The present invention relates to a method for coating a titanium-based substrate, the relative coatings and the relative implants for biomedical applications.

Titanium (Ti) and its alloys are widely used as starting materials for the construction of implants for dental and orthopaedic applications, thanks to their excellent mechanical properties and corrosion stability. In order to increase osseointegration, the surface of the titanium is generally functionalized. A method commonly used in this field, capable of effectively changing the chemistry of the surface of a metal implant, is the coating of the same with bioactive ceramic layers based on calcium phosphate (CPC, Calcium Phosphate Ceramic).

This approach allows the mechanical resistance of the metal to be combined with the biocompatibility and bioactivity of the ceramic [1]. The surface roughness of the metal substrate also plays a decisive role in osseointegration processes; in this respect, the titanium substrates are generally previously treated in order to increase their surface roughness in a controlled manner. The techniques industrially used for this purpose are surface blasting (with alumina or titania particles) and/or acid attack - ac d etching.

In order to improve the adhesion between the substrate of Ti (or its alloys) and the bioactive coatings based on calcium and phosphorous, the introduction of dense and compact ceramic inter-layers is also known [2]. These reduce the discrepancy between the thermal expansion coefficients of the metal and the calcium derivative deposit, and also favour the adhesion of the bioactive component, thanks to the availability of -OH surface sites. Among the various ceramics, crystalline titania (Ti0 ₂ ) [2] is generally used for producing these intermediate layers, due to its known properties of biocompatibility, bioactivity and chemical affinity with respect to existing materials.

Furthermore, the presence of a compact layer of titania allows the concomitant increase in the corrosion resistance of the metal substrate itself [2] . The reference cited [2] therefore describes a two-layer coating of a titanium substrate: the first layer is crystalline titania and the second is hydroxyapatite, both deposited by means of electrophoretic deposition: a negative aspect of a coating produced by electrophoresis is linked to the inability of effecting a fine control of the thicknesses obtained within the desired range of values. In the article cited [2], layers of Ti0 ₂ are produced with a thickness of 4 microns (4,000 nanometers). At these values, the difference in thermal expansion coefficient between the titanium substrate and the coating layer in Ti0 ₂ becomes important with a consequent degradation of the mechanical properties causing a deterioration in the coating- substrate adhesion characteristics.

The Applicant proposes to enhance the characteristics of titanium substrates and in particular implants for biomedical applications, obtained starting from said titanium substrate through an optimum functionalization of the surface of the substrate itself, thanks to the identification of an innovative coating method.

An objective of the present invention is therefore to provide a particularly efficient and reliable coating method which allows titanium-based substrates to be coated, improving the biocompatibility and bioactivity of the substrate itself, contemporaneously increasing the corrosion resistance.

A further objective of the present invention is to identify suitably coated titanium- based substrates for the construction of implants for improved dental and/or orthopaedic applications in terms of biocompatibility and bioactivity.

An object of the present invention therefore relates to a method for coating a titanium-based substrate characterized in that it comprises the following steps: a) deposition on the titanium-based substrate of a first compact intermediate layer based on crystalline titania (Ti0 ₂ ) by means of a vapour deposition technique;

b) deposition on the substrate coated with the intermediate layer obtained at the end of step a) of a second homogeneous discontinuous layer based on phosphorous and calcium derivatives by means of a spray pyrolysis technique;

c) possible post-synthesis thermal treatment.

A further object of the present invention relates to a titanium-based substrate coated by a first compact, intermediate layer, based on crystalline titania (Ti0 ₂ ) and a second homogeneous discontinuous layer, based on derivatives of phosphorous and calcium, for biomedical applications.

Another object of the present invention also relates to implants for dental or orthopaedic applications characterized by being composed of a titanium-based substrate coated with the method according to the present invention.

A first advantage linked to the deposition of a layer of (Ti0 ₂ ) by means of the chemical vapour deposition technique used herein (MOCVD) is the formation of a layer of Ti0 ₂ that is perfectly compliant with the substrate and with a fine control of the thickness, whereas the deposition of the second layer by means of spray pyrolysis allows the discontinuous and homogeneous deposition of CPC which increases the direct interaction of the bone and biological fluids with the CPCs themselves.

