WO2016193741A1

WO2016193741A1 - Orthopaedic and dental material

Info

Publication number: WO2016193741A1
Application number: PCT/GB2016/051634
Authority: WO
Inventors: Jason Peter MANSELL
Original assignee: University Of The West Of England, Bristol
Priority date: 2015-06-04
Filing date: 2016-06-03
Publication date: 2016-12-08
Also published as: GB201509693D0

Abstract

The present invention provides an orthopaedic material comprising a biocompatible material and at least one compound corresponding to the general formula A- [P(O)(OH)₂]_n wherein A means A₁, A₂ or A₃, and wherein A₁ is a linear, branched or cyclic, saturated or unsaturated hydrocarbon residue with 2-70 carbon atoms, which may be interrupted by one or more oxygen atoms, and comprising at least one fluorine, A₂ is a linear, branched or cyclic, saturated or unsaturated hydrocarbon residue with 2-70 carbon atoms, A₃ is a linear, branched or cyclic, saturated or unsaturated hydrocarbon residue with 2-70 carbon atoms, which may be interrupted by one or more oxygen atoms, and comprising at least one sulphur, and n is 1 or 2; the compound being covalently bound to a surface of the biocompatible material via a phosphate oxygen, wherein when the compound has the general formula A₂[P(O)(OH)₂]_n at least one additional compound having the formula A₁[P(O)(OH)₂]_n or A₃[P(O)(OH)₂]_n is bound to the A₂[P(O)(OH)₂]_n compound via a hydrophobic interaction between the A₁ or A₃ and A₂ residues. Orthopaedic implants comprising the material and methods of manufacturing the material are also provided.

Description

Orthopaedic and Dental Material

Field of Invention

The present invention is directed to an improved orthopaedic implant material, particularly a biocompatible material with at least one compound corresponding to the general formula A-[P(0)(OH)2]_n bound to a surface of the biocompatible material via a phosphate oxygen, and methods of manufacturing the same.

Background to the Invention

Developing novel surface finishes to encourage osteoblastogenesis is a continuing theme of contemporary bone biomaterials research. Enhancing human osteoblast (hOB) formation and maturation at prosthetic surfaces is predicted to secure superior implant integration and longevity. Finding ways of fabricating unique substrates includes coating bone biomaterials with small biological agents. Importantly the selected molecules should target hOBs and their bone marrow-derived stromal cell (BMSC) precursors. Particularly attractive are agents that participate in signalling cooperation or "cross-coupling" with key molecules central to bone formation, health and homeostasis. Candidate factors fulfilling these criteria are the simple bioactive lysophosphatidic acids (LPAs) and/or their more stable, phosphatase-resistant analogues, for example (3S)l-fluoro-3-hydroxy-4-(oleoyloxy)butyl-l-phosphonate (FHBP) and/or (2S)-l-oleoyl-2-0-methyl-glycero-3-phosphothionate (OMPT).

Skeletal cells, including hOBs and BMSCs, are targets for LPA (Blackburn & Mansell 2012). Of particular relevance to bone formation is the discovery that LPA co- operates with the active metabolite of vitamin D3, 1,25-dihydroxy vitamin D3 (1,25D); the co-stimulation of hOBs with LPA/FHBP/OMPT and 1,25D synergistically enhances their maturation (Gidley et al. 2006, Mansell et al. 2010, Mansell et al. 2012, Lancaster et al. 2014), an event associated with the provision of a mechanically robust, mineralised matrix. The signalling cross-coupling that occurs between LPA and active vitamin D3 metabolites culminates in synergistic increases in alkaline phosphatase (ALP), an enzyme absolutely essential for bone matrix calcification (Whyte 2010). These features of LP A, its small size and ability to cooperate with 1,25D make it an especially desirable molecule for the functionalisation of titanium (Ti) and hydroxyapatite (HA), two widely used bone biomaterials. The only other molecules reported to co-operate 1,25D in stimulating hOB maturation are TGF-β (Bonewald et al. 1992) and epidermal growth factor (Yarram et al. 2004). Their larger size and greater cost make them less desirable contenders for biomaterial conjugation and they are less likely to withstand conventional sterilisation protocols compared to the simpler LP As. In addition to its reported effects on hOBs, monopalmitoyl LP A (16:0 LP A), has been reported to inhibit virulence factor production and biofilm formation of the human opportunistic pathogen Pseudomonas aeruginosa (Laux et al. 2002). 16:0 LP A, along with several other phospholipids, e.g., dipalmitoyl phosphatidyl serine, dipalmitoyl phosphatidic acid and monomyristoyl phosphatidic acid, have been shown to sensitize otherwise resistant P. aeruginosa isolates to the actions of betalactams. It was also reported that 16:0 LP A and monopalmitoyl phosphatidyl choline were antimicrobial against a range of gram negative and gram positive bacteria including Staphylococcus aureus, a species often associated with bone implant failures through sepsis. Collectively LP A- functionalised devices could be beneficial in two important ways; the enhancement of early osseointegration by promoting hOB maturation at the surface and secondly, deterring the initial attachment and spread of bacteria known to be associated with implant failures through sepsis.

However, it is known, e.g. from WO2012/156746, that in developing an LPA- functionalised surface it is important to ensure the correct alignment of LPA molecules in relation to the metal surface. Phospholipids and phosphopeptides have a natural affinity for titanium, which has been exploited, for example, in solid phase extraction systems to remove phospholipids from complex biological samples such as foodstuffs (Calvano et al 2009). However, the phospholipid-Ti interaction is via the polar head group and such a configuration is unsuitable in the generation of LPA- signalling supports, as it is the phosphate group that binds to cell surface receptors to initiate cell signalling (Tigyi & Parrill 2003). As described in WO2012/156746, to ensure LPA is appropriately bound it is necessary to bind the tail section of the phospholipid to the metal surface. However, in order to overcome the natural affinity of the polar head group it is necessary to use complex and time consuming chemical methods, often involving harmful reagents such as piranha solution.

There is therefore a need to provide an improved LPA-functionalised orthopaedic material utilising faster, safer and cheaper methods of providing the same.

