BIOCOMPATIBLE IMPLANTS MADE OF NANOSTRUCTURED TITANIUM WITH ANTIBACTERIAL PROPERTIES

Abstract

A new titanium-based implant is disclosed, which is formed by a titanium coating manufactured with biomaterials with applications in osseous implantology. The nanotopographical characteristics of these implants inhibit bacterial adhesion and the formation of a bacterial biofilm on the surface, whilst simultaneously presenting suitable properties for the adhesion, stretching and proliferation of bone-forming cells. Moreover, the invention comprises a method for manufacturing the implant by means of oblique-incidence techniques and the use thereof in osseous implantology.

Claims

1. An implant that comprises a titanium coating deposited on a substrate, wherein: the substrate comprises a biomaterial with a root mean square roughness lower than 5 nm on a surface area of 4 μm.sup.2, the coating has a purity greater than 95% and comprises nanostructured titanium formed by metallic titanium and a titanium oxide layer, and the nanostructured titanium has a nanocolumnar form, wherein the diameter of the nanocolumns ranges between 30 and 100 nm, the height ranges between 100 and 300 nanometres, and the space between the nanocolumns ranges between 50 and 150 nanometres, with a nanocolumn tilt angle with respect to the vertical of the substrate ranging between 0° and 30°.

2. The implant according to claim 1, wherein the substrate biomaterial comprises at least one of the following materials: commercially-pure medical-grade titanium with a purity greater than 99%, for periodontal implants, or medical-grade metallic alloys, for orthopaedic, cranial and maxillofacial applications.

3. The implant according to claim 2, wherein the substrate biomaterial comprises Ti6Al4V.

4. The implant according to claim 2, wherein the substrate biomaterial is shaped into structures that comprise discs, bolts, nails, rods, osteosynthesis plates and other fracture fixation devices.

5. A process for obtaining the implant of claim 1, which comprises depositing the coating on the substrate using glancing-angle (GLAD) techniques.

6. The process according to claim 5, wherein the deposition is performed in a cathode sputtering system.

7. The process according to claim 6, wherein the cathode sputtering system comprises a magnetron.

8. The process according to claim 7, wherein the deposition comprises the following steps: a) introduction of the substrate into the cathode sputtering system chamber, b) closing of the chamber and creation of a vacuum, c) introduction of gas into the chamber, d) electromagnetic excitation of the gas particles present in the chamber by means of a source, e) collision of the particles present in the chamber against a titanium target, and f) deposition of the material detached from the target on the substrate, wherein the product of multiplying the operating pressure (P.sub.g) by the target-substrate distance (L) fulfils the ballistic regime condition for the sputtering of Ti, given by p.sub.gL<12 Pa cm, and the substrate forms a tilt angle greater than 60° with respect to the target.

9. The process according to claim 8, wherein the vacuum reached is lower than 10.sup.−4 Pa, and the chamber fulfils the condition that the L/d quotient is greater than 3.5, where d is the diameter of the target and L is the target-substrate distance.

10. The implant defined according to claim 1, wherein the implant is an osseous implant.

11. The implant according to claim 10, wherein the osseous implant is a temporary or a permanent implant.

Description

DESCRIPTION OF THE FIGURES

[0073] FIG. 1: Images of the implant obtained by means of scanning electron microscopy (the images on the left are cross-sections, those on the right are bird's eye views), which present nanocolumns obtained under different deposition conditions by means of glancing-angle cathode sputtering. A) GLAD angle 70° and operating pressure in the chamber, or argon pressure, 0.15 Pa. B) GLAD angle 80° and argon pressure 0.15 Pa. C) GLAD angle 85° and argon pressure 0.15 Pa. D) GLAD angle 60° and argon pressure 0.5 Pa.

[0074] FIG. 2: Images of surfaces that do not present nanocolumns, obtained by means of electron microscopy (on the left, cross-sections; on the right, bird's eye views), obtained under different conditions by means of glancing-angle cathode sputtering: A) GLAD angle 60° and argon pressure 0.15 Pa. B) GLAD angle 60° and argon pressure 1 Pa.

[0075] FIG. 3: X-ray diffraction diagrams of nano-Ti6Al4V (above) and Ti6Al4V (below) samples, acquired by means of grazing incidence (Ω=0.5°). The stars (*) indicate the diffraction corresponding to the Ti6Al4V alloy. The Miller indices for the rutile phase of TiO.sub.2 are indicated.

