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    • 专利摘要:
    Biocompatible nanostructured titanium implants with antibacterial properties. A new implant based on titanium is described, formed by a titanium coating manufactured on biomaterials with application in bone implantology. The nanotopographic characteristics of these implants inhibit bacterial adhesion and the formation of bacterial biofilm on the surface, simultaneously presenting adequate properties for adhesion, extension and proliferation of bone-forming cells. Additionally the invention comprises a method of manufacturing the implant by oblique incidence techniques and its use in bone implantology. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2552278A1
    申请号:ES201430616
    申请日:2014-04-25
    公开日:2015-11-26
    发明作者:José Miguel GARCÍA MARTÍN；Alberto PALMERO ACEBEDO；Rafael ÁLVAREZ MOLINA；María VALLET-REGÍ；Daniel Arcos Navarrete；Isabel Izquierdo Barba
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad Complutense de Madrid；
    IPC主号:
    专利说明:

    SECTOR OF THE TECHNIQUE AND OBJECT OF THE INVENTION
    The invention is framed in the field of coating materials and adhesives for medical, medical engineering or sanitary engineering applications in the broadest sense. The invention is also framed in the field of nanomaterials with medical application.
    The object of the invention is an implant composed of a titanium coating deposited on a substrate comprising a biomaterial of surgical choice with osseointegrating and inhibiting properties of bacterial biofilm formation, as well as methods of manufacturing the implants by means of oblique incidence techniques. , preferably by sputtering and its use in bone implantology.
    STATE OF THE TECHNIQUE
    Titanium and its alloys are widely used in bone implantology due to its exceptional biocompatibility, excellent mechanical properties and corrosion resistance. This high corrosion resistance lies primarily in the rapid formation of a layer of titanium oxide on its surface, known as dry or passivated corrosion of titanium. The formation of titanium oxide occurs spontaneously by oxidation of the metal surface when it comes into contact with atmospheric oxygen. The passivation layer gives titanium-based implants excellent anticorrosive properties and osseointegrating properties.
    Most infections in metal orthopedic implants are caused by bacteria of the Staphylococcus type. Among them Staphylococcus aureus (S. aureus) represents the main pathogenic species in the case of metal biomaterials such as stainless steel, GrGo, Ti and their alloys. When bacteria adhere to the surface of the implant, they secrete an extracellular matrix of polymeric nature called biofilm, which gives them a high resistance to antibiotics (F. G6tz et al, Molecular Microbiology 43, 1367, 2002). The formation of this biofilm almost always forces the removal of the prostheses, because otherwise the infection of the implant can lead to chronic infections and, in extreme cases, even to the death of the patient due to septicemia.
    At present, the problem of infection in implants is attempted to be prevented with several strategies, for example, by implanting polymethylmethacrylate rosaries loaded with broad-spectrum antibiotics. This strategy has the limitation that the rosaries require a second intervention to be removed. On the other hand, once the implant has been infected, the strategy to follow is the systemic administration of high doses of antibiotics with the consequent toxicity problems. In any case, when the bacteria manage to form the bacterial biofilm, they become very resistant to antibiotic treatment systemically and the implant must be removed to avoid chronic infections and septicemia. For all this, address the problem of infection from its first stage
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    avoiding bacterial adhesion, represents an important advance in the prevention of infection of bone implants.
    With regard to orthopedic and dental implants, the formation of nanostructures by various techniques (anodization, vapor deposition, etc.) has been described in the field in order to favor the adhesion of bone-forming cells (osteoblasts) as well as to improve their behavior once attached (differentiation, extracellular matrix formation, etc.). Thus, for example, nanostructured coatings on implants have been synthesized by various techniques and with different shapes, an example are TiO :: nanotubes manufactured by anodizing techniques (M. Ma, et al, J. Biomedical Material Research Part A 100, 278 , 2012), but also nanostructured hydroxyapatites by hydrolysis of solid precursors, metal alloys obtained by low-temperature powder metallurgy, titanium nanostructures obtained by chemical nanotopography or titanium nanostructures obtained by surface oxidation.
