesses of the samples were measured from the cross sections (n = 3, 10 measurements per sample, 50X objective lens).

[0099] Field emission scanning electron microscopy (FESEM, Nova NanoSEM 450 FESEM; FBI, Hillsboro, OR, USA; secondary electron (SB) mode at 5 kV) was used to assess the morphologies of the samples. Carbon tape and Pt coating (Emitech K575x Pt Sputter Coater; Quorum Technologies Ltd, Laughton, East Sussex, UK) were used to enhance the electrical conductivity of the samples. FESEM and energy dispersive spectroscopy (EDS, Bruker Corp,, Billerica, MA, USA; secondary electron (SE) mode at 15 kV) were used to detect phosphorus (P) on the surface and cross sections of the bone-like films. Carbon tape and carbon coating (DCT Desktop Carbon Coater; Vac Coat Ltd., London, United Kingdom) were used to enhance the electrical conductivity for EDS analyses.

[0100] Laser Raman microspectroscopy (Raman) (inVia 2, Renishaw pic, Wotton-under-Edge, Gloucestershire, UK) was used to determine the phase composition of a selected area of the cross section of the films (Raman mapping). StreamLine™ mapping in static mode was selected for higher resolution. The maximal peak for CaCO3 (1085 cnr ¹ ) and C-Ap (960 cm ^-1 ) was used to differentiate between the two phases. A green diode laser (532 nm) was used as the light source. Key parameters include resolution (1 ,5 to 1 .7 cm ^-1 ), spot size (approximately 1 μm), grating (1800 grooves/mm), spectral range (Raman shift 0 to 1800 cm ^-1 ) and magnification (1 ,000 X).

[0101] The experimental set-up for the formation of the CaCO3 primary layer involved placing a 40 mL solution of Ca(HCO3)2 in a polystyrene petri dish. A Kapton® polylmide film with a hollow centre (approximately 10 mrn x 10 mm) was placed on the air-solution Interface to encourage evaporation/CO2 degassing and crystal nucleation/growth In the centre. An incandescent reflector lamp (125 W) was positioned at a fixed distance from the air-solution interface to achieve the target temperature of approximately 40°C, as measured by a thermocouple positioned at the top of the sample cell.

[0102] The in situ X-ray diffraction (XRD) experimental set-up for conversion to a bone-like film involved placing a precursor CaCO3 film at the air-solution interface of a polycarbonate container with PBS (10 niM, pH = 7.6, 37.5 mL). The ratio of air-to-solution volume in the container was designed to match laboratory conditions. A piece of polyimide film with a hollow centre (approximately 10 mm c 10 mm) was placed on the air-solution interface to maintain centre positioning of the CaCO3 film. The container was covered with a final layer of Mylar® polyethylene terephthalate (PET) film with the aim of preventing evaporation. Self-fusing silicone tape was used to seal the container. An infrared (IR) lamp (275 W) was positioned at a fixed distance from the air-solution interface to achieve the target temperature of approximately 60 °C, as measured by a thermocouple positioned at the top of the sample cell. This temperature was used to prevent deformation of the polycarbonate container from overheating, 'which was observed at 80 °C.

[0103] The CaCO3 primary layer is formed by an evaporation/CO2 degassing process, in contrast, the method for converting at least some of the primary layer to apatite Is intended to avoid evaporation or to minimise evaporation. CaCO3 primary layer

[0104] in one embodiment, a free-standing CaCO3 film is formed at the air-solution interface of a solution of Ca(HCO3)2 by evaporation and CO2 degassing. The resultant films are sufficiently thick (approximately 40 μm thickness x 8.5 mm diameter) and robust for handling, distinguishing them from previously reported rnineral-only disparate fractal aggregates and particles of calcite formed at the air- water interface which also used a Ca(HCO3)2 solution. In some of the embodiments described herein, CaCO3 films are converted to bone-like films with a layered biphasic microstructure consisting of a carbonate-rich hydroxyapatite core and remnant carbonate primary layer.

[0105] The inventors have observed that the CaCO3 primary layer is made up of intergrown calcite crystals (see Fig. 2 and Fig. 3). The air-solution interface of the Ca(HCO3)2 solution has a relatively high saturation state due to evaporation and CO2 degassing. Accordingly, the inventors have noted that CaCO3 heterogeneously nucleates at the air-solution interface and continues to grow into crystal aggregates that eventually converge and intergrow to form the CaCO3 primary layer. The primary layer has an anisotropic microstructure consisting of a flat air surface and a rough water surface. The primary layer is held at the air-solution interface by surface tension.

[0106] In a preferred embodiment, a processing temperature of approximately 40 °C is used to expedite and favour the precipitation solely of the calcite CaCO3 polymorph, which is favoured at higher temperatures. As the skilled person will know, like vaterite, aragonite is another polymorph of CaCO3.

[0107] Fig. 4(a,b) shows cross-sectional micrographs of the samples imaged by 3D confocal microscopy. They show that the CaCO3 primary layer consists of polycrystalline aggregates with an anisotropic microstructure, where the air surface is relatively flat and the solution surface is rough. SEM micrographs of the air surface are shown in Fig. 5(a-c) and the results are consistent with 3D confocal microscopy; the air surface appears relatively flat across multiple magnifications. The samples exhibited a relatively low surface roughness of Ra = 3.2 ± 0.7 nm. Conversely, 3D confocal micrographs of the cross section show that the solution surface appears to be rough (Fig. 4(a,b)).