Summary of the Invention

According to a first aspect of the invention there is provided an orthopaedic material comprising a biocompatible material and at least one compound corresponding to the general formula (I):

A-[P(0)(OH)₂]„ (I) wherein A means Ai, A₂ or A3, and

wherein Ai is a linear, branched or cyclic, saturated or unsaturated hydrocarbon residue with 2-70 carbon atoms, which may be interrupted by one or more oxygen atoms, and comprising at least one fluorine,

A₂ is a linear, branched or cyclic, saturated or unsaturated hydrocarbon residue with 2- 70 carbon atoms,

A3 is a linear, branched or cyclic, saturated or unsaturated hydrocarbon residue with 2- 70 carbon atoms, which may be interrupted by one or more oxygen atoms, and comprising at least one sulphur, and

n is i or 2;

the compound being covalently bound to a surface of the biocompatible material via a phosphate oxygen,

wherein when the compound has the general formula A₂[P(0)(OH)₂]_n at least one additional compound having the formula Ai[P(0)(OH)₂]_n or A3[P(0)(OH)₂]_n is bound to the A₂[P(0)(OH)₂]_n compound via a hydrophobic interaction between the Ai or A3 and A₂ residues. Binding of compounds of formula A2[P(0)(OH)2]_n (such as alkyl phosphonic acids) has been described. However, these compounds are biologically inert. The inventors have found that a compound of formula Ai[P(0)(OH)2]_n or A3[P(0)(OH)2]_n provides a bioactive coating on the surface of the biocompatible material, thereby providing an orthopaedic material that enhances hOB formation and maturation, while simultaneously reducing bacterial attachment. The orthopaedic material can be stored stably under ambient conditions and can withstand autoclaving. The orthopaedic material also retains its bioactivity even after recycling, showing improved hOB maturation compared to uncoated biocompatible material. Surprisingly, this bioactivity can be achieved without need for the complex and time consuming chemical methods described in WO2012/156746 (used to ensure binding of the tail section of the phospholipid to the metal surface).

The hydrocarbon residues of Ai, A2 and/or A3 may comprise 10-30 carbon atoms, preferably 10-25 carbon atoms, such as 15, 16, 17, 18, 19 or about 20 carbon atoms.

In preferred embodiments of the invention, when A is Ai or A3, n is 1.

The orthopaedic material of the invention may comprise a coating of Ai[P(0)(OH)2]i and/or A3[P(0)(OH)2]i covalently bound to a surface of the biocompatible material via phosphate oxygen, thereby forming a first layer of the coating. Optionally, the orthopaedic material may comprise a second or further layer of Ai[P(0)(OH)2]i and/or A3[P(0)(OH)2]i bound to the compounds of the first layer via a hydrophobic interaction between the Ai and/or A3 residues. In alternative embodiments of the invention, the orthopaedic material may comprise a first layer of A2[P(0)(OH)2]_n covalently bound to a surface of the biocompatible material via phosphate oxygen, thereby forming a first layer of the coating, and a second or further layer of Ai[P(0)(OH)₂]i and/or A₃[P(0)(OH)₂]i bound to the compounds of the first layer via a hydrophobic interaction between the Ai and/or A3 and A2 residues. Preferably the orthopaedic material comprises a first layer of A2[P(0)(OH)2]_n and a second or further layer of Ai[P(0)(OH)₂]i. The first layer of the coating is bound to a surface of the biocompatible material via a covalent bound utilising a phosphate oxygen. Without being bound by theory, the inventors believe that this binding may be monodentate, bidentate or tridentate, as described in Paz, 201 1.

Hydrophobic interactions between the Ai, A₂ and/or A3 residues refer to a non- covalent binding. Hydrophobic interactions are well known in the art and would be familiar to a person skilled in the art. Examples of such interactions can be seen e.g., in lipid bilayers.

Compounds of formula Ai[P(0)(OH)₂]i or A₃[P(0)(OH)₂]i may be LP A receptor agonists. Seven surface receptors for LPA are known. Of these, LPA receptors 1-4 (known as LPAl, LPA2, etc.) are considered to be the most significant for skeletal cells. hOBs express LPAl and LP A3 while marrow stromal cells express LPAl and LPA4, with low variable expression of LPA2 and LP A3. In the present invention agonists of LPAl and LP A3 are preferred, more preferably agonists of LPAl . Suitable compounds may be pan-agonists, i.e. agonists of more than one of, or all of, LPA1-4.

The skilled person may determine whether or not a compound is capable of acting as an LPA receptor agonist using a number of methods known in the art, for example, such as is disclosed in Gidley et al. By way of example, the skilled person may prepare a culture of MG63 cells as outlined in section 2.2 of Gidley et al., treat cells with a combination of D3 and the compound which is being investigated for LPA receptor agonist activity and detect alkaline phosphatase (ALP) expression as described in section 2.7 of Gidley et al. An increase in ALP expression under these conditions, the increase being inhibited by Ki 16425 (a known LPA receptor antagonist), is indicative of the compound being an LPA receptor agonist. Detection of Erk phosphorylation in the cells, as described in section 2.9 of Gidley et al., provides a further supporting indication of LPA receptor agonist activity of the compound. The compound of formula Ai[P(0)(OH)2]i may be l-fluoro-3-hydroxy-4-butyl-l- phosphonate (FHBP), and/or the compound of formula A₃[P(0)(OH)₂]i may be (2S)- l-oleoyl-2-0-methyl-glycero-3-phosphothionate (OMPT). The compound of formula A₂[P(0)(OH)₂]i may be an alkyl phosphonic acid (APA) or an alkyl bisphosphonic acid. For example, the compound of formula A₂[P(0)(OH)₂]i may be octadecylphosphonic acid (ODPA).

In preferred embodiments of the invention the orthopaedic material comprises a first layer of APA, such as ODPA, and a second or further layers of FHBP and/or OMPT. Alternatively, the orthopaedic material may comprise a first layer of FHBP and/or OMPT and, optionally, a second or further layer of FHBP and/or OMPT. In preferred embodiments of the invention the first layer comprises ODPA and/or FHBP with a second or further layer of FHBP.

The biocompatible material may comprise one or more of titanium, titanium alloy, stainless steel, tantalum, a tantalum alloy, polyethylene, ceramics (e.g. aluminium oxide or calcium phosphates, such as hydroxyapatite), a natural polymer material (e.g. collagen-, fibrin-, agarose- or chitosan-based material) or a saturated aliphatic polymer material (e.g. poly(lactic acid), poly(glycolic acid), poly(lactic-coglycolide)), or derivatives of any of these. Materials comprising an oxide layer on the surface may be especially suitable. Preferably the biocompatible material comprises titanium.