[0076] FIG. 4: Fourier transform infrared (FT-IR) spectrum of nano-Ti6Al4V (below) and Ti6Al4V (above), obtained by means of attenuated total reflectance (ATR).

[0077] FIG. 5: SEM images of: (A) Ti6Al4V; (B) nano-Ti6Al4V, where the surface marked with an ellipse indicates an estimate of the size of the osteoblast; (C) nano-Ti6Al4V, where the surface marked with a circle indicates an estimate of the size of S. aureus; (D) SEM image of a cross-section of nano-Ti6Al4V, showing the nanocolumns.

[0078] FIG. 6: AFM images of: A) Ti6Al4V, and B) nano-Ti6Al4V. The greyscale on the right indicates the height of the motifs, which has a maximum of 46 nm and 380 nm for Ti6Al4V and for nano-Ti6Al4V, respectively.

[0079] FIG. 7: Evaluation of the surface wettability: A) Photograph of a drop of water on a Ti6Al4V sample; (B) image of a drop of water on a nano-Ti6Al4V sample; (C) evolution of the contact angle as a function of time for both samples.

[0080] FIG. 8: (A) Adhesion of the osteoblasts after 90 minutes on nano-Ti6Al4V and Ti6Al4V samples. (B) Mitochondrial activity (MTT assay) after three days of culture on Ti6Al4V and nano-Ti6Al4V.

[0081] FIG. 9: SEM images obtained after 24 hours of culture with osteoblastic cells on a substrate of (A) and (C), Ti6Al4V; and (B) and (D), nano-Ti6Al4V. In C), some of the anchors formed by the cells are indicated by means of ellipses.

[0082] FIG. 10: Count of S. aureus colonies formed after 90 minutes of culture on nano-Ti6Al4V and Ti6Al4V surfaces. The * indicates statistically significant differences, p<0.05.

[0083] FIG. 11: Confocal fluorescence microscopy images after 90 minutes of culture with live and dead S. aureus bacteria, (A) and (B), Ti6Al4V; (C) and (D), nano-Ti6Al4V.

[0084] FIG. 12: SEM images of samples of (A) Ti6Al4V and (B) nano-Ti6Al4V after 24 hours of culture with S. aureus. The inset in (a) is the surface of a Ti6Al4V sample prior to the culture.

EMBODIMENTS OF THE INVENTION

Example 1: Implant Obtained by Coating Deposition Using Glancing-Angle Cathode Sputtering on a Biomaterial

[0085] In this example, we indicate how the implant was formed.

[0086] Using glancing-angle cathode sputtering, a coating was deposited which was formed by nanostructured titanium on a mechanically mirror-polished Ti6Al4V alloy disc (root mean square roughness lower than 5 nm measured on a surface area of 4 μm.sup.2), 1 cm in diameter and 2 mm thick. The chamber had a base pressure (prior to the introduction of the gas) lower than 5×10.sup.−7 Pa (ultra-high vacuum) and the target-substrate distance was 22 cm. The 5-cm-diameter, 5-mm-thick target used was made of titanium with a purity of 99.999%, and, on the upper part, had a cylindrical chimney 5 cm in diameter and 9 cm in length (this chimney primarily serves to prevent cross-contamination with other targets in the chamber, but, moreover, contributes to the collimation of the atomic flow, by directing the flow of material towards the surface of the substrate). The L/d parameter had a value of 4.4. During the deposition, the pressure in the reactor, or operating pressure in the chamber, was given by an argon gas pressure ranging between 0.15 and 3 Pa, and the DC excitation had a constant power of 300 W. The temperature of the substrate was maintained below 350 K. The tilt angle ranged between 0° and 85°. The process was performed under the ballistic regime, fulfilling the condition that p.sub.dL be lower than 12 in all cases.

[0087] The implant obtained was observed using SEM; in Table 1, we may observe when the nanocolumns are formed as a function of the operating pressure in the chamber, which is caused by the inert gas introduced, and the tilt angle of the substrate with respect to the vertical of the substrate:

TABLE-US-00001 TABLE 1 List of coatings obtained by means of glancing-angle cathode sputtering. Tilt angle P (Pa) 0° 45° 60° 70° 80° 85° 0.15 X X X C C C 0.5 X X C 1 X X X 1.5 X X X 3 X

[0088] The cells containing the letter C indicate those situations wherein nanocolumns were observed, and the letter X indicates those situations wherein nanocolumns were not formed.

