WO2011039513A1

WO2011039513A1 - Devices inhibiting bacterial attachment

Info

Publication number: WO2011039513A1
Application number: PCT/GB2010/001839
Authority: WO
Inventors: Nicholas William Botterill; David Malcom Grant
Original assignee: Eminate Limited
Priority date: 2009-09-30
Filing date: 2010-09-30
Publication date: 2011-04-07

Abstract

A device has a surface provided with a coating of amorphous calcium phosphate having a thickness of 1 nm to 10000 nm and is characterised in that the coating of amorphous calcium phosphate incorporates silicon. The coating is able to resist colonisation by bacteria. Devices in accordance with the invention may be implantable, medical devices such as orthopaedic implants.

Description

DEVICES INHIBITING BACTERIAL ATTACHMENT

The present invention relates to devices (e.g. medical devices or implants or surgical instruments) and more particularly to such devices having surface characteristics which inhibit the attachment of bacteria to the device such as, in the case of a medical device, to reduce the possibility of bacterial infection in the patient arising from the device. The invention is particularly applicable (but not limited to) orthopaedic implants.

Implantable devices such as orthopaedic implants have been used successfully for many years. Orthopaedic implants may, for example, be used as a replacement for a worn or otherwise damaged body part, such as a hip joint or a knee. Further examples of orthopaedic implants are bone fixation devices (e.g. pins) to assist in the healing of fractured limbs or joints.

In spite of the successful use of implants over many years there is a significant risk of infection which can be a serious complication to surgery. The problem is aggravated as a result of bacterial resistance to anti-bacterial agents which would otherwise be used to control or eliminate the infection. For example, in June 2003, orthopaedic related infection had become such a concern that the Chief Medical Offer for the UK announced mandatory surveillance was to be carried out in all NHS Trusts to monitor infection rates in hip/knee joint replacement and open long bone fracture fixation surgeries. Infection rates in joint replacements are between approximately 1 % and 10% depending on reporting agency and area although it is generally accepted that the incidence of infections is under-reported. Medicare data puts the rate at 3.5% and 12.8% for primary and revisions respectively. Infection associated with surgical implants is therefore a serious problem and for some operations such as Total Knee replacements (TKR) the improvements in surgical techniques are at such a level that the prime mode of failure of TKR is likely to be infection.

Orthopaedic implants frequently have a coating designed to promote a bioactive response to aid the bone tissue-implant interface. Many designs are based on coatings deposited by various physical and chemical methods (see references 1-29). These are often based on crystalline phases of calcium phosphate such as hydroxyapatite produced by plasma spraying (e.g. US Patent 5763092) where importance is given to degree of crystallinity of the coating or modifications that combine pores (e.g. US Patent 6764769) or mixtures with other crystalline phases such as those based on metal oxides (US Patent Specification 2005/0019365). These coatings all provide a crystalline component for the required chemical, physical and biological stability over the period of tissue integration which can be from months to years.

Our earlier PCT Application No. PCT/GB2009/000805 (published on 1^st October 2009 as WO 2009/118531 , the disclosure of which is incorporated herein by reference) discloses medical devices (e.g. hip implants) in which the body contacting surfaces of the device are provided with a coating of amorphous calcium phosphate having a thickness of 1 nm to 10000 nm and an average dissolution rate calculated as the time taken to remove 95% of the coating of at least 1 nm h-¹ as determined in accordance with ASTM Standard P1926-03). The underlying rationale of the invention disclosed in our aforementioned PCT application is that the dissolution of the surface coating of amorphous calcium phosphate (by body fluids) is sufficiently fast to prevent the attachment of bacteria, thus preventing them forming a biofilm and keeping the cells in a planktonic (non-biofilm) state in which they are more vulnerable to the body's defences and, in the case of an implant, any antibacterial agent applied with the implant as per standard operating procedure.