The orthopaedic material may comprise 10-100% surface area coverage of the compound of formula (I), for example 20-95% or 30-70%) of the surface area of the biocompatible material may be covered. Preferably the compound of formula (I) covers any surfaces of the orthopaedic material that come into direct contact with bone and/or bone marrow when the material is implanted into a patient. The orthopaedic material of the invention may be formed as an orthopaedic implant device or portion thereof, i.e., an implant device or portion thereof may be formed from (or at least partially using) the orthopaedic material according to the invention. Composite devices comprising the orthopaedic material in combination with other materials are envisaged. An orthopaedic implant device is any device intended for insertion, on a temporary or permanent basis, into a body to replace or repair or abut a component of the body which is formed from bone. By way of non-limiting example, the device may be a replacement joint (e.g., a knee, hip or knuckle) or portion thereof, a replacement long bone (e.g., tibia, fibula, radius, ulna, femur or humerous) or portion thereof, or a pin, bolt or screw intended for engagement with or insertion into or through a bone. The body may be an animal, for example a mammal such as a human, cat, dog or horse.

In embodiments of the invention the orthopaedic implant device may be a dental implant, such as a screw or a cylinder for insertion into the jaw. Said implants can be inserted into prepared bony sockets of the jaw to serve as replacement roots for missing teeth. The screws or cylinders may be from about 5 mm to about 20 mm in length, preferably from about 8 mm to about 16 mm in length. An attachment known as an abutment may be fitted to the top of the dental implant, thereby forming an external connection for the new replacement tooth (crown) or teeth (bridge or denture). Successful osseointegration of known dental implants usually takes 3-6 months but this could be reduced by introducing the coatings of the present invention.

The present invention also provides a method of surgery in which an orthopaedic implant device of the invention is inserted into the body of a patient. Suitable surgical techniques for inserting the device will be familiar to orthopaedic surgeons. According to a further aspect of the invention there is provided a method of manufacturing an orthopaedic material of the invention, the method comprising immersing or coating a biocompatible material with at least one compound of formula (I). The biocompatible material may be in the form of biocompatible material item, such as an orthopaedic implant device or a disc for use in cell culture, for example, a titanium item. In embodiments of the invention the biocompatible material is baked at a temperature of at least 120°C prior to immersing or coating the biocompatible material with the compound of formula (I). For example, the biocompatible material may be baked at a temperature of about 180°C. Baking may be carried out for about 24 to about 120 hours, preferably for about 72 hours. Preferably baking induces oxide formation on the surface of the biocompatible material to "functionalise" the material.

Immersing or coating may comprise exposing the biocompatible material to a solution of the compound of formula (I) for a period of 12-48 hours, preferably for about 24 hours. The method of exposure is not particularly limited and may include, for example, spraying, dipping or steeping.

Following coating or immersion of the biocompatible material to form the orthopaedic material, the orthopaedic material may be baked at a temperature of at least 120°C. For example, the orthopaedic material may be baked at a temperature of about 180°C. Baking may be carried out for about 5 to about 48 hours, preferably for about 24 hours.

After baking the orthopaedic material may be rinsed and then immersed or coated with a further compound of formula (I). Rinsing may be carried out using any suitable solvent solution, such water or a solvent solution comprising ethanol. In embodiments of the invention the orthopaedic material may be rinsed first in an alkaline solvent solution, such as a potassium carbonate and ethanol solution, before being rinsed in water.

In an embodiment of the invention a method of manufacturing an orthopaedic material may comprise the steps of: (i) baking the biocompatible material at a temperature of about 180°C for about 72 hours, (ii) coating or immersing the biocompatible material in an alkane phosphonic acid solution, (iii) baking the coated biocompatible material at a temperature of about 180°C for about 24 hours, and (iv) coating or immersing the coated biocompatible material in an LP A receptor agonist solution. The LP A receptor agonist solution of step (iv) may be a 0.5 to 10μΜ or 1 to 5μΜ solution of l-fluoro-3- hydroxy-4-butyl-l-phosphonate (FHBP) or a 0.5 to ΙΟμΜ or 1 to 5μΜ solution of 1- oleoyl-2-methyle-sn-glycero-3-phosphothionate (OMPT).

Brief Description of the Drawings

The invention will now be described in relation to specific embodiments in which:

Figure 1. shows a stylised summary of an embodiment of the invention featuring titanium surface modification using an alkane phosphonic acid and an analogue of lysophosphatidic acid. A. Titanium (Ti) surfaces are initially steeped in a ImM solution of octadecylphosphonic acid (ODPA). B. The polar head group of the phosphonic acids react with available hydroxyl residues at the metal surfaces forming robust Ti-O-P bonds leaving acyl chain extensions perpendicular to the metal surface. These fatty acyl chains provide sites for further surface modifications. C. ODPA- preconditioned surfaces are then bathed in an aqueous solution of the LPA analogue, FHBP, and the molecules allowed to coalesce with fixed ODPA residues at the Ti surface. D. Depicted how the surface may appear with bioavailable FHBP tucked into fixed ODPA in a manner resembling the formation of phospholipid membranes commonly found in nature. Figure 2 shows titanium functionalisation with FHBP, a lysophosphatidic acid analogue. Titanium (Ti) surfaces were either steeped in a ImM solution of octadecylphosphonic acid (ODPA) followed by immersion in FHBP (2μΜ) or taken straight into an FHBP solution. In both instances recovered discs were rinsed and then seeded with human (MG63) osteoblasts in the presence of ΙΟΟηΜ 1,25D. Unmodified, control (CTRL) Ti surfaces were also seeded with osteoblasts, with 1,25D present at lOOnM. Cells were left on these surfaces for three days and the discs processed for total alkaline phosphatase (ALP) activity. The data, expressed as the fold change of enzyme activity relative to the CTRL Ti group, clearly indicate enhanced total ALP activity for both of the modified surface types. Groups (1) and (2) refer to independent experimental runs. In each instance the data represent the mean ± the standard deviation from 4 replicates. Figure 3 shows FHBP-functionalised titanium withstands autoclaving. Solid titanium (Ti) discs were directly steeped in aqueous 2μΜ FHBP and the samples left at ambient temperature. Control Ti specimens were simply left in tissue culture-grade water. Modified discs were subsequently split so that half went through an autoclave cycle and the remainder left under ambient conditions. Sample discs were then seeded with MG63 osteoblasts in the presence of ΙΟΟηΜ 1,25D and the samples left for three days under conventional cell culturing conditions prior to an analysis of total alkaline phosphatase (ALP). Increasing concentrations of p-nitrophenol (p- P) generated from p-nitrophenylphosphate reflect raised ALP and therefore enhanced osteoblast maturation. The data clearly indicate that autoclaved Ti-FHBP withstands autoclaving and supports l,25D-induced cellular maturation. In each instance the data represent the mean ± the standard deviation from 4 replicates.