    On the other hand, nanostructures on Ti that inhibit bacterial adhesion have also been prepared (D Campoccia, Biomaterials 34, 8533, 2013; K. Anselme, Acta Biomaterialia 6, 3824, 2010). For example, in some cases, TiO :: present on the surface has demonstrated some bactericidal capacity after being irradiated with UV light.
    There are several papers that describe nanotopographies that preserve their behavior with respect to osteoblasts but inhibit bacterial colonization simultaneously (Colon et al. J Biomedical Materíals Research. A 78, 595, 2006 and Ploux L. et al, Langmuir 25, 8161, 2009). In both cases, the studies are done on materials without clinical application so far. In M. Kazemzadeh-Narbat et al, Biomaterials 34 5969 2013 both effects are achieved (biocompatibility with osteoblasts and antimicrobial activity) using titanium obtained by anodizing processes, but based on the incorporation of drugs in the grown layers, so that their Antimicrobial activity is not due to the grown nanostructure, but to the incorporated medicine.
    A technique widely used in microelectronics that allows the formation of nanostructures with a wide variety of properties such as topographies, compositions, etc., is the so-called cathodic spraying (or sputfering in English) (P.J. Kelly et al, Vacuum 56, 159, 2000). This technique has been well known for decades for growing very compact thin sheets with low roughness. In this, a solid block, also called white, of a particular material (in this case titanium) is placed inside a reactor or vacuum chamber with an inert gas inside (normally argon gas is used). By injecting electromagnetic power through the target through an excitation source, a gaseous plasma is generated, rich in energy ions, which pulverize the surface of the target, emitting atoms in a direction preferably perpendicular to the target, with kinetic energies of the order of 10 eV. These atoms, upon reaching a surface inside the reactor parallel to the target, called a substrate, accumulate and agglomerate on a surface, generating a thin film. Depending on the operating pressure in the chamber, which is due to the inert gas introduced, the energy with which these atoms reach the surface can be controlled. At high pressures (above 1 Pa under standard conditions), the collisions are numerous, so the atoms reach the substrate with low energy (tenths or hundredths of eV). At low pressures, however, the arrival energy is very similar to that of the target's output (ballistic regime), generating highly compact thin films.
    5 Sputtering is a widely used process in thin film formationon materials, in fact sputtering is used industrially in multipleApplications: manufacture of hard drives, mirrors, internal coatings ofgrocery bags, etc. Faced with techniques that involve some kind of chemical reaction,As chemical synthesis, anodization, photolithography, etc., sputtering is a
    10 vacuum technique, so that no aggressive waste is generated with the environment, and it is also energy efficient since it allows manufacturing at low temperatures (room temperature). Compared to other physical techniques in vacuum, such as thermal evaporation or assisted evaporation by electron gun, cathodic spraying is widely used in industry and allows the growth of
    15 nanostructured material on large surfaces with different morphologies.
    In recent years, cathodic spraying is also being used in oblique angle geometries, this is the so-called sputfering glancing angle or GLAD sputfering in the literature) (JC Sit el al, Journal o, Maferials 20 Research 14 (4), 1197, 1999). In this case, after the generation of the plasma in the chamber, the substrate where the atoms accumulate is no longer placed parallel to the target, but forming with it an angle of inclination greater than 60 °, the so-called GLAD angle, which causes that the atoms reach the substrate with oblique incidence. This configuration induces shadow processes on the surface of the growing thin layer that generate
    25 inclined structures.
    So far no coatings on bone implants have been prepared with a biomaterial that can be used to coat implants or prostheses and that are formed by Ti nanotopographies that simultaneously allow adhesion and proliferation of
    30 osteoblasts, but inhibit bacterial colonization. These studies have always been done separately with other techniques and most of them, with bacteria that do not imply a significant incidence in prosthetic infections.
    EXPLANATION OF THE INVENTION
    A first aspect of the invention is an implant having a titanium coating deposited on a substrate characterized in that
    - The substrate comprises a biomaterial with an average quadratic roughness lower than
    40 5 nm in an area of 4 IJm2, - the coating has a purity greater than 95% and comprises nanostructured titanium formed by metallic titanium and a layer of titanium oxide, - the nanostructured titanium has a nanocolumnar shape where the diameter of the nanocolumns is 30 to 100 nm and the height varies between 100 and 300 nanometers, the
    The space between nanocolumns varies between 50 and 150 nanometers with an inclination of the nanocolumn with respect to the vertical of the substrate between 0 ° and 30 °.