Reference is made in this earlier application to the possibility of incorporating additional elements in the amorphous calcium phosphate layer to control dissolution behaviour, but without more specific exemplification. The present invention is based on the disclosure in our prior PCT application and more particularly to modifications that may be made to the amorphous calcium phosphate layer that provide significant improvement in the ability of a surface coating comprised of amorphous calcium phosphate to inhibit bacterial colonisation on the device. According to a first aspect of the present invention there is provided a device having a surface provided with a coating of amorphous calcium phosphate having a thickness of 1 nm to 10000 nm characterised in that the coating of amorphous calcium phosphate incorporates silicon. By the term "amorphous" we mean that the calcium phosphate coating does not display any crystalline peaks in its X-Ray diffraction spectrum but rather a broad amorphous "hump". The silicon in the coating may be present in the form of silicon or a silicon compound (e.g. silica). The amount of silicon present in the coating will depend on the environment to which the coating is to be subjected. Generally the amount of silicon (expressed as elemental silicon even though the silicon may be present in compound form) in the coating will be a maximum of 15% with the minimum usually being 0.01 % by weight, these amounts being based on the total weight of the coating. More typically, the maximum amount of silicon in the coating will be 10% by weight and more usually a maximum of 5% by weight based on the total weight of the coating. Coatings for use on medical devices (a preferred embodiment of the invention - see below) will generally comprise less than 1 % by weight of silicon based on the total weight of the coating.

In accordance with the invention, a medical device has a thin coating (1-10000 nm) coating of amorphous calcium phosphate incorporating silicon which may be either in elemental or compound form. Our studies have been based on surface coatings of (i) amorphous calcium phosphate incorporating silicon, and (ii) amorphous calcium phosphate without silicon and correlating the rates at which calcium ions are able to dissolve therefrom with the ability of the coating to resist colonisation by bacteria. Surprisingly, we have found that surface coatings of amorphous calcium phosphate incorporating silicon which release calcium into solution at (or at about) the same rate as amorphous calcium phosphate coatings (without silicon) have significantly improved properties with regard to inhibition of colonisation by bacteria. Consequentially the effect obtained by the incorporation of silicon cannot be attributed solely to dissolution. We are unable, at present, to explain the reason why the incorporation of silicon provides significant improvements. This could be due to its chemical form such as forming a silica matrix or being incorporated into the amorphous calcium phosphate as silicates or due to some unreported activity of silicon or perhaps the silicon causes the amorphous calcium phosphate to be removed in stages, clusters or in a different way than amorphous calcium phosphate on its own which upsets the bacterial attachment. Whatever the reason the result is dramatic and unexpected based on the previous results with amorphous calcium phosphate alone.

The amorphous calcium phosphate has a thickness of 1 nm to 10000 nm. Preferably the thickness of the amorphous calcium phosphate coating is 10 nm to 5000 nm, more preferably 20-1000 nm. Thickness determination may be effected using a ball crater method in which a large spherical ball erodes a crater and the crater is then measured from above using high resolution optical microscopy or, for very thin coatings, scanning electron microscopy. Using the geometry of a sphere and the diameters of the crater on the top of the thin coating and the bottom of the thin coating the thickness can be calculated. Thickness measurements can also be determined and/or cross checked using X-section transmission electron microscopy in which the coating can be measured directly.

The calcium:phosphorous ratio in the amorphous calcium phosphate may, for example, be in the range of (0.1 to 5):1 , preferably (0.4 to 2):1 most preferably (1 to 2):1.

The invention is applicable to a wide range of devices and particularly to medical devices, although non-medical applications are also envisaged where anti biofouling is advantageous. The invention is applicable particularly to orthopaedic and dental implants and examples include prosthetic hips, ankles, shoulders and knees as well as other orthopaedic devices such as bone plates, percutaneous devices (e.g. pins, fracture fixation devices, dental pins and cannulae). Although the invention is applicable particularly to orthopaedic implants, it may also be applied to other devices. These include non-orthopaedic implants and devices such as stents (e.g. cardiovascular or urological stents), artificial grafts, catheters, shape memory devices, surgical instruments and needles.