This Is consistent with SEM micrographs of the solution surface across multiple magnifications (Fig, 5(d-f)).

[0108] The SEM micrographs of the air surface in Fig. 5(a-c) show that it is comprised of clusters of small crystallites (<5 μm), large crystals (<4G μm), and void areas between some crystals. Without wishing to be bound by any theory, the inventors believe that nucleation is favoured over growth when the saturation state of a solution is high, whereas crystal growth/aggregation is favoured when the saturation state is low. The saturation state of a solution of Ca(HCO3)2 is the highest in relation to CaCO3 at the beginning of the evaporation/CO2 degassing process. Hence, the clusters of small crystallites may represent initial nucieation sites and smaller crystals from early in the process. The larger crystals probably form later in the process when the saturation state decreases, and crystal growth is favoured.

[0109] The SEM micrographs of the solution surface in Fig. 5(d-f) show that the solution surface is comprised of largo intergrown rhombohedral crystals in the range of approximately 10-60 μm. The morphology of the crystals is characteristic of caicite, with corresponding sharp rhombohedral terminations. SEM micrographs in Fig. 5(d-f) show that the packing density of the grains at the solution surface is relatively lower. Foliated surfaces also are observed on some crystals in high magnification SEM micrographs (Fig. 5(f)). Other high magnification SEM micrographs in Fig. 5(c) show that the air surface is comprised of caicite crystals that do not nucleate exclusively on the (104) plane, indicating a multiplicity of nucleating planes and growth habits. 3D confocal micrographs of the cross section in Fig. 4(b) show that large caicite crystals can extend through the entire thickness of the film, which, has an average thickness of 37.7 ± 2.5 μm and a maximal thickness of 52.4 ± 6.1 μm. [0110] The highest saturation state in the solution of Ca(HCO3)2 is observed at the air-solution interface due to evaporation and CO2 degassing. Fig. 3(a) shows a schematic diagram of nucieation and cluster formation during the early stages of primary layer formation. Heterogeneous primary nucieation occurs at the air-solution Interface and clusters of small crystallites form due to van der Waals forces. The crystals naturally favour (104) plane development and this morphological orientation is enhanced by re-orientation due to meniscus forces.

[0111] Fig. 3(b) shows a schematic diagram of the crystal growth phase of the process. Without wishing to be bound by any theory, it is believed that the saturation state of the solution decreases and so crystal growth becomes favoured over nucieation with increasing time. Hence, interface crystals grow downward and laterally into the solution. Intergrowth between Interface crystals within the same aggregate occurs probably due to differences In size and orientation. The accumulation of crystals results In gradual weight gain that deforms the air-solution interface and gives rise to gravity- altered capillary forces. However, the similar orientations of the crystals on the outer edges of the crystal aggregates may cause electrostatic repulsion between crystal aggregates that overcome the effects of gravity-altered capillary forces, i.e., capillary forces < electrostatic forces (Fig. 3(b)).

[0112] SEM micrographs of the air surface in Fig. 5(a,b) show that crystals on the outer edge of clusters grow large relative to those in the centre, probably due to greater access to solution and more space for growth. Subsequent-growth crystals are formed upon secondary nucieation on the interface crystals. Fig. 5(f) shows SEM micrographs of the solution surface at high magnification.

The subsequent-growth crystals are oriented randomly in various planes. This may be attributed to the variegated orientations of the foliated features of some interface crystals, on which subsequent- growth crystals grow.

[0113] Fig. 3(c) shows a schematic diagram of processes that occur during the final film formation phase. The aggregates of the present invention converge and intergrow. The multiple levels of crystal intergrowth (within aggregates and between aggregates) owing to different crystal sizes and orientations significantly enhances the robustness and integrity of the mineral film.

[0114] The CaCO3 primary layer can be held at the air-solution interface by surface tension throughout the entire formation process despite the high true density of caicite of 2710 kg/m ³ . This characteristic is advantageous for conversion to a layered biphasic structure having a primary layer comprising a CaCO3 film and a secondary layer or coating of apatite on, and in contact with, the primary layer.

Bone-Like films

[0115] Free-standing mineral films exhibit favourable properties for fundamental in vitro bone research and applied bone tissue engineering as bone mineral accounts for ~60 wt% of adult bone mass. Free-standing mineral films that have a layered diphasic structure of crystalline minerals have not been synthesised before. A mineral-only film that does not contain organic additives or a template/substrate would be advantageous for certain applications. The films also have thermal stability across a wider temperature range, compared to organics (e.g., gelatine and silk fibroin) which typically have lower melting temperatures. In some embodiments, the present invention describes the first free-standing crystalline mineral film that has a layered biphasic structure. It also does not contain an organic phase/template and does not need a final heat treatment step to convert an amorphous mineral to a crystalline phase.