    The substrate biomaterial can have at least one of the following materials:
    s - Commercially pure titanium with purity greater than 99% medical grade for periodontal implants, - Medical grade metal alloys such as CrCo, stainless steel, Ti6AI4V for orthopedic, cranial and maxillofacial applications
    The substrate biomaterial can be formed in structures comprising discs, screws, nails, stems, osteosynthesis plates and other fracture fixation devices, generally made of stainless steel.
    10 Another aspect of the invention is the method of obtaining the implant which comprises depositing the coating on the substrate using oblique incidence techniques (GLAD), preferably in a sputtering system.
    The sputtering can be magnetron type.
    The procedure may comprise the following stages:
    15 20 a) b) c) d) e) f)introduction of the substrate in the cathode spray system chamber, closing of the chamber and realization of vacuum, introduction of gas into the chamber, electromagnetic excitation of the gas particles present in the chamber by means of a source, collision of the particles present in the chamber against a titanium target, deposit of the material detached from the target on the substrate,camera
    25 characterized in that the product of the operating pressure (Pg) by the white substrate distance (L), fulfills the ballistic regime condition for Ti spraying, given by pgL <12 Pa cm; and the substrate forms an inclination angle greater than 60 ° with the target.
    In a particular case, the vacuum reached is less than 10 4 Pa, in the chamber it is satisfied that the quotient Ud, where d is the diameter of the blank and L the white-substrate distance is greater than 3.5.
    30 The third aspect of the invention is the use of the implant in bone implantology.
    Another aspect of the invention is a method of human or animal treatment comprising the following steps:
    -insert the implant by surgery in a human or animal body, preferably by orthopedic, cranial, dental and / or maxillofacial surgery.
    35 In a particular embodiment, the implant may be temporary or permanent use.
    Detailed description of the invention
    40 The invention is based on the fact that the inventors have achieved a new type of titanium coatings on metal biomaterials of surgical importance that have a
    selective behavior against bacteria and osteoblasts. The nanotopographic characteristics of these coatings inhibit bacterial adhesion and the formation of bacterial biofilm on the surface, simultaneously presenting adequate properties for adhesion, extension and proliferation of bone-forming cells.
    An object of the invention is an implant comprising a titanium coating deposited on a substrate characterized in that
    - The substrate comprises a biomaterial with an average quadratic roughness lower than
    10 5 nm in an area of 4 IJm2, - the coating has a purity greater than 95% and comprises nanostructured titanium formed by metallic titanium and a titanium oxide layer, - the nanostructured titanium has a nanocolumnar shape where the diameter of the nanocolumns is from 30 to 100 nm and the height varies between 100 and 300 nanometers, the
    15 space between nanocolumns varies between 50 and 150 nanometers with an inclination of the nanocolumn with respect to the vertical of the substrate between 0 ° and 30 °,
    The substrate is decisive in the formation of the coating, since its purity, material, topography, etc. influence the structural properties of the coating that is
    20 deposited on top. If a suitable substrate is not used, the coating with the necessary properties to function as an implant is not formed.
    In a particular embodiment the substrate biomaterial comprises at least one of the following materials: commercially pure titanium with purity greater than 99% medical grade, for periodontal implants, medical grade metal alloys such as CrCo, stainless steel, Ti6AI4V for orthopedic, cranial and maxillofacial applications
    30 These biomaterials are of great importance in the manufacture of modular joint prostheses. Ti6AI4V alloys are the biomaterial of choice to manufacture the prosthesis component implanted in the medullary canal of the bone, as it has excellent osseointegration and an elastic module around 110 GPa, which allows mechanical load from the material to the bone to be transmitted. This value is half with
    35 with respect to stainless steel and CrGo. The lower rigidity of the Ti6AI4V avoids situations of protection against loading, which are associated with bone loss in the region surrounding the implant and with subsequent loosening of the prostheses. On the contrary, for those components that are part of the friction torque in joint prostheses, CrCo or stainless steel alloys are the ones of choice due to their better behavior.