Figure 4 shows human osteoblast maturation on ODPA/FHBP-functionalised hydroxyapatite. Solid hydroxyapatite (HA) discs were initially steeped in lmM octadecylphosphonic acid (ODPA), rinsed, and then steeped in different concentrations of FHBP (1, 5 and ΙΟμΜ). Recovered samples were rinsed and subsequently seeded with MG63 human osteoblasts in the presence of ΙΟΟηΜ 1,25D. Cells were left on these surfaces for three days and the discs processed for total alkaline phosphatase (ALP) activity through the quantification of p-nitrophenol (p- P) from p-nitrophenyl phosphate. The data depicted clearly indicate a dose- dependent effect on MG63 maturation with an initial steeping solution strength of ΙΟμΜ supporting no influence on cellular differentiation. Conversely when discs were exposed to the lower concentrated solutions the recovered samples have a more positive effect on MG63 maturation (*p<0.01 versus control HA), with the Ι μΜ solution yielding the best (**p<0.001 versus control HA) response. In each instance the data represent the mean ± the standard deviation from 4 replicates.

Figure 5 shows recycled ODPA/FHBP-functionalised solid HA is still able to support l,25D-induced osteoblast maturation. Solid hydroxyapatite (HA) discs were initially steeped in lmM octadecylphosphonic acid (ODPA), rinsed, and then steeped in 2μΜ FHBP. Recovered samples were rinsed and subsequently seeded with MG63 human osteoblasts in the presence of lOOnM 1,25D. Cells were left on these surfaces for three days and the discs processed for total alkaline phosphatase (ALP) activity through the quantification of p-nitrophenol (p- P) from p-nitrophenyl phosphate. On completion of the assay the discs were cleaned under running tap water with an electric toothbrush, rinsed in molecular biology-grade water, plunged into 70% ethanol, recovered, equilibrated in tissue culture medium and seeded with cells again in the presence of lOOnM 1,25D. This cycle was repeated a further two times. Even after a 4th reuse there is clear evidence of heightened osteoblast maturation compared to control surfaces (*p<0.001 versus control (CTRL)-4th reuse HA). Collectively the data support the robust attachment of a biologically active LP A receptor agonist to solid HA. In each instance the data represent the mean ± the standard deviation from 4 replicates.

Figure 6 shows human osteoblast maturation at FHBP-functionalised hydroxyapatite. Solid HA discs were immersed in molecular biology-grade water (HA control) or in aqueous solutions of FHBP (250 & ΙΟΟΟηΜ). Recovered samples were rinsed and subsequently seeded with MG63 human osteoblasts in the presence of lOOnM 1,25D.

Cells were left on these surfaces for three days and the discs processed for total alkaline phosphatase (ALP) activity through the quantification of p-nitrophenol (p- P) from p-nitrophenyl phosphate. Compared to the control HA samples there is clear evidence of cellular maturation for the FHBP-functionalised materials (*p<0.01 in both instances).

Figure 7 shows (a) Average roughness coefficient and (b) surface free energy of unmodified titanium and FHBP-modified titanium discs (* - p<0.05).

Figure 8 shows fluorescent microscopy images of S. aureus on (a) titanium, (b) ODPA treated titanium and (c) 10 μΜ FHBP titanium; and (d) percentage area covered and (e) colony counts of S. aureus attached on treated and untreated titanium discs (*- p<0.05, ** - p<0.01 and *** - pO.001). Figure 9 shows (a) Crystal violet staining and (b) colony counts of S. aureus biofilms on treated and untreated titanium discs (*- p<0.05, ** - p<0.01 and *** - p<0.001).

Figure 10 shows Silver-stained SDS-PAGE gels of (a) 24, (b) 48, (c) 72 and (d) 96 hour biofilms; and western blots of (a) GCSH and (b) TRX.