[0118] In some embodiments, the precursor CaCO3 primary layer is at least partially converted to a bone-like mineral film consisting of a primary caicite layer and a carbonate- rich hydroxyapatite secondary layer. This process is driven by dissolution -recrystallisation at the air-solution interface of a phosphate buffered saline solution (PBS, 80 °C). The temperature (80 °C) represents a balance between CaCO3 dissolution (inversely correlated with temperature increase) and carbonate- rich hydroxyapatite precipitation (positively correlated with temperature increase). The temperature and immersion duration results in partial conversion which demonstrate the mechanism and show that a biphasic film can be achieved. This method uses the air-solution interface analogously to a physical boundary to introduce a layered biphasic structure to the films. The microstructure of the bone-like films also parallels the surface structure of bone nanocrystals, which are comprised of an apatite core and an outer hydrated layer of labile units such as carbonates.

[0117] Fig. 4(a-d) shows cross-sectional micrographs of the samples imaged by 3D confocal microscopy. They indicate that bone-like films retain the anisotropic microstructure of CaCO3 films, 'where the air surface is flat, and the solution surface is rough. SEM micrographs of the air surface are shown in Fig. 5(g-l) and the results are consistent 'with 3D confocal microscopy; the air surface appears relatively flat across multiple magnifications. SEM micrographs at higher magnification show that the air surface is comprised of caicite crystals and carbonate-rich hydroxyapatite sheets,

[0118] In some embodiments, the crystal faces of remnant caicite crystals at the air-solution interface of bone-like films exhibit higher surface roughness than caicite crystal faces at the air surface of CaCO3 films (Ra = 4.4 ± 3.7 nm > Ra = 3.2 ± 0.7 nm). The surface roughness of the carbonate-rich hydroxyapatite region is much higher at Ra = 86.5 ± 30.4 nm.

[0119] In some embodiments, at least some of the CaCO3 primary layer is maintained at the air- solution interface throughout the entire conversion process, with the solution surface’s being oriented downwards. The air surface remains relatively flat because it is not exposed to the PBS solution that facilitates the dissolution-recrystallisation reactions that enable significant crystal growth. The surface roughness of caicite crystal faces on bone-like films is higher than that of caicite films. The difference is attributed to humidity-induced dissolution-recrystallisation reactions (i.e., water vapour in a sealed container during heat treatment) and precipitation (I.e., small volumes of PBS that leak onto the air surface). [0120] SEM micrographs (Fig, 5(j-l)) of the solution surface show that if is comprised of micron-sized rosette clusters of sheets that radiate from a central nucleus, resembling carbonate-rich hydroxyapatite. The outward growth of the rosette clusters likely reduces the area of void space that exists between precursor calcite crystals, leading to a slight reduction in the topographical roughness. Clusters of rosette sheets are arranged in the general outline of precursor calcite rhombohedra in some areas, as observed in Fig. 5(k). This suggests that the roughness is driven by the topography of the precursor CaCO3 primary layer and newly formed rosette dusters. The air surface of the bone- like film has a roughness of Ra = 2.3 ± 0.3 μm, and the solution surface of the bone-like film has a roughness of Ra = 7.4 ± 1 .9 μm.

[0121] Raman spectral maps for a region of the cross section of bone-like films are shown in Fig. 4(e-f). The white areas In Fig. 4(e) correspond to calcite at the air surface and white areas in Fig. 4(f) correspond to carbonate-rich hydroxyapatite at the solution surface. SEM/EDS micrographs of the cross-sectional region are shown in Fig. 4(g-j) and it indicates that the air surface is comprised of areas with and without phosphorous (P), whereas P is detected across the entire solution surface. The regions without P are attributed to remnant rhombohedral calcite crystals from the precursor CaCO3 primary layer, whereas the P-containing regions represent carbonate-rich hydroxyapatite rosette clusters, which form the secondary layer. The solution surface is immersed in PBS for dissolution-recrystallisation reactions that facilitate conversion from the calcite primary layer to the carbonate-rich hydroxyapatite secondary layer, while the crystals at the air surface are relatively protected from the solution.

[0122] However, the void areas on the air surface of the precursor CaCO3 primary layer are filled with PBS during the conversion process. Consequently, carbonate-rich hydroxyapatite sheets are observed around the outer edges of the calcite crystals at the air surface in regions that previously may have been void space. EDS images of the air surface in Fig. 4(h,j) and the SEM Image of the solution surface in Fig. 5(i) show that the apatite sheets in general penetrate the sharp rhombohedral terminations of calcite crystals, indicating that crystal growth begins at the surface and proceeds to the core.

[0123] SEM micrographs of the solution surface in Fig. 5(j,k) are characterised by rosette clusters of sheets in the range of approximately 15-30 μm. The high magnification image in Fig. 5(l) shows that the rosette clusters exhibits outward lateral and vertical growth. The average thickness of the bone- like mineral films increased marginally from approximately 37.7 ± 2.5 μm for the CaCO3 primary layer to approximately 4Q.6 ± 2.8 μm for the carbonate-rich hydroxyapatite films, further suggesting the outward growth of these sheets. A measurement of the air surface yields a Ca/P ratio of 5.22 ± 1 .93, whereas the solution surface has a Ca/P ratio of 1 .63 ± 0.04, which is similar to that of bone. The SEM images show that smaller precursor calcite grains that were randomly oriented have been converted to carbonate- rich hydroxyapatite. This leaves a larger proportion of crystals oriented parallel to the (104) plane (when assessed by X-ray diffraction (XRD)). Conversely, the solution surface has a greater proportion of carbonate-rich hydroxyapatite. It was found that no intermediate phases form during the conversion of the CaCO3 primary layer to the carbonate-rich hydroxyapatite secondary layer.