Medical devices in accordance with the invention will generally have their body contacting surfaces provided with the thin coating of amorphous calcium phosphate incorporating silicon. By "body contacting surfaces" we mean those surfaces of the device which (when "used", e.g. implanted) will be in contact with body tissue, which term as used herein includes but is not limited to bone. In the case, for example, of implants which in use are wholly intra-corporeal then all exterior surfaces of the prosthesis will generally be provided with the thin coating of calcium phosphate containing silicon, except possibly articulating surfaces of prosthetic joints. However the invention also extends to implants which will be partially extra-corporeal, in which case those surfaces of the implant which will be extra-corporeal need not be provided with the calcium phosphate coating. Although the invention is considered to have particular application in the field of medical devices other uses are envisaged. Thus, for example, a surface coating of calcium phosphate containing silicon may be provided on a non-medical device where bacterial growth and/or formation of a biofilm is to be inhibited. Thus, for example, the invention will find use in the provision of coatings on filters and other surface in contact with the environment where anti biofouling is advantageous. The material forming the "structural" component of the device (e.g. implant) is not critical, subject of course to the proviso that the device has the required strength for its purpose. Thus, for example, the device may be of a metal, metal alloy or ceramic or polymeric material or composite. Examples of specific materials include, but are not limited to, titanium and its alloys, cobalt chrome alloys and stainless steel alloys, NiTi based shape memory alloys, crystalline ceramics such as hydroxyapatite, titania, alumina, zirconia, degradable and non degradable polymers such as polyglycolide, polylactide, copolymers of glycolide and lactide, polycaprolactone and polymethylmethacrylate, polyethylene and polytetrafluorethylene. Particularly in the case of medical devices, the substrate onto which the amorphous calcium phosphate coating containing silicon is deposited may have a bioactive or optimised implant surface which (it will be appreciated) is exposed once the calcium phosphate coating has been dissolved. Such a bioactive or optimised implant surface may take any form known in the art and is not critical to the present invention. Thus, for example, a substrate forming an orthopaedic implant may have a surface promoting a bioactive response to aid the bone tissue - implant interface. Examples have been given above (see also references (1-30)) and include crystalline phases of calcium phosphate such as hydroxyapatite produced by plasma spraying or modifications that combine pores or mixtures with other crystalline phases such as those based on metal oxides.

The underlying bioactive surface can be already present as on an existing commercial implant such as managed topographies, roughened, beaded or wired surfaces. In terms of chemistries these surfaces often have beneficial natural oxides such as titania or additional stable bioactive coatings such as crystalline bioceramics. These chemistries have defined crystalline structures with long term stabilities (greater than several days to years).

The amorphous calcium phosphate coating incorporating silicon (e.g. in the form of silicon oxide or other compound of silicon) may be deposited by Physical Vapour Deposition (PVD). Preparative Example 1 (below) gives full details as to the manner in which the PVD procedure may be effected to produce surface coatings in accordance with the invention to which reference should be made for the purposes of the following description.

The dissolution rate of the coating can be controlled readily by changes to the PVD conditions of power density, partial pressures of gases, chamber geometry.target geometry and bias. Process gases may be inert only such as argon or a combination of inert and reactive gases such as argon and oxygen. For example the silicon content in the coating, as exemplified in Preparative Example 1 (Invention), can be readily modified by varying the respective power densities to the relative targets that provide the source of silicon and calcium phosphate during the deposition process. Varying the number and composition of the targets or using a composite target or targets would provide alternative routes to produce such coatings that have amorphous calcium phosphate containing silicon. Other parameters such as temperature, sample bias and target to sample distance, can all affect composition and structure of the coating. Variation of thickness of the coating can be readily achieved through control of the above parameters over set periods of deposition time in a similar manner to that presented in Preparative Example 2 (Comparative).

The PVD method may be effected using two separate targets, one comprising hydroxyapatite and the other comprises silicon or a silicon-containing compound (e.g. silica). Alternatively the method may be effected using a composite target which comprises both hydroxyapatite and either silicon or a silicon-containing compound (preferably silica) in a predetermined ratio to achieve the desired coating composition. An alternative method for creating such coatings would be achieved by using a similar arrangement to that presented in Preparative Example 1 but with two key differences, namely the substitution of a Silicon (Si) target in place of the Silica (S1O2), and the provision for an additional piezo controlled mass flow control gas valve connected to a supply of pure oxygen and in turn to the capacitive manometer pressure transducer controlled feedback system in such a way that the relative partial pressures of Argon and Oxygen (and thus the total chamber pressure) could be controlled.

In this modification, identical process conditions to those presented in Preparative Example 1 would be employed, except a controlled flow of 36 seem of Argon gas and 5 seem of Oxygen would be introduced into the chamber via the aforementioned feedback system in order to raise the chamber partial pressure to circa 1 x10^"3 Torr. A further alternative method for creating such coatings would be achieved by using a similar arrangement to that presented in Preparative Example 1 , but by the use of composite targets which would contain both Hydroxyapatite and Silica or Silicon in a predetermined ratio to achieve the desired coating composition.