In conclusion, both layers, Ti0 ₂ (which is bioactive and not reabsorbable) and CPC (more bioactive, reabsorbable, and which acts as a germ with respect to bone development) act synergically allowing a more rapid (due to the CPC) and long- lasting (thanks to Ti0 ₂ ) osseointegration.

Finally, possible post-synthesis thermal treatment allows the materials to be crystallized in the most performing phases.

The process according to the present invention allows an improved substrate/implant to be obtained in terms of osseointegration, as it provides the contemporaneous presence on the surface of both materials: CPC for a rapid osseointegration and Ti0 ₂ for a long-lasting effect. In this way, at the end of the natural reabsorption process of the CPC, the surface areas that already expose Ti0 ₂ as a result of the discontinuous and homogeneous deposition of CPC, will already have an osseointegration in an advanced state, not starting from zero, however, as would be the case, on the contrary, if Ti0 ₂ were exposed only after the reabsorption of the CPC according to what is described by the processes and substrates of the state of the art.

Within the present description, titanium-based substrates (or titanium substrates or substrates in titanium) preferably refer to substrates composed of titanium or titanium alloys, but also zirconium oxide, 316, 316L stainless steel and cobalt- chromium alloys.

Phosphorous and calcium derivatives preferably refer to phosphorous and calcium salts, such as, for example, calcium phosphate salts (generically defined as "CPC", calcium phosphate ceramic) with a suitable chemical composition, morphology and crystallinity, preferably hydroxyapadte Ca ₅ (P0 ₄ ) ₃ OH (meant as minimum formula) (HA), tricalcium diphosphate Ca ₃ (P04)2 (TCP), calcium phosphate monoacid (CaHP0 ₄ ), calcium phosphate diacid (Ca(H ₂ P0 ₄ ) ₂ ), calcium pyrophosphate (Ca ₂ P ₂ 07), calcium metaphosphate (Ca(P0 ₃ ) ₂ ), calcium phosphite (CaHP0 ₃ ), calcium hypophosphite (Ca(H ₂ P0 ₂ ) ₂ ) and/or relative combinations thereof.

Compact layer means a layer of non-porous material, free from cracking, which acts as a barrier.

Homogeneous and discontinuous layer refers to a layer in which the deposition is effected in islands, i.e. areas of the substrate in titanium wherein the surface layer is the layer based on phosphorous and calcium derivatives and areas of the substrate in titanium, wherein the surface layer is the intermediate crystalline titanium-based layer (Ti0 ₂ ), i.e. not coated with the second layer. In any case, the discontinuity is a homogeneous discontinuity which is obtained thanks to the specific deposition method used in step b) of the coating method according to the present invention. Figure 6 schematically shows the structure of the substrate according to the present invention, with the evident presence of islands based on phosphorous and calcium derivatives.

Step a) of the coating method according to the present invention, in which a first compact intermediate layer based on crystalline titania (Ti0 ₂ ) is deposited on the titanium-based substrate, is preferably effected by means of chemical vapour deposition (Metal Organic Chemical Vapour Deposition, MOCVD). This deposition technique, in fact, allows a homogeneous, conformal coating to be created (also of nanostructured surfaces), that can be reproduced and with controlled thicknesses. Furthermore, the chemical vapour deposition technique is at the same time characterized by lower costs with respect to other deposition techniques frequently used (for example PVD - Physical Vapour Deposition). Step b) of the coating method according to the present invention, in which a second homogeneous discontinuous layer based on phosphorous and calcium derivatives, preferably salts of calcium phosphates, is deposited on the product obtained at the end of step a), is carried out by means of a spray pyrolysis technique. This deposition technique, in fact, allows a porous and discontinuous material, with a defined and modulable stoichiometry, high deposition rates and reduced process costs, to be simultaneously produced.

From 10 to 90%, more preferably from 30 to 70%, of the surface of the intermediate layer is preferably homogeneously and discontinuously coated with the layer based on calcium and phosphorus derivatives.

The coating method according to the present invention can also comprise post- synthesis thermal treatment (step c) in order to crystallize the materials in the most performing phases.

The present invention therefore particularly relates to a method for coating a titanium-based substrate which comprises the following steps:

a) deposition on the titanium-based substrate of a first compact intermediate layer based on crystalline titania (Ti0 ₂ ) by means of chemical vapour deposition;

b) deposition on the substrate coated with the intermediate layer obtained at the end of step a) of a second discontinuous layer based on phosphorous and calcium salts by means of spray pyrolysis techniques; c) post-synthesis thermal treatment.