[0124] To summarise, precursor CaCO3 films were converted to bone-like films by partial conversion to carbonate-rich hydroxyapatite In PBS at the air-solution interface. The CaCO3 primary layer is oriented with the air surface facing up during the conversion step to limit exposure to PBS and retain a surface layer of calcite. Fig. 6(a) shows a schematic diagram of the experimental set-up for the process. Constituent crystals of bone-like mineral films are categorised as interface crystals, with nucleation sites at the air-solution interface, or subsequent-growth crystals, which nucleate on existing crystals. Fig. 6(b) shows a schematic diagram for the dissolution-recrystallisation reactions that underpin the conversion mechanism. The SEM micrographs of bone-like mineral films In Fig. 5(k,i) suggest that apatite sheets nucleate and grow on the surface of calcite crystals and proceed toward the core.

Conversion to Apatite

[0125] PBS is a water-based solution primarily comprised of phosphate salts. Without wishing to be bound by theory, the inventors propose that calcium ions dissolve from the CaCO3 primary layer upon contact with PBS, The calcium ions become saturated with respect to carbonate-rich hydroxyapatite and bind with phosphate and carbonate ions in PBS. The calcite surface dissolution results in the formation of surface defects, which become active sites for the nucleation and growth of the carbonate-rich hydroxyapatite secondary layer. This dissolution/recrystallisation reaction is irreversible because carbonate-rich hydroxyapatite has a lower solubility product than calcite. The inventors have observed no intermediate phase formation during the process,

[0126] Fig. 6(e,d) show a schematic diagram of the conversion process at different time periods. Conversion of the CaCCfe primary layer to the carbonate-rich hydroxyapatite secondary layer Is a time-driven process for surfaces exposed to PBS, SEM/EDS micrographs in Fig, 4(e-g) show that large crystals that extend through the film are converted partially to carbonate-rich hydroxyapatite around the perimeter of the crystal (approx. 10 μm). Void areas within the precursor CaCO3 primary layer expedite the conversion by increasing the surtace area exposed to PBS. The carbonate-rich hydroxyapatite rosette clusters are comprised of sheets that radiate from a central nucleus and the voids between the sheets also enable PBS infiltration. The calcite crystals at the Interface do not convert to apatite because they are relatively protected from exposure to PBS and there is limited void space due to the high degree of intergrowth between adjacent interface crystals.

Calcium-Based Mineral Coatings

[0127] The following experimental information relates particularly to the first, and third to ninth aspects of the invention as defined above. [0128] Coatings for orthopaedic implants that are unidirectionaliy and bidirectionally applied to the surface of such implants are used commonly. However, these coating methodologies have limited applicability to the complex structural features characteristic of state-of-the-art Implants. In preferred embodiments, the present invention provides a multidirectional technique that encapsulates or coats a suitable substrate with a mineral film, in some embodiments, the Invention relates to coating a precursor primary layer of CaCO3 onto a suitable substrate, followed by conversion of at least some of the primary layer into apatite.

[0129] in some embodiments, the CaCO3 primary layer Is coated onto the substrate through the sequential steps of: surface roughening: oxygen plasma treatment, and/or corona discharge; immersion in a solution containing Ca(HCO3)2 and suspended CaCO3-NPs; and CSA of CaCO3-NPs.

[0130] In a preferred embodiment, the surfaces of the substrate were roughened to enable particle trapping. Fig. 8(a)i-iii, (c)i-iii show the microtopography of ridges-and-valleys on uncoated PEEK- OPTIMA™ HA (polyether ether ketone doped with hydroxyapatite) and PEEK-OPTIMA™ (polyether ether ketone) that was introduced by hand-grinding. Oxygen plasma treatment subsequently was applied to enhance surface wettability and to deposit oxygen functional groups.

[0131] Fig. 8 shows SEM/EDS micrographs of uncoated and coated PEEK-OPTIMA™ HA and PEEK-OPTIMA™. Fig. 8(a)(i-iv) shows uncoated PEEK-OPTIMA™ HA at various magnifications. Fig. 8(a)(i) shows the surface pattern of microscale ridges-and-valleys topography, and Fig. 8(a)(ii) shows EDS analyses of Ca on the surface. Fig. 8(a)(iii) shows HA visible in crevices, and (iv) Identifies crevices with embedded HA and nanotopographical features visible at high magnification. Fig. 8(b)(i- iv) shows coated PEEK-OPTIMA™ HA at various magnifications. Fig. 8(i) shows the general undulations of ridges-and-vaiieys surface topography and large granular protrusions (<20 μm), and (ii) shows increased magnification highlighting said undulations. Fig. 8(iii) shows a magnified image of the coating comprised of rosette clusters (approximately 1 μm), and (iv) shows a high magnification image with rosette dusters comprised of nanopiatelefs and adjacent voids. Fig, 8(c)(i-iv) shows uncoated PEEK-OPTIMA™ at various magnifications, where (i) displays the surface pattern of microscale ridges-and-vaiieys topography and (ii.iii) show swarf from roughening. Fig. 8(iv) shows the nanotopographical features visible at high magnification. Fig. 8(d) (i-iv) shows coated PEEK- OPTIMA™ at various magnifications, with observations being the same as for coated PEEK- OPTIMA™ HA (Fig. 8(b)(i-iv)).