Such an arrangement involving composite targets would offer the advantage over separate target methods in that multiple composite targets could be mounted in the physical vapour deposition system (up to four in the Teer Coatings UDP650 for example), which could be powered simultaneously by four separate RF power generation systems; such an arrangement would allow for much higher total power densities to be applied to the magnetrons and thus enhanced coating deposition rates, thereby reducing the overall process time to allow for more efficient incorporation of the coating step into existing manufacturing processes.

In preferred embodiments, increased sputtering rates to increase deposition time can be achieved utilising magnetron sputtering and multiple targets.

A particular advantage of this invention is that the required thin coating to be deposited can be deposited in relatively short times making it commercially attractive.

The PVD methodology can deposit the amorphousthin coating onto any metallic or ceramic substrate and many polymeric substrates. Therefore this invention has advantages in that the coating can be applied readily to all existing metallic implants (such as those based on titanium alloys, cobalt chrome alloys and stainless steel alloys) and on top of any bioactive ceramic coatings that are applied to such implants such as crystalline Hydroxyapatite or Tricalcium phosphate that are used for bone integration. The PVD methodology with suitable jigging allows easy deposition onto 3-D objects and the thin coating conforms with the existing surface.

The coating can be applied readily to any external shape and is ideally suited to be applied to existing orthopaedic implants providing an easy to apply and safe antimicrobial coating which does not include any pharmacological elements or antibacterial agents that may harm the implant's integration and reduces usage of antibiotics. The invention will be illustrated with reference to the following non-limiting Examples and the accompanying drawings, in which: Fig 1 is a plot of calcium dissolution rates for various thicknesses of coating representing the result of Test Example 3 below;

Fig 2 is a comparison of the ability of 100 nm and 400 nm coatings of amorphous calcium phosphate on titanium coupons to inhibit bacterial colonisation as compared to a titanium coupon control, the data being obtained in accordance with Test Example 4 below; and

Fig 3 is a comparison of the ability of a 50 nm amorphous calcium phosphate coating containing silica on a titanium coupon to inhibit bacterial colonisation as compared to a titanium coupon control, the data being obtained in accordance with Test Example 4 below.

Preparative Example 1 (Invention) This example illustrates use of Physical Vapour Deposition to deposit thin coatings of amorphous calcium phosphate with Silica doping onto 12 mm diameter coupons of a commercially produced medical grade Ti6AI4V titanium alloy with a grit blast finish (typical of hip stem implants). A commercial physical vapour deposition (PVD) system (Teer Coatings

UDP650) was used to generate the amorphous coatings for this Example, comprising a vacuum chamber equipped with a serial pumping system (rotary and diffusion pumps). Gas pressure within the system was controlled using a feedback system comprising a capacitive manometer pressure transducer and piezo controlled mass flow control gas valve. Samples were held on a stainless steel jig which was capable of rotating at speeds of between 0.5 and 5 rpm and was coupled to a pulsed direct current (DC) bias power supply. A 57 mm diameter Hydroxyapatite target, was mounted on a magnetron on the sidewall of the vacuum chamber (such that it was immediately adjacent to the sample holder) and connected to radio frequency (RF) power supply with an automatic capacitive/inductive tuned matching network. Diametrically opposite to the Hydroxyapatite target was mounted an identical magnetron, with a 57 mm diameter Silica (Si0₂) target, which was in turn connected to a separate but identical radio frequency (RF) power supply with an automatic capacitive/inductive tuned matching network.

The titanium coupons were cleaned in an ultrasonic bath for 900 s each in acetone, methanol and distilled water before being dried in a Nitrogen gas stream. Samples were then introduced into the vacuum chamber, in which they were held on the stainless steel jig in line with the central axis of the Hydroxyapatite and Silica targets, with a separation of circa 1 10 mm. The chamber was subsequently evacuated to a base pressure of circa 5 x 10^'5 Torr.