The deposition step b) provides a spraying time and a number of spraying steps suitably regulated so that from 10 to 90%, more preferably from 30 to 70%, of the surface of the intermediate layer is homogeneously and discontinuously coated with the layer based on calcium and phosphorous derivatives.

Excessive spraying times, as also a number of sprayings higher than twenty, can in fact lead to the formation of a layer that is no longer discontinuous.

The method according to the present invention also preferably comprises a thermal treatment step c) of the coated substrate coming from step b).

Said post-synthesis thermal treatment step c) involves modifying the homogeneous discontinuous layer wherein the calcium phosphate salts, generally obtained at the end of step b) if present in amorphous form, rearrange their structure into a crystalline form (or with a greater crystallinity), forming nanodimensional calcium phosphate salts.

Furthermore, following the thermal treatment, the intermediate layer of crystalline titania can also, even partially, modify its crystalline form from anatase to rutile. Together with the phase transition, a colour variation of the material can also be observed, which can be correlated to a variation in the thickness of the layer of Ti0 ₂ .

Before being subjected to step a) for the deposition of the intermediate layer, the titanium-based substrate can be suitably processed. The titanium-based substrate can in fact be a machined titanium substrate, i.e. a titanium-based substrate in which the surface appears as it is at the end of the mechanical processings necessary for its production; or a sandblasted titanium substrate, i.e. a titanium- based substrate subjected to suitable sandblasting treatment after the mechanical processing; or a machined and/or sandblasted titanium substrate and subjected to one or more acid etching treatments (for example with hydrofluoric acid, hydrochloric acid, sulfuric acid etc. and/or combinations thereof). The various types of titanium substrates differ in their different morphological characteristics. The implementation of step a) of the coating method according to the present invention, by means of chemical vapour deposition techniques that allow a deposition of an intermediate layer of crystalline and compact Ti0 ₂ , adherent and conformal to the titanium substrate, to be obtained, requires operating under optimum conditions with respect to the deposition temperature, operating pressure, carrier gas flows, temperature of the precursors, etc. Optimum conditions for step a) of the method according to the present invention are the following.

The deposition temperature in the deposition chamber can range from 250 to 500°C, and is preferably equal to 400°C, the deposition pressure can range from 1 to 101,000 Pa, and is preferably equal to 100 Pa.

The precursor used for the deposition step of crystalline titania is titanium tetraisopropoxide (TTIP).

The use of different precursors is also possible, which, as they are characterized by a different chemistry, require a suitably adapted set of parameters (temperature, pressure, flows, quantities ...).

The temperature of the precursor can range from 20 to 100°C, and is preferably equal to 40°C. The flow of carrier gas, preferably N ₂ , which touches the precursor, can range from 50 to 300, and is preferably equal to 110 seem, for laboratory plants, whereas it can range from 500 to 6,000, preferably from 3,000 to 4,000 seem, for industrial reactors, it being understood that the larger the plant, the higher the flows must be.

A co-reagent gas can also be present (0 ₂ , 0 ₂ +H ₂ 0 or H ₂ 0 ₂ , N ₂ +H ₂ 0 or H ₂ 0 ₂ ) or dilution gas (nitrogen, helium, argon or other inert gases).

Alternatively, the TTIP can also be vaporized in a vaporization chamber and brought from the same, by means of a flow of carrier gas (nitrogen, helium, argon or other inert gases) into the deposition chamber.

The crystalline form of the titania in the coating layer deposited is preferably in the form of anatase.

The optimization of the various parameters that characterize the deposition process such as the flow of N ₂ that touches the precursor, the evaporation temperature of the precursor, the flow of N ₂ or 0 ₂ of the co-reagent gas (or dilution gas), the temperature of the deposition chamber, is fundamental for effecting the deposition of crystalline layers of titania, adhering and homogeneous, at the same time preventing reactions from taking place in homogeneous phase.

An exemplary embodiment of an optimization of the process parameters for deposition by means of MOCVD of the first coating layer on the titanium substrate using a laboratory reactor is indicated in Table 1 hereunder.