[0132] The surface-modified samples were immersed in a suspension containing Ca(HCG3)2 and CaCO3-NPs. Without wishing to be bound by theory, the inventors propose that the CaCO3-NPs, oxygen functional groups, and embedded H-Ap particulates act as nucleation sites for the formation of a surface mineral layer that is chemically bonded to the substrate. However, it will be appreciated that the mineral layer may be mechanically keyed into the substrate and/or chemically bonded to the substrate. The sample then is immersed in a suspension of CaCO3-NPs for CSA, where the nanoparticles seif-assemble toward the triple contact line of liquid (CaCO;3-NP suspension), solid (substrate), and vapour phase (air) due to capillary forces at the meniscus. A contiguous coating is deposited along the sample surface as the meniscus retracts by evaporation. A schematic diagram that illustrates this mechanism is provided in Fig 7.

[0133] in preferred embodiments, the CaCO3 primary layer coating the substrate is substantially contiguous and preferably encapsulates the substrate.

[0134] In preferred embodiments, after conversion of at least some of the CaCO3 to apatite, the mineral film remains substantially contiguous preferably encapsulates the substrate.

[0135] Planar SEM micrographs in Fig. 8(b)i-iv, (d)i-iv show that the coating is contiguous and compieteiy covers the substrate. This is consistent with SEM micrographs of the cross section of the samples shown in Fig. 9(a)i-iii, (b)i-iii, where the coating is observed along the entire surface of the substrate.

[0136] Fig. 9 shows backscattered SEM/EDS micrographs of cross sections of coated PEEK- OPTIMA™ HA and PEEK- OPTIMA™. Fig. 9(a) shows Coated PEEK-OPTIMA™ HA at various magnifications, with (i) showing a low magnification image identifying the contiguous coating with H- Ap particles contained or dispersed within the substrate. Fig. 9(a)(ii) shows a higher magnification image, which highlights H-Ap particles apposite to the coating (circled in yellow and brighter than the coating) and voids in the coating (dark spots). EDS detection of Ca in coating and substrate can be seen in Fig. 9(iii). Fig. 9(b) shows coated PEEK OPTIMA™ at various magnifications, with low magnification images showing (i) contiguous coating, (ii) dark spots visible in the coating at higher magnification, and (iii) and EDS detection of Ca in coating but not in substrate,

[0137] Fig. 9 shows an embodiment where PEEK-OPTIMA™ HA has an average coating thickness of 1.5 ± 0.6 μm and PEEK-OPTIMA™ has an average coating thickness of 2.7 ± 0.6 μm. The differences are likely driven by the precursor CaCO3-NP layer. The thicknesses of CSA-deposited films are influenced by the height of the meniscus, where extra layers of particles are accommodated with increasing height. The exposed hydroxyapatite on the surface of PEEK-OPTIMA™ HA increases hydrophilicity and the lower contact angle between the liquid and solid results in a thinner deposit. Other controlling factors influencing the contact angle include temperature and velocity of solution retraction. Comparison of the Ra before and after coating revealed a 2.4-fold increase in surface roughness for PEEK-OPTIMA™ HA and a 3.2-fold increase for PEEK-OPTIMA™.

[0138] Low magnification SEM micrographs in Figure 8(b)i-ii, (d)i-ii show that the coating reflects the general undulations of the ridges-and-valleys microtopography of the substrate. This is consistent with the SEM micrographs of the cross sections shown in Figure 9(a)ii, (b)ii, where the profile of the coating roughly follows that of the substrate. The combined effect of ridges-and-valleys microtopography and rosette clusters in the coating can provide higher surface roughness on coated substrates compared to uncoated substrates. Rapid ( 10 min) ‘Cold-Sintering’ Treatment

[0139] Biomimetie Ca-P coatings made by immersion in simulated body fluid (SBF) are of significant interest to the orthopaedics field because they are comprised of bone-like apatite, which readily bonds with bone. However, the precipitation kinetics represents a limitation, where coating deposition can take days if not weeks. In some embodiments, the present invention uses a novel step to reduce the time needed for coating precipitation to approximately 10 minutes. The solution also permeates interstices in the structure and so acts as a carbonate binder upon precipitation within pores, fissures, voids, and asperities in substrates. This cold sintering treatment could provide significant enhancement of the adhesion strength of the coating.

[0140] In some embodiments, the present invention involves the preparation of a solution of Ca(HCO3)2 and the heterogeneous precipitation of CaCO3 on a template of CaCO3-NPs.

[0141] CaCO3 solubility is very limited in water under atmospheric conditions (approximately 10 mg/L) but can be increased with the addition of CO2, the solubility of which increases with pressure. CaCO3-NPs were dissolved in a batch reactor at and pressurised to the CO2 saturation pressure (6 MPa) to form a solution of Ca(HCO3)2 that achieves CaCO3 solubility of approximately 1 ,250 mg/L at approximately 25 ^o C.