[0089] FIG. 1 shows several representative cases of nanocolumns observed by means of Scanning Electron Microscopy, or SEM, whereas FIG. 2 shows cases wherein the nanocolumns were not formed.

[0090] In the case of the obtainment of nanostructured titanium in nanocolumnar form, the nanocolumns obtained have a diameter ranging between 30 and 100 nm, a separation ranging between 50 and 150 nanometres, and an inclination with respect to the vertical of the substrate ranging between 0° and 30°.

Example 2: Use of the Implant in Osseous Implantology

[0091] In this example, we show that the implant obtained under the conditions of Example 1 have osseointegrative properties that inhibit the formation of a bacterial biofilm.

[0092] The implant was obtained following the process of Example 1, using an argon pressure of 0.15 Pa and a GLAD angle of 80°. The temperature of the substrate was maintained below 350 K.

[0093] In this particular case, the surface of the implant is the surface of the coating, and is formed by nanostructured titanium which forms nanocolumns with dimensions ranging between 100 and 300 nm in height, and between 30 and 100 nm in diameter. The nanocolumns grow during deposition on the Ti6Al4V surface, and cover the surface with a high degree of density, i.e. a high degree of nanomotifs per unit surface area, with a mean space of 100 nm.

[0094] X-ray diffraction studies were performed (represented as X-ray diagrams or XRD) using a Philips X'Pert Model diffractometer in the 2θ range of 20-80. In order to obtain information, preferably about the surface of the disc, the grazing incidence method was used, with a grazing angle w of 0.5°. FIG. 3 shows the X-ray diffraction diagrams obtained by means of grazing incidence for a commercial Ti6Al4V substrate (hereinafter Ti6Al4V) without the coating and for the coating of the invention, or nano-Ti6Al4V. The diffraction maxima of Ti6Al4V may be assigned to the hexagonal phase α-Ti (the main phase of Ti6Al4V alloys) with a P63/mmc space group. The X-ray diffraction diagram for nano-Ti6Al4V shows the diffraction maxima pertaining to the α-Ti phase, jointly with a secondary rutile TiO.sub.2 phase with a P42/mm space group. Fourier transform infrared (FT-IR) spectra were obtained using a Thermo Nicolet Nexus spectrophotometer equipped with a Goldengate attenuated total reflectance (ATR) device. FIG. 4 shows the spectrum of Ti6Al4V and nano-Ti6Al4V, and in both samples we may observe absorption bands corresponding to Ti—O—Ti bonds within a wide range of frequencies, between 950 and 500 cm.sup.−1, which is indicative of the TiO.sub.2 layers on the surface of Ti6Al4V and nano-Ti6Al4V. Moreover, bands corresponding to the tension mode of the O-H bond are observed, which may be assigned to the presence of Ti—OH groups on the surface.

[0095] Finally, phonon mode bands (between 1100 and 1400 cm.sup.−1) of Al.sub.2O.sub.3 are observed on the substrate; their presence is characteristic of the surface of the Ti6Al4V alloy. This band does not appear in the nano-Ti6Al4V material, which indicates that the substrate has been effectively coated with the titanium nanocolumns. The presence of diffraction maxima in the X-ray diagram corresponding to a rutile-type TiO.sub.2 phase in nano-Ti6Al4V and the presence of absorption bands in the infrared spectrum attributable to Ti—O—Ti bonds demonstrate the presence of a TiO.sub.2 layer that would be coated with the Ti nanocolumns grown on the Ti6Al4V substrate.

[0096] The structure of the implant may be seen in FIG. 5. To this end, measurements were taken using SEM. The initial surface of Ti6Al4V does not present any roughness perceptible by SEM (FIG. 5A), which corresponds to a mirror-polished surface. However, at the same scale, the nano-Ti6Al4V surface appears to be completely covered by nanoroughness as a result of the deposition of Ti on the Ti6Al4V substrate, due to the growth of nanocolumns. The Atomic Force Microscopy, or AFM, measurements for both surfaces (FIG. 6) show the difference in roughness; for Ti6Al4V, it was 3 nm (root mean square value, or RMS) on a surface area of 4 μm.sup.2, whereas, for nano-Ti6Al4V, the measured roughness value was 57 nm on a surface area of 4 μm.sup.2.