Table 1

Deposition temperature 400°C

Ti precursor Titanium

tetraisopropoxide (TTIP)

Aldrich >97%

Carrier flow N ₂ - 110 seem

TTIP temperature 40 °C

Co-reagent Not present

Deposition pressure 100 Pa

Process time Variable in relation to the

desired thickness

The thickness of the first coating layer in crystalline titania influences the performances of the final material. The thickness of the first intermediate layer preferably ranges from 50 to 2,000 nm, and is more preferably equal to 160-200 nm.

This thickness allows the formation of an optimum layer of crystalline titania to protect the underlying titanium from possible corrosive actions, without particularly modifying however the intrinsic morphology of the substrate and allowing a good adhesion of Ti/Ti0 ₂ .

In order to also evaluate the modifications obtained in terms of surface structure, the different types of titanium substrate coated with crystalline titania via MOCVD were also subjected to morphological analysis with a scanning electron microscope (SEM) and compositional analysis with the use of an EDX microprobe.

Figure 1 indicates and compares, by way of example, the SEM images obtained for the machined substrates and for the sandblasted substrates before and after the deposition process of Ti0 ₂ via MOCVD i.e. before and after effecting step a), according to the experimental laboratory parameters indicated in Table 1. More specifically, figures la) and lc), lb) and Id) show, at different magnifications, the typical morphology of the machined and sandblasted substrates, respectively, as they appear before the deposition of Ti0 ₂ . The images le) and lg) refer to the surface of the machined titanium substrate, at different magnifications, following the application of step a) of the method according to the present invention. The images If) and lh) refer to the surface of the sandblasted titanium substrate, at different magnifications, following the application of step a) of the method according to the present invention.

Both types of substrates, after deposition of the first coating layer of crystalline titania via MOCVD (step a)), have a homogeneous surface morphology (figures le- lh), in which the surface morphology typical of the original substrate is still visible, at low magnifications (compare figure la with le and figure lb with If). At high magnifications, on the other hand, the surface morphology characteristic of the layer of titania, consisting of grains with a size of about 100 nm, can be observed (compare figure lc with lg and figure Id with lh). Analysis of the composition and spatial distribution of the different elements using an EDX microprobe confirms the presence, for all of the sample analyzed, of Ti and Oxygen atoms.

Figure 2 indicates the X-ray diffraction patterns effected on titanium substrates (machined, sandblasted and sandblasted + acid etching) without the deposit of Ti0 ₂ (figure 2a) and the same following the deposition process of titania via MOCVD (as described in step a) of the method according to the present invention). The diffraction patterns shown in figure 2a are typical of metal titanium, whereas the diffraction patterns in figure 2b reveal that deposition by means of the MOCVD technique induces the formation of a crystalline layer of Ti0 ₂ in anatase (A) and/or rutile (R) phase in relation to the starting substrate and under the growth conditions. The implementation of step b) of the coating method according to the present invention, by means of spray pyrolysis techniques, which allow a deposition to be obtained of a homogeneous and discontinuous surface layer, of calcium phosphate salts on the intermediate layer of crystalline titania obtained at the end of step a), requires operating under optimum conditions with respect to the distance between nozzle and substrate, the spraying pressure of the carrier gas flow (for example nitrogen), the kind and concentration of spraying solution, the number of sprayings, the temperature of the substrate on which the deposition is effected, the spraying time, etc.

The optimum conditions for step b) of the method according to the present invention in a possible embodiment are the following.

A first example of a set of parameters that can be adopted using a laboratory apparatus can be the following: the distance between nozzle and substrate can range from 5 to 50 cm, and is preferably equal to 25 cm, the spraying pressure of the flow of carrier gas, preferably nitrogen, (but not limited thereto), can range from 0.2 to 5 bars, and is preferably equal to 1 bar, the temperature of the substrate can range from RT to 500°C, and is preferably equal to 300°C.

The calcium and phosphorous precursors for effecting step b) are chemical compounds of calcium and phosphorous, respectively, solubilized and mixed in a stoichiometric ratio which is such as to allow, following reaction, the formation of one of the bioactive phases previously mentioned. The solvent to be used is preferably water, but ethanol or 1-propanol or 2-propanol can also be used.