    40 friction
    In a more particular embodiment, the substrate biomaterial is shaped in the form of implantable devices comprising discs, screws, nails, stems, osteosynthesis plates as well as other fracture fixation devices.
    The coating may have additional layers of material, for example a layer between the coating and the substrate that facilitates adhesion of the nanostructured titanium grown thereon.
    5 The surface of the implant is the surface of the coating and is given by the nanocolumn formation of the nanostructured titanium. This surface inhibits the formation of bacterial biofilm. Likewise, bacterial adhesion on the coating is much lower than that observed in uncoated biomaterials. Specifically, the microbiological studies carried out in this invention with S. aureus show that the coatings
    10 are effective surfaces to inhibit the adhesion of this pathogen. The inhibition of bacterial adhesion prevents the subsequent formation of colonies and the subsequent development of bacterial biofilm.
    Regardless of the cell type, prokaryotic in the case of bacteria or eukaryotic in
    In the case of osteoblasts, there are a number of common stages for surface colonization by both types. Cell adhesion to a substrate is a process that is carried out sequentially by the cell in different stages: i) surface recognition, ii) formation of initial contacts, and subsequent development at focal points, and ¡ji) cell expansion and development (S. Faghilhi, el al, Journal of
    20 Biomedical Materials Research, Parl A 91 656 2009). In this case, the possibility of controlling the surface topology, not only in the size of the nanocolumns but also in their spacing and surface chemistry, allows to design surfaces with a selective behavior based on the differential characteristics between both types of cells .
    Osteoblasts are cells with lateral dimensions between 10 and 50 IJm. They have a flexible cell membrane that allows them to adapt to different nanotopographies. Osteoblasts require prior adsorption of integrins on the surface of material to adhere to it. The coatings described in this invention show that adsorption 30 of integrins is sufficient to facilitate the adhesion, extension and proliferation of osteoblasts, with the same efficiency as they do on polished surfaces of medical grade Ti6AI4V. On the contrary, bacteria do not need prior adsorption of surface proteins to colonize them. On the other hand, bacteria generally have a characteristic shape and are less deformable. In particular S. aureus shows about 35 dimensions of 1 IJm in diameter and its bacterial wall is much stiffer than the membranes of eukaryotic cells. These differential characteristics with respect to osteoblasts, result in S. aureus having a high sensitivity to nanotopography. The roughness and the short distance between nanocolumns exert a double effect on S. aureus that limits their adhesion. On the one hand, bacteria have access to a very limited number of anchor points, since their small size and stiffness only allows them to come into contact with the upper area of a limited number of nanocolumns to develop focal points of adhesion. On the other hand, the air trapped between the nanocolumns exerts a low wettability effect similar to that of the lotus leaf, which further hinders the adhesion of S. aureus and the development of the polymer matrix
    Extracellular 45 that generates the bacterial biofilm.
    Surprisingly, while under the conditions of this work, S. aureus develops a bioñlm in samples of commercial grade medical Ti6AI4V substrates after several days of culture (3 days for the particular case of a Ti6AI4V substrate), the implant surface , which in this particular case is the surface of the coating deposited on
    5 those same substrates, or nano-Ti6AI4V, show a selective behaviorDepending on the type of cell. While osteoblasts adhere and proliferate overthe surface of nano-Ti6Al4V in the same extension as on Ti6AI4V, the adhesion of S.aureus is strongly impeded and biofilm formation inhibited when grownon the nanostructured surface.
    Regarding the behavior against osteoblasts, the nanostructural characteristics of the nanostructured titanium, such as the high density of nanocolumns and the short spacing between them (with an average value of 100 nm), do not modify their behavior with respect to polished surfaces of Ti6AI4V . In this sense, the largest size of
    15 osteoblasts, their flexibility and the adhesion of integrins on the passivation layer of titanium oxide that presents the nanocolumns, allow excellent cell development on these coatings, analogous to that of medical grade Ti6AI4V alloys.
    Together, this property of the coatings on the substrates, allows the
    20 osseointegration of the implants, avoiding the possible infection that may arise in a relatively short period of time after the implant, and which generally requires the removal and replacement of the prostheses.