Examples

Materials & Methods

Titanium surface functionalisations - Solid titanium discs (12.7 mm diameter and 2.5 mm thickness - check) were a generous gift from Conn (Cirencester, UK). Unless stated otherwise, all reagents were of analytical grade from Sigma-Aldrich (Poole, UK). The alkane phosphonic acid, octadecylphosphonic acid (ODPA) was prepared as a ImM solution in anhydrous tetrahydrofuran (THF) in glass. Stocks of FHBP (Tebu- bio, Peterborough, UK) were prepared in 1 : 1 ethanoktissue culture grade water to a final concentration of lOmM and stored at -20 °C. The first step in Ti- functionalisation involved baking samples at 180°C for 3 days to encourage further Ti02 formation, once cooled, specimens were steeped in the ODPA solution (5 discs in 10ml using glass universal tubes) for 24 hours. Recovered samples were dried at ambient temperature before being baked at 180°C for 24 hours to convert ODPA to the phosphonate (Hanson et al. 2003). After this baking step a rinsing solution comprising 5ml 1.5M K2C03 in 10ml ethanol (sufficient for 20 Ti discs) was used to displace unbound/loosely bound ODPA by shaking (300RPM), at ambient temperature for 20 minutes. The solution was discarded and the samples rinsed three times in molecular biology grade water (MBW). These Ti samples were then placed into sterile 24-well tissue culture plates for exposure to either 0.8ml MBW or FHBP (2, 5 & 10μΜ in MBW). After 24 hours the discs were turned over and left for a further 24 hours at ambient temperature. Once incubated the sample discs were transferred to clean 24-well plates and given a single rinse with MBW (1ml per disc) before being rinsed twice in phenol red-free Dulbecco's modified Eagle medium/F12 nutrient mix (PRF-DMEM/F12, Gibco, Paisley, Scotland). These control and modified Ti specimens were then ready for immediate osteoblast seeding. In some instances after the MBW rinse, Ti samples were rinsed twice in 70% ethanol, allowed to dry in a tissue culture cabinet and subsequently shipped out for microbiological and physiochemical evaluation. Physicochemical characterisation of FHBP-functionalised Ti - To determine changes in surface roughness and surface energy, unmodified titanium discs and titanium discs modified with ImM ODPA followed by 0.1, 0.5, 1, 2, 5 and 10 μΜ of FHBP were prepared as previously outlined. Surface roughness was determined using a Surftest SV-2000 (Mitutoya, Hampshire, UK). Three sample surfaces were tested for each group in triplicate and surface roughness determined as the average roughness coefficient, in μπι. Surface free energy was determined according to the BS EN 828:2013 standard using the sessile drop method with water, ethylene glycol, glycerol and hexadecane on the surface of the samples. Responses of human (MG63) osteoblasts to phosphonate-functionalised Ti - Human osteoblast-like cells (MG63) were cultured in conventional tissue culture flasks (250 mL, Greiner, Frickenhausen, Germany) in a humidified atmosphere at 37 °C and 5 % C02. Although osteosarcoma-derived, MG63 cells exhibit features in common with human osteoblast precursors or poorly differentiated osteoblasts. Specifically, these cells produce type I collagen with no or low basal osteocalcin (OC) and alkaline phosphatase (ALP). However, when MG63s are treated with 1,25D, OC expression increases and when the same cells are co-treated with 1,25D and selected growth factors, e.g., LP A, the levels of ALP markedly increase, a feature of the mature osteoblast phenotype. Consequently, the application of these cells to assess the potential pro-maturation effects of novel materials is entirely appropriate. Further, the MG63 continues to be a widely used cell line in biomaterials research. Cells were grown to confluence in DMEM/F12 supplemented with sodium pyruvate (1 mM final concentration), L-glutamine (4 mM), streptomycin (100 ng/mL), penicillin (0.1 units/mL) and 10 % v/v foetal calf serum (Gibco, Paisley, Scotland). The growth media (500 mL final volume) was also supplemented with 5 mL of a lOOx stock of non-essential amino acids. Once confluent, MG63s were subsequently dispensed into blank 24-well plates (Greiner, Frickenhausen, Germany) or plates containing either control (ODPA treated) or ODPA/FHBP-modified Ti discs. In each case, wells were seeded with 1 mL of a 4 x 104 cells/mL suspension (as assessed by haemocytometry) in PRF-DMEM spiked with 1,25D (from a ΙΟΟμΜ stock in ethanol) to a final concentration of 100 nM. For the porous Ti samples, cells were seeded in the same growth medium but devoid of phenol red to eliminate any interference with the ALP assay described below. Cells were then cultured for 3 d, the media removed and the cells processed for total ALP activity to ascertain the extent of cellular maturation.

In a separate study, the potential of recycling the lipid-functionalised Ti was explored to ascertain whether the modified metal was still able to support an osteoblast maturation response. Briefly, discs that had already been seeded with cells and processed for total ALP activity were recovered, rinsed under running tap water and scrubbed with a toothbrush to remove any remaining cellular debris. Once rinsed, the samples were immersed in 70 % aqueous ethanol and left for 2-3 min before being rinsed several times with sterile PBS. Washed discs were then placed into clean, multi-well plates, rinsed with DMEM/ F12 nutrient mix and subsequently seeded with MG63 cells as described above. A second repeat of this step was performed to explore how three independent uses of the same functionalised discs could support the maturation ofMG63 osteoblasts to 1,25D.

Total ALP activity - An assessment of ALP activity is reliably measured by the generation of p-nitrophenol (p- P) from p-nitrophenylphosphate (p- PP) under alkaline conditions. Briefly, the MTS/PMS reagent was removed and the monolayers incubated for a further 15 min in fresh phenol red-free DMEM/ F12 to remove the residual formazan. Following this incubation period, the medium was removed and the monolayers lysed with 0.1 mL of 25 mM sodium carbonate (pH 10.3), 0.1 % (v/v) Triton X-100. After 2 min, each well was treated with 0.2 mL of 15 mM p-NPP (di- Tris salt, Sigma, UK) in 250 mM sodium carbonate (pH 10.3), 1 mM MgC12. Lysates were then left under conventional cell culturing conditions for 1 h. After the incubation period, 0.1 mL aliquots were transferred to 96-well microtitre plates and the absorbance read at 405 nm. An ascending series of p-NP (25-500μΜ) prepared in the incubation buffer enabled quantification of product formation. To ensure that the ALP activity was only associated for cells attached to Ti and not to the surrounding plastic, the sample discs were transferred to clean multiwell plates and then processed for ALP activity. Attachment and growth of a clinical S. aureus isolate to ODPA/FHBP-modified Ti - Joint aspirates and excised soft tissue were obtained from revision surgeries of infected knee replacements. Swabs of these samples were taken and cultured on blood agar under aerobic (5% C02) conditions at 37 °C. S. aureus isolates were identified as gram positive clustered cocci, γ-haemolysis, catalase and coagulase positive, oxidase negative, non-lactose fermenting and with a yellow/gold colony appearance. After 24 hours culture, S. aureus colonies were isolated from the agar and stored either at -80 °C or at 4 °C on Tryptone Soya Agar (TSA) slopes for later use. The clinical S. aureus isolate was cultured in 20 mL of Tryptone Soya Broth (TSB) at 37 °C, 5% C02 for 24 hours. The suspension was centrifuged at 3000rpm for 5 minutes and the pellet was resuspended in 20mL phosphate buffered saline (PBS). The pellet was centrifuged again at 3000rpm for 5 minutes and resuspended in PBS to give an absorbance at 600nm of 0.08 to 0.1 (approximately 1 x 107 CFU/mL).

To determine rates of bacterial attachment, uncoated titanium discs; titanium discs treated with 1 mM ODPA; and titanium discs treated with ImM ODPA followed by 0.1, 0.5, 1, 2, 5 and 10 μΜ of FHBP were prepared as previously outlined. The discs were placed on a 24 well plate and 1 mL of a lxlO⁷ CFU/mL bacterial suspension in PBS pipetted onto the surface of the discs. After 1, 2, 6, 12 and 24 hours incubation at 37°C, 5% C0₂ the discs were transferred onto a sterile 24 well plate and gently washed with lmL solution of 0.85% NaCl to remove non-adherent bacteria. 1 of syto9 and 1 of propidium iodide was added to lmL of sterile water (LIVE/DEAD® BacLight™ Bacterial Viability stain, Life Technologies, Paisley, UK) and 20 μΐ_^ of this solution added to the surface of the discs, covered with a sterile glass coverslip and left at room temperature for 5 minutes. Five random images of the disc surface were taken using a fluorescent microscope at xlO magnification at emission/excitation wavelengths of 485/530-630 respectively. Bacterial coverage was quantified using a macro written in ImageJ that calculates the percentage of the image occupied by fluorescence. The experiment was repeated however at each time point the samples were vortex mixed in ImL of PBS for 30 seconds to remove adherent bacteria. The suspensions were serially diluted and spiral plated on TSA for colony counting.