A necessary feature of said precursors is their ability to decompose cleanly, i.e. without leaving residues. Calcium and phosphorous precursors include, but are not limited to, Ca(N0 ₃ ) ₂ , Ca(OH) ₂ , (CH ₃ COO) ₂ Ca, (NH ₄ ) ₂ HP0 ₄ , (NH ₄ )H ₂ P0 ₄ , H ₃ P0 ₄ , H ₃ P0 ₃ , etc.

In a possible embodiment, the reagents used are Ca(NO3) ₂ *4H ₂ 0 and (NH ₄ ) ₂ HP0 ₄ , in such quantities as to obtain the right Ca/P ratio with respect to the desired final phase (1.67 if hydroxyapatite is to be obtained, from 0.5 to 2.3 in the case of calcium phosphate salts, whereas outside these ranges, mixtures of different calcium phosphate salts are obtained).

The spraying time must be defined in relation to the specific deposition characteristics (spray pyrolysis apparatus, solution used, etc.). It must be selected so as to allow a homogeneous and discontinuous coating of the titania surface effected during step a). In order to obtain the desired deposit, variable- or multiple-time spraying processes can also be considered.

A longer spraying time and/or more successive spraying steps obviously result in a greater coating of the titania surface.

Another parameter to be considered is the possibility of operating keeping the substrate in rotation in order to allow a greater homogeneity of the deposit.

A possible example of optimization of the process parameters for step b) for deposition, by means of spray pyrolysis, of the second discontinuous coating layer on the titanium substrate coated and coming from step a) using a laboratory spray pyrolysis apparatus, is indicated in Table 2 hereunder.

Table 2

Parameters used during the spray pyrolysis step b) of calcium phosphate salts Distance nozzle-substrate = 25 cm

Rotation substrate

Temperature of substrate = 295-310°C

Pressure = 1 bar

Spray time: 2 sec, with delay time: 60 sec

Solution hydroxyapatite precursor 0.1M

Number of sprayings = 1 spraying or more than one depending on the desired coating degree

The optimum conditions at a laboratory level for effecting step b) of the coating method according to the present invention by means of spray pyrolysis techniques allow a homogeneous and discontinuous surface layer of calcium phosphate salts to be obtained on the intermediate layer of crystalline titania obtained at the end of step a), therefore allowing a double-layered coating of crystalline titania/calcium phosphate salts to be obtained on the titanium-based support.

Finally, the optimum conditions for a possible post-synthesis thermal treatment c) in order to modulate the crystallinity of the deposits and tribological properties of the double-layer system thus obtained, are the following: operating in air at a temperature that can range from 300 to 800°C, and preferably equal to 600°C, for a time that can range from 30 minutes to 24 hours, and preferably equal to 6 hours. The titanium substrates, coated with a first intermediate layer of crystalline titania having a thickness of about 160-200 nm, at the end of step a) of the deposition method according to the present invention using the MOCVD deposition technique, were then subjected to step b) for deposition by means of spray pyrolysis of the second homogeneous and discontinuous coating layer of calcium phosphate salts under the conditions indicated in the previous Table 2.

Figure 3 shows SEM images relating to the surfaces of machined substrates (figure 3a) and sandblasted substrates (figure 3b) following the applications of steps a) and b) of the method according to the present invention, using the process parameters indicated in Tables 1 and 2. SEM analysis shows that the surfaces of the samples have areas with different morphological characteristics. The images, in fact, clearly show wide areas with a morphology characteristic of crystalline titania, as already observed in figures le-lh, and newly highlighted areas, that can be attributed to the compounds of calcium phosphates. Finally, EDX analysis confirms the presence of calcium and phosphorous.

Some of the samples, coming from step b) of the method according to the present invention and therefore with a coating of crystalline titania/calcium phosphate salts, were subjected to a post-synthesis thermal treatment step at 600°C for 6 hours (step c). Figure 4 shows SEM images relating to the surfaces of machined substrates (figure 4a) and sandblasted substrates (figure 4b) following the applications of steps a), b) and c) of the method according to the present invention, using the process parameters indicated in Tables 1 and 2. SEM analysis shows that the surfaces of the samples have areas with different morphological characteristics as already revealed in the previous figures 3. The images of figure 4 clearly show wide areas with a morphology characteristic of crystalline titania, as already observed in figures le- lh and 3a and 3b, and areas attributable to the compounds of calcium phosphates. Unlike the images of figures 3a and 3b, the areas relating to the deposit of calcium phosphate salts now show a nanodimensional structure with grains of a few tens of nanometers and a significant increase in the porosity. Finally, EDX analysis confirms the presence of calcium and phosphorous.