    The second object of the invention is a method for obtaining the implant, hereafter
    A method, which comprises depositing the coating on the substrate using oblique incidence techniques (GLAD), preferably in a sputtering system.
    The sputtering system can be magnetron type, that is to say, at least,
    A magnetron, that is, uses magnets that concentrate the ionization of the gas in the vicinity of the target, which makes ionization in the rest of the vacuum chamber more rare and allows working with lower gas pressures necessary to obtain the structure of the gas covering.
    In a particular embodiment the deposit is made in a sputtering system and comprises the following steps:
    a) introducing the substrate into the cathode spray system chamber b) closing the chamber and performing 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 blank, f) deposit of the material detached from the white on the substrate,
    P201 430616
    characterized in that the product of the operating pressure (Pg) by the white substrate distance (L), fulfills the condition of ballistic regime for the pulverization of Ti, given by pgL <12 Pa cm; and the substrate forms an inclination angle greater than 60 ° with the target.
    In a particular embodiment, the procedure is characterized in that the vacuum reached is less than 10-4 Pa, in the chamber it is satisfied that the ratio LId is greater than 3.5, d being the diameter of the blank and L the white-substrate distance
    The choice of GLAD tank conditions is critical as the same nanostructures are not formed. The sputtering technique in oblique incidence allows the formation of nanocolumns whose dimensions and spacing depend on the operating pressure in the chamber, the inclination of the substrate with respect to the flow of atoms from the target, the geometry of the deposit system, of the substrate and the duration of the deposit.
    When working at low pressures, the titanium atoms that will form the nanostructured material reach the surface with great energy, making the nanocolumnal structures grow with great aspect ratio and decrease the effective horizontal surface, so that bacteria have less surface to anchor. Similarly, the value of the LId ratio ensures that the beam from the target that will form the nanostructured titanium is sufficiently collimated to form the nanocolumns.
    The titanium targets used in sputtering are usually of high purity, usually higher than 99% since it makes no sense to work in a vacuum reactor if the blank then has impurities in most applications.
    The sputtering can generate the electromagnetic excitation by means comprising at least one of the following: DC (continuous excitation), RF (alternating excitation in the range of radio frequencies) or pulsed DC (excitation with pulses of direct current).
    The deposition of the coating on the substrates results in a surface topography formed by nanocolumns. The nanostructured titanium grows during deposition on the surface of the substrate covering the surface with nanocolumns with a high degree of density, i.e. a large degree of nanomotives per unit area, with an average nanocolumn spacing of 100 nm.
    The implant formed has a series of advantages over the state of the art: the proposed technique (sputtering with low pressures and oblique incidence) is more efficient than other techniques such as anodizing not only from the energy point of view, since it leads to conducted at room temperature and without generating waste, but also because it allows more precise control over the morphology of the coating and avoids chemical treatments; it also allows the coatings to grow on large surfaces of varied shapes, and by an industrially scalable process; when carried out at room temperature, it allows its synthesis on surfaces that can only be processed at low temperatures, such as polymeric materials; inhibits bacterial adhesion and
    formation of bacterial biofilm on the surface and has adequate properties for adhesion, extension and osteoblast proliferation.
    The third object of the invention is the use of the implant in bone implantology since it is
    5 allows simultaneous behaviors against osteoblasts and bacteria,specifically against S. aureus which is the main cause of prosthesis infectionmetallic
    Another aspect of the invention is a method of human or animal treatment comprising the following steps:
    - Insert the implant by surgery in a human or animal body, preferably by orthopedic, cranial, dental and / or maxillofacial surgery.
    In a particular embodiment, the implant may be temporary or permanent use.
    15 DESCRIPTION FIGURES:
    Figure 1: Images of the implant obtained by scanning electron microscopy (the images on the left are cross-sections, on the right, zenithal views) that present nanocolumns obtained in different deposition conditions by
    20 sputtering in oblique incidence. A) GLAD angle 70 ° Y chamber operating pressure or argon pressure 0.15 Pa. B) GLAD angle 80 ° Y argon pressure 0.15 Pa. E) GLAD angle 85 ° Y argon pressure 0.15 Pa. D) GLAD angle 60 ° And argon pressure 0.5 Pa.