Once base vacuum was achieved, a controlled flow of 41 seem of Argon gas was introduced into the chamber via the aforementioned feedback system in order to raise the chamber partial pressure to circa 1 x10³ Torr. RF power (13.56 MHz) at a density of 3.8 Wcm^"2 was then applied to the Hydroxyapatite target, and at a density of 0.1 1 Wcm^'2 to the Silica target, whilst simultaneously a pulsed DC bias voltage of -25 V (frequency 250 kHz pulse width 500 ns) was applied to the sample holder, thereby striking a plasma within the process chamber. The sample holder was rotated continually at a speed of 2 rpm such that samples passed through the individual plasmas of the Hydroxyapatite and Silica targets. Such conditions were maintained for a period of 14400 s before DC and RF power were switched off and samples allowed to cool for a period of 900 s before the chamber was vented to atmosphere to facilitate sample removal.

The above described procedure resulted in the formation on the coupon of a 50 nm thick amorphous coating of calcium phosphate with less than 1 % by weight_silica. The X-ray diffraction spectrum of the coating displayed a broad "hump" confirming the amorphous nature of the coating.

Preparative Example 2 (Comparative)

This example illustrates use of Physical Vapour Deposition to deposit thin coatings of amorphous calcium phosphate without silica having thicknesses of 50 nm, 100 nm and 400 nm onto 12 mm diameter coupons of a commercially produced medical grade Ti6AI4V titanium alloy with a grit blast finish (typical of hip stem implants). A commercial physical vapour deposition (PVD) system and sample cleaning regime identical to that presented in Example 1 was employed to generate the amorphous coatings for this work (save that no silicon dioxide target was present). Samples were introduced into the vacuum chamber, in which they were held on the stainless steel jig, immediately adjacent to the Hydroxyapatite target, with a separation of circa 110 mm. The chamber was subsequently evacuated to a base pressure of circa 5 x 10^"5 Torr.

Once base vacuum was achieved, a controlled flow of 41 seem of Argon gas was introduced into the chamber via the aforementioned feedback system in order to raise the chamber partial pressure to circa 1 x10^"3 Torr. RF power (13.56 MHz) at a density of 3.8 Wcm^"2 was then applied to the Hydroxyapatite target, whilst simultaneously a pulsed DC bias voltage of -25 V (frequency 250 kHz pulse width 500 ns) was applied to the sample holder, thereby striking a plasma within the process chamber. Such conditions were maintained for a period of 1800 s to generate coatings of 50 nm thickness, 3600 s to generate coatings of 100 nm thickness, or 14400 s to generate coatings of 400 nm thickness before DC and RF power were switched off and samples allowed to cool for a period of 900 s before the chamber was vented to atmosphere to facilitate sample removal.

Test Example 3

The procedure of Test Protocol 1 (see Appendix) was carried out on the coated samples produced in accordance with Preparative Example 1 (Invention, 50 nm coating) and those produced in Preparative Example 2 (Comparative 50 nm, 100 nm and 400 nm coatings) to compare dissolution rates.

The results are shown in Fig 1 which illustrates the cumulative amount of calcium released for the four different coatings repeated either n = 3, 6 or 9 times.

As can be seen from Fig 1 increasing the thickness of coating from 50 to 100 nm does little to effect the dissolution rate of calcium in the first 100 minutes. However increasing the thickness to of the amorphous calcium phosphate coating to 400 nm does make a significant change to the dissolution rate of Ca.

Examining the 50 nm amorphous calcium phosphate with silica coating, then the dissolution behaviour initially in the first 15 minutes tends to follow the 400 nm coating then follows a smilar trend to that of the 50 nm amorphous calcium phosphate coating.

In view of the similarity of that initial dissolution behaviour between, on the one hand, the 50 nm amorphous calcium phosphate with silica coating and, on the other hand, the 400 nm amorphous calcium phosphate coating without silica these two coating types were selected for the purpose of investigating their relative abilities to inhibit bacterial colonisation (see Test Example 4 below). Test Example 4

This Example demonstrates the ability of coated substrates produced in accordance with Preparative Example 1 (Invention) having a 50 nanometer coating of amorphous calcium phosphate containing silica to inhibit bacterial colonisation as compared to the coated substrates produced in accordance with Preparative Example 2 (Comparative) having 100 nm and 400 nm coatings of amorphous calcium phosphate (without silica) and uncoated titanium coupons (control).