To assess biofilm formation, an overnight culture of S. aureus was prepared in TSB and adjusted to an absorbance at 600nm of 0.08 to 0.1. The suspension was serially diluted to produce a suspension of lxlO³ CFU/mL in TSB. Treated and untreated titanium discs were placed in a 24 well plate and ImL of the diluted suspension added to each well following incubation at 37 °C, 5% C02 for 24, 48, 72 and 96 hours, with broth changes every 24 hours. At each time point the broth was carefully removed, ensuring the biofilm was not disrupted and the disc washed gently with PBS twice to remove any non-adherent bacteria. 2 mL of methanol was added for 15 minutes to fix the biofilm followed by 2 mL of a 0.1% crystal violet solution for a further 15 minutes prior to two washes in PBS. 2 mL of 30% acetic acid solution was added to each well for 15 minutes to dissolve the biofilm and the absorbance of the solution measured at 580nm. The experiment was repeated however at each time point the samples were vortex mixed in ImL of PBS for 30 seconds to detach the biofilm and the suspensions serially diluted and spiral plated on TSA for colony counting.

To investigate S. aureus protein secretion, 24, 48, 72 and 96 hour biofilms were cultured on the titanium discs as previously described. At each time point the broth was carefully removed, ensuring the biofilm was not disrupted and the disc washed gently with PBS twice to remove any non-adherent bacteria. The discs were vortex mixed in 0.5mL of PBS for 30 seconds followed by sonication on ice at 50% intensity for 30 seconds using a SLPe sonifier (Branson Ultrasonics, Connecticut, USA) to remove the biofilm. The protein sample was centrifuged at 3000rpm to remove cell debris and the supernatant diluted 1 : 1 in Laemmli sample buffer containing 5% 2- mercaptoethanol and heated at 95°C for 5 minutes. 20 uL of the sample was separated on a mini-protean TGX gel, with pre-stained Kaleidoscope standard, at 200V for 30 minutes in a BioRad Mini-Protean Tetra Cell (BioRad, Hertfordshire, UK). Gels were stained using BioRad' s Silver Stain Plus Kit according to the manufacturer's instructions. 48 hour biofilms were also separated on another gel and proteins transferred onto nitrocellulose membranes for western blotting with glycine cleavage system protein H (GCSH, which is part of the glycine cleavage system and degrades glycine) and thioredoxin (TRX, a protein expressed in response to oxidative stress) antibodies followed by an alkaline phosphatase secondary antibody and 5-bromo-4- chloro-3'-indolyphosphate/nitro-blue tetrazolium (BCIP/NBT) staining.

Results

Physicochemical characterisation of ODPA/FHBP-functionalised Ti - The roughness of the titanium surfaces was not significantly altered as a result of FHBP-modification (Figure 7). Although a slight reduction in surface free energy was observed due to FFIBP-modification of the titanium discs, this was only significant for samples treated with 1 μΜ FHBP (Figure 7b). Attachment and growth of a clinical S. aureus isolate to ODPA/FHBP-modified Ti - S. aureus attachment to the surface of untreated and ODPA-modified titanium discs (Figures 8a and b respectively) was found to be greater than on FFIBP-modified discs (Figure 8c). The surface modifications however were not bactericidal towards the clinical strain of S. aureus employed. When analysing the area covered by bacteria using ImageJ, there were significant reductions in bacterial load as a result of FFIBP- modification, particularly at concentrations of 1, 2, 5 and 10 μΜ (Figure 8d). ODPA however had no effect on bacterial load when compared to the untreated titanium discs. The results of this analysis are in agreement with the results from the colony counts (Figure 8e). A parabolic relationship between FFIBP concentration and bacterial attachment was observed, whereby 1, 2 and 5 μΜ FFIBP concentrations were most effective at inhibiting bacterial attachment. The rate of attachment increased for all samples throughout the 24 hour culture period, demonstrating the initial rate of attachment of S. aureus to be inhibited at the surface of the FHBP-modified titanium. Subsequent attachment would be predicted to increase as a result of cell attachment and cell-cell interactions. Biofilm formation on treated and untreated discs was determined as a function of crystal violet staining (Figure 9a) and colony counts (Figure 9b). Similar to the attachment results, ODPA-modification did not significantly alter biofilm formation when compared to untreated titanium. FFIBP-modified titanium however was found to hinder biofilm formation for all time points when staining with crystal violet (Figure 9a). This effect was significant for 1, 2 and 5 μΜ FFIBP concentrations for all time points. A similar parabolic trend was observed with the colony counts (Figure 9b) however the reductions in biofilm formation were only significant for 1, 2 and 5 μΜ FHBP concentrations at 48 hours and 1 μΜ FHBP at 72 hours.

Silver staining of the SDS-PAGE gels for the 24 hour biofilms yielded low protein levels (Figure 10a). Faint bands were observed at 19 kDa however, with 1, 2 and 5 μΜ FFIBP-modified titanium demonstrating the most intense bands. This increase in intensity for low molecular weight bands (14 and 19 kDa) on 1, 2 and 5 μΜ FHBP- modified discs was also observed for 48, 72 and 96 hour biofilms (Figures 10b, c and d respectively). Interestingly, plain titanium discs had less protein present in the biofilm than FUBP-coated discs. Western blotting for GCSH confirms the presence of the low molecular weight protein within the biofilm, particularly on titanium discs coated with 1, 2, 5 and 10 μΜ FHBP (Figure lOe). Very low levels of TRX were detected (Figure lOf) and therefore the band at 19 kDa is thought to consist of a different protein.

Discussion Biomaterials that have the capacity to enhance hOB formation and maturation are particularly appealing in a bone regenerative context. One way of improving host cell responses to existing materials, such as titanium, includes attaching small, robust biological agents known to target hBMSCs and hOBs. If the biomaterial modification can also hinder the initial attachment of bacteria then they are more likely to reduce the infection risk of implantable devices. Such dual-action biomaterials for either orthopaedic or dental applications have not been forthcoming. In this particular study we coated orthopaedic-grade titanium with FHBP, a phosphatase-resistant LP A analogue which we now report as exhibiting this novel dual-action.