    25 Figure 2: Images of surfaces that do not have nanocolumns obtained by electron microscopy (on the left, cross-sections, on the right, overhead views) obtained under different conditions by sputtering on oblique incidence: A) GLAD angle 60 ° and argon pressure 0.15 Pa. B) GLAD angle 60 ° And argon pressure 1 Pa.
    30 Figure 3: X-ray diffraction diagrams of nano-Ti6Al4V (top) and Ti6AI4V (bottom) samples acquired by flush incidence (0 = 0.5 °). The stars (*) indicate the diffraction maxima corresponding to the Ti6AI4V alloy. Miller indices are indicated for the rutile phase of Ti02 •
    Figure 4: Infrared spectrum with Fourier transform (IR-FT) of nano-Ti6Al4V (below) and Ti6AI4V (above) obtained by attenuated total reflectance (ATR).
    Figure 5: SEM images of: (A) Ti6AI4V, (8) nano-Ti6AI4V, the selected area with a
    40 ellipse indicates an estimate of the size of the osteoblast. (C) nano-Ti6AI4V; the area selected 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.
    Figure 6: AFM images of: A) Ti6AI4V B) nano-Ti6AI4V. The gray scale on the right indicates the height of the motifs, and has a maximum of 46nm and 380 nm for Ti6AI4V and nano-Ti6Al4V respectively.
    5 Figure 7: Evaluation of surface wettability A) Photograph of a water drop
    on a sample of Ti6AI4V (B) Image of a drop of water on a sample of nano-Ti6AI4V. (C) Evolution of the contact angle as a function of time for both samples.
    10 Figure 8: (A) Osteoblast adhesion after 90 minutes on samples of nanoTi6AI4V and Ti6AI4V. (B) Mitochondrial activity (MTT test) after three days of culture on Ti6AI4V and nano-Ti6AI4V.
    Figure 9: SEM images obtained after 24 hours of culture with osteoblast cells on a substrate of (A) and (C) Ti6AI4V; and (B) Y (D) nano-Ti6AI4V. In C) some of the anchors formed by the cells are indicated with ellipses.
    Figure 10: Count of S. aureus colonies formed after 90 minutes of culture on nano-Ti6Al4V and Ti6AI4V surfaces. With * indicates statistically significant differences 20 p - <0.05
    Figure 11: Confoeal fluorescence microscope images after 90 minutes of culture with live and dead S. aureus (A) and (B) Ti6AI4V bacteria; (C) and (D) nano-Ti6AI4V.
    25 Figure 12: SEM images of samples of (A) Ti6AI4V and (B) nano-Ti6Al4V after 24 hours of culture with S. aureus. The inset in (a) is the surface of a Ti6AI4V sample before cultivation.
    EXAMPLES OF REALIZATION OF THE INVENTION
    Example 1: Implant obtained by the sputtering coating deposit in oblique incidence on a biomaterial.
    This example indicates how the implant was formed.
    35 Using a cathodic spray in oblique incidence, a coating formed by nanostructured titanium has been deposited on a mirror-polished Ti6AI4V alloy disk (mean quadratic roughness less than 5 nm measured in an area of 4 IJm2), 1 cm in diameter and 2 mm thick The chamber has a base pressure (previous
    40 at the introduction of the gas) less than 5 x 10.7 Pa (ultra-high vacuum) and the white substrate distance is 22 cm. The white of 5 cm in diameter and 5 mm thick used was titanium with a purity of 99.999%, and it had a cylindrical chimney of 5 cm in diameter and 9 cm in length (this chimney is mainly used to avoid cross contamination with others white in the chamber, but also helps the flow collimation
    Atomic directing the flow of material towards the surface of the substrate). The parameter Ud had a value of 4.4. During the tank a pressure in the reactor or chamber operating pressure was used, given by the argon gas pressure between 0.15 and 3 Pa and a
    OC excitation with constant power of 300 W. The substrate temperature was maintained below 350 K. The angle of inclination varied between O 'and 85'. It has been operated within the ballistic regime, complying that PdL is less than 12 in all cases.