[0142] In preferred embodiments, the substrate is immersed In the solution for a short period of time (i.e. , approximately 10 minutes) and immediately transferred to an oven at approximately 130 ^o C, which is the temperature of maximal thermal conductivity of H2Q, The existing CaCO3-NP layer on the sample enables heterogeneous nucleation and growth, CaCO3 and CO2 exhibit inverse solubility with temperature so heating accelerates CaCO3 nucleation/precipitation and CO2 degassing.

[0143] Several morphological features of the coatings can be attributed to the “cold sintering” step. SEM micrographs In Fig. 8(b)iii-iv, (d)iii-iv show rosette clusters (approximately 1 μm) formed from the method. They may have derived from micron-sized calcite precursor crystals formed by rapid nucleation and precipitation during immersion in Ca(HCO3)2 , Fig. 9(a)ii, (b)ii shows SEM micrographs of the cross section, where voids (dark spots) are observed throughout the coating. Voids also are observed on the planar surface of each sample, as shown in Fig. 8(b)iv, (d)iv. These voids likely formed when calcite precipitated around GG2 bubbles that nucleated on the sample surface.

[0144] Large granular protrusions (<20 μm) were observed on the planar surface of each coated sample in SEM micrographs, shown in Fig. 8(b)i, (d)l). This is consistent with the SEM micrographs of the cross sections in Figure 9(a)ii, (b)il, where some regions are uncharacteristically thicker. These large granular protrusions may be attributed to crystals that nucleated and grew at the air-water interface because it exhibits the highest saturation state. Conversion to Nanocrystalline Carbonate Apatite

[0145] H-Ap (Caio(P04)6(OH)2) is one of the most common types of Ca-P coatings used for orthopaedic implants, but its Ca/P ratio Is 1 .67 while bone mineral has a Ca/P ratio in the range ~1 .5- 1 .6. Hence, one advantage of the present C-Ap coatings Is that the Ca/P ratio is in same range as bone mineral. A second advantage is that, relative to H-Ap, C-Ap also has a solubility at pH levels characteristic of the osteoclastic resorption process, enabling it to be bioresorbed. A third advantage is that disordered nanotopographical features provide osteoinductive cues that are conducive to new bone formation.

[0146] In some embodiments, the substrate coated with the CaCO;3 primary layer is immersed in PBS (10 mM, pH 7.6) and held at about 80°C for approximately 24 hours. Near-physiological pH favours the formation of C-Ap and the ionic strength of P In the PBS solution is conducive to calcium- deficiency. The selected temperature represents a balance between CaCO3 dissolution and C-Ap precipitation. CaCO3 dissolution is inversely related with temperature and C-Ap precipitation is positively correlated with increases in temperature. Hence, higher temperatures accelerate C-Ap precipitation kinetics but limits dissolution of the CaCO3 primary layer.

[0147] The uncoated PEEK-OPTIMA™ HA has a Ca/P ratio of approximately 2.21 ± 0.40 while the coated substrate had a Ca/P ratio of approximately 1 .62 ± 0.07. Similarly, coated PEEK-OPTIMA™ has a Ca/P ratio of approximately 1 ,56 ± 0.06. This is consistent with the backscattered SEM micrographs of the cross section of PEEK-OPTIMA™ HA shown in Fig, 9(a)ii, where the brighter appearance of the embedded H-Ap particles suggests that the coating has a lower Ca/P ratio.

[0148] SEM micrographs of the planar surfaces of the coatings are provided in Figures 8(b)iv, (d)iv. These show a nano platelet-like morphology, consistent with other studies of C-Ap. The complete conversion of CaCO3 to C-Ap has been reported to occur by immersion in PBS at 37 ^o C for 9 days. Without wishing to be bound by theory, the Inventors propose that the dissolution of CaCO3 at the coating surface results in defects that become active sites for C-Ap nucleation. The reaction Is irreversible because C-Ap would be more thermodynamically stable and has a lower solubility product than calclte. The rate of nucleation is favoured over growth in the early stages of the process when the absolute concentration of Ca ions is higher owing to CaCO3 dissolution, whereas growth and associated aggregation are favoured in the later stages, when conversion from CaCO3 to C-Ap is further advanced.

[0149] In preferred embodiments, the C-Ap nanoplatelets in the coating are comparable to those of bone mineral: 1) width approximately 200-500 nm (bone mineral platelet length <30-50 nm), 2) thickness approximately 15-20 nrn (bone mineral platelet ~2 nm), 3) Ca/P ratio approximately 1 .5, same range as bone mineral; and 4) carbonate substitution in lattice. Tribological Properties of the Coating

[0150] Intervertebral spacers are implanted by mechanical insertionai techniques such as Inline tapping of the prothesis into the prepared disc space. The scratch test was used to assess the adhesion strength of the present C-Ap coating because It Is a semi-quantitative technique that closely simulates the frictional stresses that occur during implantation and Hertzian contact pressure is a key indicator of coating performance.

[0151] Failure is defined as the point along the middle of the scratch, where the substrate is exposed first. The tribological properties of embodiments of the present invention at failure are shown in Table 1 . The results show that the coating on PEEK-OPTIMA™ HA exhibits lower critical load but higher contact pressure than PEEK-OPTIMA™. The lower critical load of PEEK-OPTIMA™ HA largely results from the thinner coating since the stylus penetrates through the coating at an earlier stage. Most notably, the contact pressures for the coating on PEEK-OPTIMA™ HA (327 MPa) and PEEK- OPTIMA™ (284 MPa) are an order of magnitude greater than those reported for biomimetic Ca-P coatings on polymeric substrates, as shown in Fig. 11.