[0097] The contact angle was measured by means of the sessile drop method, in a CAM 200 KSV contact angle equipment at 25° C., taking photographs every 1 second. The contact angle studies (FIG. 7) indicate a significant increase in hydrophobicity following the coating process. The initial contact angles, after 1 second, were 56° and 102° for Ti6Al4V and for nano-Ti6Al4V, respectively. The contact angle for nano-Ti6Al4V remained constant with time, which indicates low wettability, indicative of hydrophobic surfaces, whereas the contact angle for Ti6Al4V decreased to 44° during the first 8 seconds, which is indicative of the high wettability characteristic of Ti6Al4V alloys.

Culture of Osteoblasts

[0098] Prior to the in vitro culture of osteoblasts, the samples were sterilised and dried at 150° C. for 12 h. A human osteosarcoma (HOS) cell line was used, obtained through the European Collection of Cell Cultures (ECACC, no. 87070202). The cells were cultured in complete medium, composed of Dulbecco's modified Eagle medium (DMEM) (Sigma Chemical Co., St. Louis, USA) supplemented with 2 mM L-glutamine (Gibco, Invitrogen Corporation, USA), 100 U ml.sup.−1 penicillin (Life Technologies Limited, Scotland), 100 g ml.sup.−1 streptomycin (Life Technologies Limited, Scotland) and 10% foetal bovine serum (FBS) (Gibco, Invitrogen Corporation, USA), at 37° C. in a humid atmosphere containing 95% air and 5% CO.sub.2. The HOS cells were routinely trypsinised and subcultured. Subsequently, the HOS cells were seeded in different 24-well plates with a seeding density of 2.5×10.sup.5 cells per ml in complete medium, under a CO.sub.2 atmosphere (5%) at 37° C., for different periods of time for each of the assays.

Statistics

[0099] The data obtained from the osteoblast and bacterial cultures are expressed as the mean±standard deviation of experiments performed on three different samples. The statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software, version 11.5. The statistical comparisons were performed by means of analysis of variance (ANOVA). The differences between the groups were determined by means of post-hoc evaluation using Scheffe's test. For all the statistical evaluations, a difference value was considered to be statistically significant for p<0.05.

Cell Adhesion of Osteoblasts

[0100] In order to study the adhesion of osteoblasts on the surface of the implant, i.e., in this case, the surface of the coating, the samples were incubated under standard culture conditions for 90 min. Subsequently, the samples were washed three times with PBS; thereafter, the cells were separated by means of a trypsin treatment for 10 min. Following centrifugation, the cells were resuspended in PBS and counted in a Neubauer chamber. FIG. 8 indicates the in vitro biocompatibility results performed with HOS on the surface of the coating. To this end, the results of the initial adhesion (90 minutes) and the proliferation of the HOS cells following 3 days of culture were considered, through the quantification of the mitochondrial activity. The data of FIGS. 8A and 8B are expressed as the mean values±standard deviation of measurements taken on three different samples. The initial adhesion of the osteoblasts (90 minutes) does not show significant differences between T16Al4V, nano-Ti6Al4V, and the control (plastic of the culture plate).

Cell Proliferation of Osteoblasts

[0101] The cell proliferation was determined on the basis of the cellular mitochondrial activity. To this end, the HOS cells were seeded on the surface of the material in 24-well plates, with a density of 10.sup.5 cells per ml in complete medium, and incubated under standard conditions. The cell proliferation was determined using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, USA) for different periods of time following the seeding. The plastic of the culture plate was used as a control. The quantitative determination was performed in a UV-VIS spectrophotometer, taking a reading at 570 nm. The mitochondrial activity is directly related to the absorbance at said wavelength. The mitochondrial activity of HOS was almost identical for both surfaces and did not show differences with respect to the control following 3 days of culture, as may be observed in FIG. 8B.

Osteoblastic Cell Stretch Assays

[0102] The degree of cell stretch and the morphology of the osteoblasts were observed by means of SEM microscopy. The adhered cells were washed three times in PBS and fixed with 2.5% glutaraldehyde (50% wt., Sigma-Aldrich, USA) in PBS for 45 min. The samples were dehydrated by slowly replacing the medium, using ethanol series with an increasing concentration (30%, 50%, 70%, 90%), for 30 min, with a final dehydration in absolute ethanol for 60 min, which allowed for drying of the samples at room temperature under vacuum. The Ti6Al4V and nano-Ti6Al4V samples were mounted on specimen holders and coated with gold for viewing in the SEM.