    The implant obtained was observed using SEM, in table 1 they can be observed when nanocolumns are formed depending on the operating pressure in the chamber, which is due to the inert gas introduced, and the angle of inclination of the substrate with respect to the vertical of the substratum:
    Inclination angle
    P (Pa) OR'Four. Five'60 '70 '80 '85 '
    0.15 XXXandandand
    0.5 XXand
    one XXX
    1.5 XXX
    3 X
    Table 1: List of coatings obtained by sputtering in oblique incidence.
    Cells containing the letter C indicate the situations where nanocolumns have been observed, the letter indicates X situations where nanocolumns have not been formed.
    Figure 1 shows several representative cases of nanocolumns observed by scanning electron microscopy (or SEM), while in Figure 2 cases where nanocolum is not formed are indicated.
    In the case of obtaining nanostructured titanium with nanocolumnar form, the nanocolumns obtained have a diameter of 30 to 100 nm, a separation between 50 and 150 nanometers, and an inclination with respect to the vertical of the substrate between 0 ° and 30 °.
    Example 2: Use of the implant in bone implantology.
    This example shows how the implant obtained under the conditions of Example 1 has osteointegrating properties that inhibit the formation of bacterial biofilm.
    The implant has been obtained according to the procedure of Example 1 using an argon pressure of 0.15 Pa and a GLAD angle of 80 °. The substrate temperature was kept below 350 K.
    In this particular case, the surface of the implant is the surface of the coating and is formed by nanostructured titanium that forms nanocolumns with dimensions in the range of 100 to 300 nm in height and between 30 to 100 nm in diameter. The nanocolumns grow during the deposit on the surface of Ti6AI4V covering the surface with a high degree of 12
    density, i.e. a large degree of nanomotives in the area unit with an average space of 100 nm.
    Studies have been performed by X-ray diffraction (represented in ray diagrams
    5 X or DRX) with a Philips Model X'Pert diffractometer in the range 28 of 20 -80. To obtain information preferably from the surface of the disk, the flush incidence method was used using a flush angle w of 0.5 °. The X-ray diffraction diagrams obtained by flush incidence are shown in Figure 3 for a commercial Ti6AI4V substrate (hereinafter Ti6AI4V) without the coating and for the coating object of the
    10 invention or nano-Ti6AI4V. The diffraction maximums for Ti6AI4V can be assigned to the hexagonal phase a-Ti (the main phase of Ti6AI4V alloys) with spatial group P63 / mmc. The X-ray diffraction diagram for nano-Ti6Al4V shows the diffraction maxima corresponding to the a-Ti phase together with a secondary phase of rutile Ti02 with spatial group P421mm. Spectra have been performed by infrared spectroscopy with
    15 Fourier transform (IR-FT) using a Thermo Nicolet Nexus Thermo spectrophotometer equipped with a Goldengate attenuated total reflectance (ATR) device. The spectrum of Ti6AI4V and nano-Ti6Al4V is shown in Figure 4, observing in both samples absorption bands corresponding to Ti-O-Ti bonds in a wide range of frequencies ranging from 950 to 500 cm-l, which is indicative of the layers of Ti02 on the surface
    20 of the Ti6AI4V and nano-Ti6AI4V. Likewise, bands corresponding to the tension mode of the O-H bond are observed, assignable to the presence of Ti-OH groups on the surface.
    Finally, on the substrate the phonon bands (between 1100 and 1400 cm-l) of the AbOJ are observed, whose presence is characteristic on the surface of the Ti6AI4V alloy. This band does not appear in the nano-Ti6Al4V material which indicates that the substrate has been effectively coated by titanium nanocolumns. The presence of diffraction maxima in the X-ray diagram corresponding to a rutile type phase of Ti02 in nano-Ti6Al4V and the presence of infrared absorption bands assignable to TiO-Ti bonds, show the presence of a Ti02 layer that would cover the nanocolumns of Ti
    30 grown on the Ti6AI4V substrate.