A 10⁷ cfu/ml S. aureus suspension was applied to the coated surface of the titanium coupons using a sterile swab and allowed to dry on at 37 °C. The coupons were then transferred to a flow cell and fresh media (Tryptone Soya Broth, Oxoid UK) flowed over the samples at a rate of 4 ml min^'1 for 90 minat 37 °C. The coupons were then removed and washed to remove non adherent bacteria. The coupons were then placed into individual tubes with a recovery medium (0.85% Sodium Chloride, 1 % Tween 20) and sonicated at 60Hz in a sonicating water bath for 10 min. The resulting sonicate was then serially diluted (1 :10) 4 times and all dilutions plated out in duplicate. Plates were incubated for 48 h, plate counts were taken and counts in log cfu/sample calculated. In order to ensure that the flow cell was not accelerating calcium release, coupons with a 100 nm coating of amorphous calcium phosphate (not containing silica) were placed in a flow cell with de-ionised water at a flow rate of 4 ml min^'14 (n=3) for 90 min at 37°C. The samples coupon were removed from the flow cell at 1 , 2, 3 and 4 h and calcium release determined using the procedure of Test Protocol 1 (see Appendix). The release profiles were then compared with those for non-exposed controls and it was found that exposure to the flow cell did not result in an accelerated calcium release. Fig 2 shows the results for comparison between uncoated titanium coupons (control) and identical titanium coupons from the same batch having 100 nm and 400 nm thick coatings of amorphous calcium phosphate produced in accordance with the procedure of Preparative Example 2. Reference is now made to Fig 3 which shows the comparison between uncoated titanium coupons and identical titanium coupons from the same batch provided with a 50 nm coating in accordance with the procedure of Preparative Example 1. Fig 3 includes the results of three repeats on different batches of uncoated titanium coupons and those coated with amorphous calcium phosphate containing silica. The latter produced mean log differences of 1.35, 2.02 and 2.01 log cfu/sample respectively. This is a clear quantitative demonstration of the effectiveness of the coating comprised of amorphous calcium phosphate containing silica as a dissoluting layer in reducing bacteria colonisation. As shown in Fig 2, the reduction in bacterial colonisation produced by 100 nm and 400 nm coatings of amorphous calcium phosphate (not containing silica) was significantly less.

From the data presented in Fig 1 showing similarity of initial dissolution of the 50 nm amorphous calcium phosphate coating with silica and the 400 nm amorphous calcium phosphate coating without silica, significant difference in bacterial colonisation would not be expected. However, the presence of silica has a dramatic effect on the bacterial colonisation with a drop of two orders of magnitude observed on the amorphous calcium phosphate with silica sample as can be seen in Fig 3. The bacterial study was repeated three times as was the dissolution test (n=9) to confirm this result. This could not have been predicted from the Ca dissolution results. The mechanism is not known and it may be physical such as related to the dissolution mechanism of the amorphous calcium phosphate being different due to the silica network or presence of silicon within the amorphous calcium phosphate which prevents colonisation, or chemical in that in the combination of the silica or silicon with the dissolution products of amorphous calcium phosphate combine to prevent bacterial colonisation. APPENDIX:

Test Protocol 1

This protocol describes the procedure employed in Example 4 for determining rates of calcium dissolution from the coated samples produced in accordance with Preparative Example 1 (Invention) and Preparative Example 2 (Comparative).

3ml aliquots of de-ionised water containing approximately 0.5pg/ml of potassium internal standard were introduced into the wells of a standard polystyrene 12 well tissue culture plate which was then sealed with an adhesive strip to reduce evaporation. The aliquots were conditioned overnight at 37°C. Post conditioning, the coupons were introduced into a well and then moved to previously unused wells at time -points of 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 210 and 240 minutes. After the four hour elution testing, each sample was then left overnight at 37°C in a fresh 3ml aliquot in order to confirm the total Ca present on the coupon . All test solutions were acidified with 3μΙ of 2M nitric acid prior to calcium quantification using ion chromatography as detailed below.

Column: Metrohm Metrosep C2 100 x 4mm 7pm

Eluent: 4m tartaric / 0.75mM dipicolinic acid

Flow-rate: 1 ml/min

Temperature: ambient

Injection loop: 50μΙ

Detection: conductivity

Run-time: 12min

Potassium 0.5pg/ml was used as an internal standard.