[0152] Table 1 : Tribological properties of coatings on PEEK-OPTIMA™ HA and PEEK-OPTIMA™ i \ i · i 1 j I ! 1

[0153] Fig. 10 shows the tribological properties of coatings on PEEK-OPTIMA™ HA and PEEK- OPTIMA™ are shown. Fig. 10(b) shows the wear path of PEEK-Optima-HA (progressive load of 1-5 N, sliding speed of 5 mm/min) with (i) display of the beginning of the scratch and distance to point of failure of 56 μm (arrow for tensile cracks). Fig. 10(li) shows the middle of the scratch, (arrows for loose particles of coating and residual coating) and (iii) shows the end of the scratch, which displays dense packing of the coating into the substrate (arrow for residual coating). Fig. 10(c) shows the wear path of PEEK-OPTIMA™ (progressive load of 1-5 N, sliding speed of 5mm/min) and (i-iii) show the same observations as Fig. 10(b)(i-iii).

[0154] Fig. 11 compares analogous adhesion strengths and/or coating performance for the present invention In contrast with biomimetic methods known in the art.

[0155] Without being bound by theory, the superior performance of coatings on PEEK-OPTIMA™

HA appears to be related to the higher Young’s modulus and chemical bonding between the coating and embedded H-Ap particulates. Advantages of the Invention are that the coating performance Is improved by the mechanical interlocking between the coating and the Irregular surface of the substrate; the intermediate base layer of discontinuous minerals, which are able to bond chemically with the substrate; and/or the nanostructure of the coating. As shown above, the nanostructure comprises larger rosette clusters of nanoplatelets that overlay a nanoparticulate template and discontinuous mineral base layer. The solution of Ca(HCO3)2 applied during the “cold sintering” step also permeates pores, fissures, voids, as well as asperities in substrates, and acts as a carbonate binder upon precipitation.

[0158] The scratch paths are shown in Fig. 10(b), (c). Backscattered SEM Images were used for enhance phase contrast between the coating (light) and substrate (dark) in order to elucidate the failure mechanisms. As observed in Fig. 10(b)i, (c)l, the coating initially was compressed by the downward motion of the indenter and arc tensile cracks characteristic of the deformation of brittle coatings is observed prior to cohesive failure. Figures 10(b), (c) show that the coatings failed by buckling mode, although there is no evidence of failure beyond the immediate indenter path. This is significant because Ca-P coatings of higher thicknesses or long SBF soaking times typically fail by delamination, exfoliation, splitting, chipping and cracking. The absence of such large-scale failure events are be due to the structure of the nanoparticulate base layer, where the absence of intergranular bonding localises failure and terminates crack propagation.

[0157] Figures 10(b), (c) show that the incorporation of the coating material in the substrate is observed throughout the post-failure region of the scratch path. This further distinguishes the present invention from biomimetic methods as this phenomenon is not observed for common Ca-P coatings of higher thicknesses or long SBF soaking times. The penetration depth of the Indenter gradually increases to a maximum of >40 μm, while the maximal asperity height of the ridges-anti-valleys microtopography is <4.0 μm for both substrates. Hence, buckled particles are available for the indenter to plough and concentrate into the pores, fissures, voids, as well as asperities in substrates of extruded PEEK (and other materials) when penetration depth > maximal asperity height.

[0158] The nanostructure of the coating may be a major contributing factor to this phenomenon as localised failure generates loose particles along the entire scratch front and the rosette clusters of disordered nanoplatelets easily disintegrate for incorporation into the surface structure. The presence of coating material throughout the post-failure region indicates retention of function (osteoconductive material for osseointegration) beyond failure for high frictional stress scenarios. This also distinguishes the invention from prior art.

[0159] in preferred embodiments, the present invention provides a C-Ap coating method that has at least one of the following advantages: low-temperature, rapid, additive-free, and/or multidirectional.

[0160] In preferred embodiments, the coating is comprised of single-phase C-Ap nanoplateiets that are surprisingly comparable to bone mineral. The nanocrystalline C-Ap coating also can encapsulate the substrate, indicating suitability with standard and state-of-the-art implants (substrate/coating roughness profiles are highly correlated (R2 = 0.92, p<0.G5)), The coating’s nanostructure also exhibits outstanding tribological performance and so can withstand frictional stresses characteristic of the spacer implantation process. The Hertzian contact pressures of approximately. 300 MPa are significant because they are an order of magnitude greater than those reported for blomirnetic Ca-P coatings on polymeric substrates.

[0181] In some embodiments, a biologic is incorporated into the mineral film during its production. In other embodiments, a biologic is incorporated into the mineral film during the conversion step, in yet other embodiments, a biologic is incorporated into the mineral film after the conversion step. In another embodiment, a biologic is incorporated into the mineral film by soaking the film In an appropriate solution containing the biologic. In yet other embodiments, the biologic is an osteo- inductive or antimicrobial compound.