Figure 5 shows, by way of example, the XRD spectra for three different types of substrates (machined, sandblasted, sandblasted + acid etching), following the application of steps a), b) and c) of the method according to the present invention. The XRD analyses show that the succession of steps a), b) and c) allows the deposition of crystalline Ti0 ₂ (in anatase and rutile phases), and crystalline phases of calcium and phosphorous salts (hydroxyapatite in the spectra indicated), regardless of the morphological characteristics of the initial substrate.

It is important to verify how the whole process (steps a), b) and c)) influences the surface roughness of the titanium substrate, modifying it altogether within 15% with respect to the initial value.

The roughness was evaluated through measurement of the parameter Ra, commonly used in medical-scientific literature for assessing the roughness characteristics of a surface. Ra represents the arithmetic mean of the absolute values of the deviations of the real profile from the average line, within the basic length L (Equation 1).

Equation 1

In order to effect correct and comparable roughness determinations, reference was made to the standard ISO 3274: 1996 and the standard ISO 4288: 1996.

Another property acquired by titanium substrates coated with the method according to the present invention is linked to the increase in the hydrophilic characteristics of the surface (wettability). In biomedical applications, in fact, such as, for example, applications in the dental field or orthopaedic field, hydrophilic surfaces guarantee a better bond with fluids and biological tissues with respect to hydrophobic surfaces.

Regarding the wettability of the surface, contact angle measurements (carried out in static mode, using water as interacting liquid and under controlled temperature and humidity conditions) have shown an increase in the wettability following the implementation of the method, subject of the present invention. The contact angle measurements have shown that it is possible to approach a superhydrophilicity condition.

In conclusion, the analyses previously discussed allowed the possibility to be verified and confirmed, of functionalizing devices for biomedical applications of titanium (or its alloys) consisting of a titanium substrate suitably prepared by means of mechanical processes or combined mechanical and chemical processes, and coated by a first intermediate layer of crystalline titania (anatase and/or rutile depending on the thermal treatment) and by a subsequent homogenous and discontinuous layer of calcium phosphate salts, possibly crystalline and/or nanodimensioned, depending on the thermal treatment.

Description of the figures

The figures representing the method according to the present invention are:

Figures la-lh: SEM micrographs, at different magnifications, of machined titanium substrates (a, c, e, g) and sandblasted substrates (b, d, f, h) before (a, c and b, d for machined substrates and sandblasted substrates, respectively) and after (e, g and f, h for machined substrates and sandblasted substrates, respectively) the deposition process of Ti0 ₂ via MOCVD according to step a) of the method according to the present invention;

Figure 2: X-ray diffraction patterns effected on titanium substrates (m: machined, s: sandblasted and s+a: sandblasted + acid etching) without the deposit of Ti0 ₂ (a) and of the same following the deposition process of titania via MOCVD (b);

Figure 3: SEM micrographs of the surface of machined titanium substrates (a) and sandblasted substrates (b) following the applications of steps a) and b) of the method according to the present invention;

Figure 4: SEM micrographs of the surface of machined titanium substrates (a) and sandblasted substrates (b) following the applications of steps a), b) and c) of the method according to the present invention;

Figure 5: XRD spectra obtained for three different types of substrates (machined, sandblasted and sandblasted + acid etching) following the application of steps a), b) and c) of the method according to the present invention. The main reflections attributable to the hydroxyapatite phase are highlighted by a rectangle;

Figure 6: schematic structure of the substrate with highlighting of the homogeneous and discontinuous layer of islands based on phosphorus and calcium derivatives. Bibliographic References :

[1] B.-D. Hahn, D.-S. Park, J.-J. Choi, J. Ryu, W.-H. Yoon, J.-H. Choi, J.- W. Kim, Y.-L. Cho, C. Park, H.-E. Kim, S.-G. Kim, Appl. Surf. Sci. 257 (2011) 7792.

[2] P. C. Rath, L. Besra, B.P. Singh, S. Bhattacharjee, Ceram. Intern. 38 (2012) 3209.