The IC system (detector, autosampler, pump, degasser) was manufactured by Metrohm.

Each solution (as obtained above) was analysed to determine the amount of calcium therein. Cumulative amounts of calcium dissolved by a particular time point were obtained by summing the amounts of calcium for the solution at that time point and all earlier time points. Thus, for example, the cumulative amount for 60 min immersion of a coupon was obtained by summing the amounts of dissolved calcium measured for the 15, 30, 45 and 60 min time point.

The protocol validated conditioning of 3 ml calcium standards in the wells of a standard polystyrene 12 well tissue culture plate with an adhesive strip and lid overnight at 37 °C. On the following day the standard solutions were acidified by the addition of 3 μΙ of 2 M nitrate acid and the calcium levels then determined by ion chromatography using the procedure outlined above The results of the validation are shown below. replicate 4ppm 2ppm 0.5ppm 0.1ppm O.OSppm

1 4.0040 2.0272 0.5014 0.0945 0.0450

2 4.0667 2.0271 0.4859 0.0960 0.0469

3 4.2236 2.0444 0.5107 0.0978 0.0487

4 0.4944

5 0.4911

6 0.4942

mean 4.0981 2.0329 0.4963 0.0961 0.0469

actual 4.1664 2.0832 0.5208 0.0947 0.0473

%

accuracy 98.36 97.58 95.29 101.49 99.02

SD 0.008676

%RSD 1.67

The validation procedure demonstrated accuracy within 5% of the true value and %RSD 1.67%. The linear correlation co-efficient for the calibration was 0.99995. The range was between 4 and 0.05pg/ml or, expressed alternatively, between 12 and 0.15 pg per coupon (since 3 ml aliquots were used). Limit of detection was approximately 0.01 pgml^"1 or 0.03 pg per coupon.

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Claims

1. A device having a surface provided with a coating of amorphous calcium phosphate having a thickness of 1 nm to 10000 nm characterised in that the coating of amorphous calcium phosphate incorporates silicon.

2. A device as claimed in claim 1 wherein the silicon is provided as a silicon compound.

3. A device as claimed in claim 2 wherein the silicon compound is silicon dioxide.

4. A device as claimed in claim 1 wherein the silicon is provided as elemental silicon.

5. A device as claimed in any one of claims 1 to 4 wherein the coating comprises up to 15% by weight of silicon (expressed as elemental silicon) based on the total weight of the coating.

6. A device as claimed in claim 5 wherein the coating comprises up to 10% by weight of silicon (expressed as elemental silicon) based on the total weight of the coating.

7. A device as claimed in claim 6 wherein the coating comprises up to 5% by weight of silicon (expressed as elemental silicon) based on the total weight of the coating.

8. A device as claimed in claim 7 wherein the coating comprises up to 1% by weight of silicon (expressed as elemental silicon) based on the total weight of the coating.

9. A device as claimed in any one of claims 1 to 8 wherein the calciurrvphosphorous ratio in the amorphous calcium phosphate is in the range of (0.1 to 5):1.

10. A device as claimed in claim 9 wherein the calcium:phosphorous ratio in the amorphous calcium phosphate is in the range of (0.1 to 2):1.

11. A device as claimed in claims 10 wherein the calcium:phosphorous ratio in the amorphous calcium phosphate is in the range of (0.4 to 2):1.

12. A device as claimed in claims 11 wherein the calcium:phosphorous ratio in the amorphous calcium phosphate is in the range of (1 to 2):1.

13. A device as claimed in any one of claims 1 to 12 wherein said coating has a thickness of 10 nm to 5000 nm.

14. A device as claimed in claim 13 wherein said coating has a thickness of 20 nm to 1000 nm.

15. A device as claimed in any one of claims 1 to 14 which is a medical device.

16. A device as claimed in any one of claims 1 to 15 which is an implantable device.

17. A device as claimed in claim 16 which is an orthopaedic implant.

18. A device as claimed in claim 17 which is a prosthetic hip, ankle, shoulder or knee, a bone plate, pin, or fracture fixation device.

19. A method of producing a device as claimed in any one of claims 1 to 18 wherein the coating is deposited by Physical Vapour Deposition.