[0103] FIG. 9 shows the surface following one day of culture of the HOS cells on the surface of the coating. The surface appears to be completely covered by the cells and shows good adhesion, proliferation and degree of stretching. The micrographs obtained at higher magnifications show the anchor elements formed by the cells. FIG. 9d shows a detailed view of the nanocolumns beneath the osteoblast layer.

Bacterial Cultures with S. aureus

[0104] The preliminary in vitro studies of bacterial adhesion were performed using an ATCC 29213 strain of Staphylococcus aureus (S. aureus) as a bacterial model under the static conditions commonly specified in the literature (Montanaro L., et al., Future Microbiology 2011, 6 (11): 1329-49). The samples were sterilised by means of dry heat at 150° C. for 12 h. The S. aureus bacteria grew to their mean logarithmic phase in Todd Hewitt (THB) growth medium (Sigma-Aldrich, USA) at 37° C., under magnetic stirring at 100 rpm, until the optical density measured at 600 nm reached 1.0. At this point, the cultured bacteria were harvested by means of centrifugation at 1500 rpm for 10 min at room temperature. They were washed 3 times with sterile PBS, maintaining the pH at 7.4, and resuspended in PBS at a concentration of 6×10.sup.8 cells.ml.sup.−1. Subsequently, they were incubated at 37° C. under magnetic stirring at 100 rpm, for different incubation times, in the presence of the biomaterials under study.

Adhesion Studies for S. aureus

[0105] The incubation time for the suspended bacteria was 90 minutes. Subsequently, the samples were aseptically removed from the bacterial suspension and rinsed three times in PBS in order to eliminate the free bacteria. The bacteria bound to the surface of the nanostructured material were quantified by means of the following method: each sample was placed in an Eppendorf tube containing 1 ml of sterile PBS. Thereafter, it was sonicated for 30 s, assuming that 99.9% of the remaining bacteria were separated from the surface. Subsequently, 100 ml of each of the products obtained following the sonication were taken, cultured on Tryptic Soy Agar (TSA) plates (Sigma Aldrich, USA) and incubated overnight at 37° C. The number of colony-forming units (CFU) resulting from the sum of the three sonication processes made it possible to determine the number of original bacteria adhered to the samples. The bacterial cultures of S. aureus grown on the Ti6Al4V surfaces (FIG. 10) did not show significant differences with respect to the control following 90 minutes of exposure. However, in the case of nano-Ti6Al4V, the adhesion of S. aureus was three times lower than that of Ti6Al4V.

Confocal Microscopy of S. aureus

[0106] Following 90 minutes of incubation in PBS, the samples were stained for 15 minutes using the Invitrogen Live/Dead BacLight bacterial viability kit. The confocal microscopy studies were performed using a Biorad MC1025 microscope. The SYTO 9 fluorescence (live bacteria, green) is excited at a wavelength of 480/500 nm, and emits fluorescence at 500 nm. The propidium iodide fluorescence (dead bacteria, red) is excited at 490/635 nm, and the fluorescence emitted nm was measured at 618. FIG. 11 shows the images obtained by means of confocal microscopy following 90 minutes of culture. The images show less bacterial adhesion on nano-Ti6Al4V, in total agreement with the count shown in FIG. 11. No significant differences were observed in the live/dead ratio when the Ti6Al4V and the nano-Ti6Al4V surfaces were compared. This fact suggests that the antibacterial activity of the coatings is exerted thanks to the anti-adhesion properties thereof, without any bactericidal effects against S. aureus being observed.

SEM Microscopy of S. aureus

[0107] The SEM study was performed by preparing the samples in a manner analogous to that described for the studies with osteoblasts. In FIG. 12, we may observe the Ti6Al4V and nano-Ti6Al4V surfaces following 24 hours of culture with S. aureus. FIG. 12A corresponds to the surface of the Ti6Al4V sample and shows the bacteria surrounded by an extracellular matrix identified as a bacterial biofilm, which covers the polished surface of the substrate. In order to highlight the presence of the bacterial biofilm, FIG. 12 contains an inset which shows the clean biofilm surface prior to the bacterial culture, whereas, on the contrary, the surface of the nano-Ti6Al4V sample shows a micrography wherein the bacteria that are present have not been able to form a biofilm, which makes it possible to view the nanostructure of the nano-Ti6Al4V sample.