When hOBs were exposed to this bio-functionalised surface their maturation response to 1,25D was enhanced. As hOBs mature they mobilise greater quantities of ALP, an enzyme we know to be absolutely necessary for the formation of a mechanically sound, calcified bone matrix (Whyte 2010). Stimulating hOB maturation at the biomaterial surface would be conducive to securing superior early osseointegration by encouraging bone matrix formation and mineralisation. In our hands we find that the greatest extent of hOB maturation occurs on those surfaces steeped in 2μΜ FHBP. Why this is optimum for supporting hOB maturation may relate to greater bioavailability. The coating itself displays evidence of stability to storage under ambient conditions. This is particularly significant as titanium implants are stored this way to keep costs to a minimum. Furthermore, when FFIBP-modified samples were "recycled" by seeding hOBs on to them for a second and third time they were still able to support better hOB maturation compared to control, unmodified metal. These findings indicate the persistence of sufficiently high enough FHBP at the titanium surface despite having been re-seeded with cells, washed and placed under 70% ethanol for weeks at a time. Another noteworthy feature of our FFIBP-functionalised titanium is its ability to withstand autoclaving.

In parallel with the hOB studies we examined the attachment and colonisation of a clinical strain of S. aureus, a pathogen highly implicated in bone implant sepsis. Surface modification with FHBP was effective at significantly reducing S. aureus attachment when compared to unmodified titanium. Interestingly, the initial modification of titanium with ODPA was not sufficient to deter bacterial attachment, demonstrating the fluorinated analogue of LP A (FHBP) was responsible for reducing bacterial adherence. Furthermore, results from surface roughness and surface energy measurements highlight that the reduction in bacterial load was not due to changes in roughness or surface energy, but rather as a results of biochemical interactions between the FHBP molecule and S. aureus. It is important to stress that antimicrobial testing of the FHBP-modified titanium discs demonstrated that ODPA and FHBP molecules, at the concentrations employed, were not biocidal towards S. aureus, rather that the modification imparts an anti-adherent property.

The efficacy of the FHBP-functionalization was found to be concentration dependant and a parabolic relationship was observed, whereby the optimum concentration of FHBP to reduce bacterial attachment was in the range of 1 to 5 μΜ. It is thought that too low a concentration of FFIBP (100 to 500 nM) will not sufficiently coat the entirety of the ODPA-functionalised titanium surface; whilst over saturation of FHBP molecules (>10 μΜ) may induce multiple layers of FFIBP and therefore alter the orientation of the lipid on the surface. Similar results were obtained for biofilms formation on the titanium surfaces. FHBP functionalization at optimum concentrations of 1 to 5 μΜ was found to reduce biofilm mass, composed of extracellular DNA, proteins and polysaccharide. This is of particular importance in preventing septic implant failure as biofilms act as a reservoir for chronic infection and are not easily eradicated by antimicrobials. Although bacterial attachment and biofilm formation at each time point were significantly lower when compared to unmodified titanium, the overall increase in attachment and biomass between time points was similar. Functionalization with FHBP is a surface modification and therefore only initial bacterial attachment to the surface is inhibited. Once the surface is preconditioned, through physiochemical forces (e.g. hydrophobic interactions, electrostatic interactions, Van der Waals forces) or protein coating (e.g. fibrinogen, vitronectin, Von Willebrand factor, platelets/thrombin), it is still a possibility that any bacteria introduced at the time of implantation could adhere to this surface. Further bacterial colonisation will likely result in the formation of a mature biofilm. In vivo, adherence may also be mediated by bacterial surface proteins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs).

It was expected that a reduction in bacterial number and biofilm mass would also result in a reduction in the amount of protein detected by SDS-PAGE. Interestingly an increase in the secretion of low molecular weight proteins was observed for biofilms cultured on the FHBP-modified samples. Particular lysophospholipids, such as monopalmitoyl LP A (16:0 LP A), have been shown to inhibit virulence factor production in Pseudomonas aeruginosa strains and increase the sensitivity of resistant P. aeruginosa isolates to ampicillin. Monopalmitoyl LPA was found to act as a chelator, removing Ca²⁺ and Mg²⁺ from lipopolysaccharide found in the cell wall, consequently destabilising the outer membrane. Membrane disruption would permit leaching of low molecular weight proteins from the bacteria as observed with the SDS-PAGE and western blot from biofilms for our FHBP-modified titanium.

In conclusion we have bio-functionalised Ti with a phosphatase-resistant analogue of LPA, FHBP. This surface finish enhanced l,25D-induced hOB maturation, as indicated by increased expression of ALP, a reliable marker of osteoblast differentiation. Of further significance are the findings of an anti-adherent property of FHBP-Ti towards S. aureus, a clinically relevant bacterial species associated with sepsis-induced implant failures. These are especially desirable features of implantable bone biomaterials that could be realised in tackling the issue of revision arthroplasty in the future.

References

Blackburn J, Mansell JP. The emerging role of lysophosphatidic acid (LPA) in skeletal biology. Bone 50 (2012) 756-762.

Bonewald LF, Kester MB, Schwartz Z, Swain LD, Kharen A, Johnson TL, Leach RJ, Boyan BD. Effects of Combining Transforming Growth FactorP and 1,25- Dihydroxyvitamin DS on Differentiation of a Human Osteosarcoma (MG-63). J. Biol. Chem. 267 (1992) 8943-8949.

Calvano CD, Jensen ON, Zambonin CG. Selective extraction of phospholipids from dairy products by micro-solid phase extraction based on titanium dioxide microcolumns followed by MALDI-TOF-MS analysis. Anal. Bioanal. Chem. 394 (2009)1453-1461. Gidley J, Openshaw S, Pring ET, Sale S, Mansell JP. Lysophosphatidic acid cooperates with 1 alpha, 25 (OH) 2D3 in stimulating human MG63 osteoblast maturation. Prost. & other Lipid Med. 80 (2006) 46-61. Lancaster ST, Blackburn J, Blom A, Makishima M, Ishizawa M, Mansell JP. 24,25- Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure human (MG63) osteoblast maturation. Steroids 83 (2014) 52-61.