Experimental Section/Methods

[0182] Preparation of PEEK substrates. 12 mm diameter rods of PEEK-OPTIMA™ and PEEK- OPTIMA™ infused with hydroxyapatite (PEEK-OPTIMA™ HA) were used as the substrates. They were cut into discs of thickness approximately 2 mm. Multidirectional hand-grinding was performed to introduce a random truss-iike lattice pattern of ridges and valleys to enhance particle trapping and mechanical locking. SIC grinding papers were used to achieve a post-grinding roughness profile (Ra) of approximately 0.3-0.4 μm. In those embodiments, oxygen plasma etching was performed at this stage by a plasma asher in order to enhance surface wettability. However, it will be appreciated that the invention may not utilise this step or an alternative method, such as corona discharge, may be used,

[0163] Preparation of CaCO3 nanoparticulate suspension. Precipitated CaCO3 nanoparticles (CaCO3-NPs) were used as the starting materials to prepare calcium bicarbonate solutions (Ca(HCO3)2) and CaCO3 nanoparticulate suspensions (CaCO3 suspensions) for coating deposition, CaCQs-NPs were caicite, with average particle size of approximately 90+15 nm.

[0164] Preparation of calcium bicarbonate solution. Ca(HCO3)2 was prepared using a batch reactor. A suspension of CaCO3 (40 mL, 0.125 g/L) was transferred to the reactor and pressurised to approximately 6 MPa at approximately 25°C for approximately 30 min in order to form Ca(HCOs)2.

The solution was stirred magnetically at 600 rpm during the entire pressurisation process. The magnetic stirrer was stopped at the beginning of the depressurisation process and Ca(HCOs)2 was removed when the pressure reached atmospheric.

[0165] Fabrication of coated PEEK, in preferred embodiments, the method of coating a substrate in a mineral film comprises the sequential steps of: CaCO ₃ primary layer formation

[0166] (1a) Nucieation of CaCO3 onto PEEK: Ca(HCO3)2 (20 mL) was combined with a suspension of CaCO3 (20 mL, 0.375 wt%). PEEK discs were immersed in the combined solution and heated in an oven at 130°C for 45 min.

[0167] (1b) Deposition of CaCO3-NPs: The samples were suspended In a suspension of CaCO3 (20 mL, 0.1875 wt%) that was evaporated in an oven at 130°C for 2-3 hours.

[0168] (2) Nucleation/precipitation of CaCO3: The samples were suspended in Ca(HCO3)2 for 10 min at 130°C in an oven and air-dried.

Conversion to C-Ap

[0169] (3) Conversion to carbonate apatite (bone-like apatite): CaCO3-coated PEEK was immersed in PBS (50 mL, 10 mM, pH 7.6) and held at 80°C for 24 hours.

[0170] All samples were rinsed with deionised water three times and ultrasonicaily cleaned in ethanol for five minutes before further characterisation.

Characterization of coatings.

[0171] Microscopy. Field emission scanning electron microscopy (FESEM) was used to assess the morphology of the coatings (secondary electron mode at 5 kV). Carbon/copper tapes and Pt coating were used to enhance the electrical conductivity. FESEM and energy dispersive spectroscopy (EDS) were used to determine the elemental composition of the Pt-coated samples surface and cross sections (backscattered mode at 15 kV). Cross sections were prepared by embedding the samples in epoxy, wet alumina blade cutting, and hand-polishing using progressive 1200 grit SiC paper, 3 μm diamond suspension, and 1 μm diamond suspension,

[0172] Coating thickness. Coating thicknesses were determined from the SEM micrographs of cross sections; Image J software was used to measure the thickness of the coatings at 10 locations across approximately 100 μm. The average coating thickness of each specimen was calculated as the average of these 10 locations. The average coating thickness for PEEK types was calculated as the average of three specimens (n=3). The average thickness of the samples was calculated as the average scratch test samples also were Pt-coated and analysed by FESEM (backscatter mode at 15 kV).

[0173] Surface roughness. The roughness profiles (Ra) were determined using a stylus profilometer. Measurements were conducted at a medium speed setting across a length of 2 mm (n = 4). The more uniform uncoated PEEK substrates were measured in three orientations (-120° rotation) but the less uniform coated PEEK samples were measured in six orientation (~60° rotation). Regression analyses were conducted using Microsoft Excel (n=12).

[0174] Tribological characterisation. The adhesion strength and contact pressure were determined using a scratch tester integrated with a 200 μm U-289 Rockwell diamond lndenter (n=3). The scratch parameters Involved a sliding speed of 5 mm/m in and three different load application rates: 5 N/rnin (1 -5 N), 10 N/min (1 -40 N), and 20 N/min (1 -40 N), Electron microscopy was used to examine the wear path and stages of coating failure. Failure was defined as the point along the middle of the scratch where the substrate is exposed, in line with previous studies.

[0175] The adhesion strength was determined as the normal load (frictional force) at the point of failure, in common with previous studies, which assessed the adhesion strength and contact pressure of hard coatings on soft substrates (for these tests, indenters of different sizes and a micro-scratch testers with <1 N load were used).

[0176] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the Invention may be embodied in many other forms in particular features of any one of the various described examples may be provided in any combination in any of the other described examples. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only where the scope of the invention is intended to be limited only by the claims set forth herein as follows.