Laux DC, Corson JM, Givskov M, Hentzer M, M0ller A, Wosencroft KA, Olson JC, Krogfelt KA, Goldberg JB, Cohen PS. Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAOl alginate, pyoverdin, elastase and LasA. Microbiology 148 (2002) 1709-1723.

Mansell JP, Barbour M, Moore C, Nowghani M, Pabbruwe M, Sjostrom T, Blom AW. The synergistic effects of lysophosphatidic acid receptor agonists and calcitriol on MG63 maturation at titanium and hydroxyapatite surfaces. Biomaterials 31 (2010) 199-206.

Mansell JP, Brown J, Knapp JG, Faul CF, Blom AW. Lysophosphatidic acid functionalised titanium as a superior surface for supporting human osteoblast (MG63) maturation. European Cells and Materials 23 (2012) 348-361.

Paz Y. Self-assembled monolayers and titanium dioxide: From surface patterning to potential applications. Beilstein J. Nanotechnol. 2 (2011) 845-861.

Tigyi G, Parrill AL. Molecular mechanisms of lysophosphatidic acid action. Progress in Lipid Research 42 (2003) 498-526.

Whyte, M.P. (2010). Physiological role of alkaline phosphatase explored in hypophosphatasia. Ann. N Y Acad. Sci. 1192, 190-200. Yarram, S.J., Tasman, C, Gidley, T, Clare, M., Sandy, J.R., and Mansell, J.P. (2004). Epidermal growth factor and calcitriol synergistically induce osteoblast maturation. Molecular and Cellular Endocrinology 220, 9-20.

Claims

1. An orthopaedic material comprising a biocompatible material and at least one compound corresponding to the general formula (I):

A-[P(0)(OH)₂]„ (I) wherein A means Ai, A₂ or A3, and

n is 1 or 2;

wherein when the compound has the general formula A₂[P(0)(OH)₂]_n at least one additional compound having the formula Ai[P(0)(OH)₂]_n or A3[P(0)(OH)₂]_n is bound to the A₂[P(0)(OH)₂]_n compound via a hydrophobic interaction between the Ai or A3 and A₂ residues.

2. An orthopaedic material according to claim 1 , wherein the hydrocarbon residue of Ai, A₂ and/or A3 comprises 10-30 carbon atoms.

3. An orthopaedic material according to claim 1 or 2, wherein when A is Ai or

4. An orthopaedic material according to claim 3, wherein the compound of formula Ai[P(0)(OH)₂]i or A₃[P(0)(OH)₂]i is an LP A receptor agonist.

5. An orthopaedic material according to claim 3 or 4, wherein the compound of formula Ai[P(0)(OH)₂] i is l-fluoro-3-hydroxy-4-butyl-l-phosphonate (FHBP).

6. An orthopaedic material according to any of claims 3 to 5, wherein the compound of formula A3[P(0)(OH)₂] i is (2S)-l-oleoyl-2-0-methyl-glycero-3- phosphothionate (OMPT).

7. An orthopaedic material according to any of claims 1 to 6, wherein the compound of formula A₂[P(0)(OH)₂]_n is an alkyl phosphonic acid or an alkyl bisphosphonic acid.

8. An orthopaedic material according to claim 7, wherein the compound of formula A₂[P(0)(OH)₂]_n is octadecylphosphonic acid (ODPA).

9. An orthopaedic material according to any of claims 1 to 8, wherein the biocompatible material comprises one or more of titanium, titanium alloy, stainless steel, tantalum, a tantalum alloy, polyethylene, hydroxyapatite, a natural polymer material or a saturated aliphatic polymer material.

10. An orthopaedic material according to any of claims 1 to 9, formed as an orthopaedic implant device or portion thereof.

1 1. A method of surgery comprising implanting the orthopaedic device of claim 10 into a patient.

12. A method of manufacturing an orthopaedic material according to any of claims 1 to 9, the method comprising immersing or coating a biocompatible material with at least one compound of formula (I).

13. A method of manufacturing an orthopaedic material according to claim 12, further comprising the step of baking the biocompatible material at a temperature of at least 120°C prior to immersing or coating the biocompatible material with the compound of formula (I).

14. A method of manufacturing an orthopaedic material according to claim 13, wherein the biocompatible material is baked at a temperature of about 180°C.

15. A method of manufacturing an orthopaedic material according to claim 13 or 14, wherein the biocompatible material is baked for about 24 to about 120 hours.

16. A method of manufacturing an orthopaedic material according to claim 15, wherein the biocompatible material is baked for about 72 hours.

17. A method of manufacturing an orthopaedic material according to any of claims 12 to 16, further comprising the step of baking the orthopaedic material at a temperature of at least 120°C.

18. A method of manufacturing an orthopaedic material according to claim 17, wherein the orthopaedic material is baked at a temperature of about 180°C.

19. A method of manufacturing an orthopaedic material according to claim 17 or 18, wherein the orthopaedic material is baked for about 5 to about 48 hours.

20. A method of manufacturing an orthopaedic material according to claim 19, wherein the orthopaedic material is baked for about 24 hours.

21. A method of manufacturing an orthopaedic material according to any of claims 17 to 20, further comprising the step of rinsing the orthopaedic material and immersing or coating the orthopaedic material with a further compound of formula (I).

22. A method of manufacturing an orthopaedic material according to any of claims 1 to 9, the method comprising the steps of: (i) baking the biocompatible material at a temperature of about 180°C for about 72 hours,

(ii) coating or immersing the biocompatible material in an alkane phosphonic acid solution,

(iii) baking the coated biocompatible material at a temperature of about 180°C for about 24 hours, and

(iv) coating or immersing the coated biocompatible material in an LPA receptor agonist solution.

23. A method of manufacturing an orthopaedic material according to claim 22, wherein the LPA receptor agonist solution of step (iv) is a 0.5 to ΙΟμΜ solution of 1- fluoro-3-hydroxy-4-butyl-l-phosphonate (FHBP) or a 0.5 to ΙΟμΜ solution of 1- oleoyl-2-methyle-sn-glycero-3-phosphothionate (OMPT).

24. A method of manufacturing an orthopaedic material according to claim 23, wherein the LPA receptor agonist solution of step (iv) is a 1 to 5μΜ solution of 1- fluoro-3-hydroxy-4-butyl-l-phosphonate (FHBP) or a 1 to 5μΜ solution of 1-oleoyl- 2-methyle-sn-glycero-3-phosphothionate (OMPT).

25. An orthopaedic material as hereinbefore described with reference to the examples.