    The structure of the implant can be seen in Figure 5. For this, measurements were made with an SEM. The initial surface of the Ti6AI4V substrate does not show appreciable roughness to the observation by SEM (Figure 5A), which corresponds to a mirror polished surface. 35 However, the surface of nano-Ti6Al4V on the same scale appears completely covered with a nanorgosity as a result of the deposition of Ti on the Ti6AI4V substrate due to the growth of nanocolumns. The measurements of atomic force microscopy (or AFM of the English Alomic Force Microscopy) of both surfaces (Figure 6) show the difference in roughness, in Ti6AI4V it is 3nm (mean square value or RMS of English
    40 Rool Mean Square) in an area of 4 J.Jm2, while in the nano-Ti6Al4V the measured roughness value is 57 nm in an area of 4 J.Jm2.
    The contact angle was measured by the drop-slope method in a CAM 200 KSV contact angle device at 25 oC taking pictures every 1 second. Studies by contact angle 45 (Figure 7) indicate a significant increase in hydrophobicity after the coating process. The initial contact angles, after 1 second, were 56 ° and 102 ° for Ti6AI4V and nano-Ti6AI4V, respectively. The contact angle for nano-Ti6Al4V
    it remained constant over time, indicating a low wettability, indicative of hydrophobic surfaces, while the contact angle of Ti6AI4V decreased to 44 ° during the first 8 seconds, indicative of a high wettability characteristic of Ti6AI4V alloys.
    Osteoblast culture
    Prior to the in vitro culture of osteoblasts, the samples were sterilized and dried dry at 150 ° for 12 hours. A human osteosarcoma cell line (HOS) obtained through the European Cell Culture Collection (ECACC, no. 87070202) was used. 10 Cells were cultured in complete medium consisting of Dulbecco's modified Eagle medium (DMEM) (Sigma Chemical Ca., SI. Louis, USA) supplemented with 2 mM Lglutamine (Gibco, Invitrogen Corporation, USA), 100 U mL-1 of penicillin (Life technologies limited, Scotland), 100 9 mL · 1 streptomycin (Life technologies limited, Scotland), and 10% fetal bovine serum (F8S) (Gibco, Invitrogen Corporation, USA) at 37 ° C in a humid atmosphere
    15 of 95% air and 5% CO2. HOS cells were routinely trypsinized and subcultured. Subsequently, the HOS cells were seeded in different 24-well plates with a seeding density of 2.5 x 105 cells per mL in complete medium under CO2 atmosphere (5%) at 37 oC for different periods of time for each of the tests.
    Statistics
    Data obtained from osteoblast and bacterial cultures are expressed as the mean ± the standard deviation of experiments performed on three different samples. Statistical analysis is carried out with the Statistical Package for the Social software
    25 Sciences (SPSS) version 11 .5. Statistical comparisons were made by analysis of variance (ANOVA). Differences between groups were determined by post hoc evaluation using the Scheffe test. For all statistical evaluations, a difference value was considered as significantly statistical for p <0.05.
    30 Osteoblast cell adhesion
    For adhesion studies of the osteoblasts on the implant surface, that is in this case the surface of the coating, the samples were incubated under standard culture conditions for 90 min. Then the samples were washed three times in PBS; after which the cells were separated by a trypsin treatment for 10 min or 35 After centrifugation the cells were resuspended in PBS and counted in a Neubauer chamber. In Figure 8, the results of in vitro biocompatibility performed with HOS on the surface of the coating are indicated. For this, the results of initial adhesion (90 minutes) and proliferation of HOS cells after 3 days of culture are considered, by quantifying mitochondrial activity. The data in Figures 8A and 88 are
    40 expressed in mean values ± standard deviation of measurements carried out on three different samples. The initial adhesion of the osteoblasts (90 minutes) does not show significant differences between Tl6AI4V, nanoTi6Al4V, and the control (culture plate plastic).
    45 Osteoblast cell proliferation
    Cell proliferation was determined based on mitochondrial cell activity. For
    the HOS cells were seeded on the surface of the material in 24-well plates with a density of 105 cells per mL in the complete medium and incubated under standard conditions. Cell proliferation was determined using the MTI (3- [4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) test (Sigma-Aldrich, USA) at different time periods after sowing . As a control, the plastic plate was used
    5 crop The quantitative determination is carried out on a UV-VIS spectrophotometer carried out at 570 nm reading. Mitochondrial activity is directly related to absorbance at said wavelength. The mitochondrial activity of HOS is almost identical for both surfaces and shows no differences with respect to the control after 3 days of culture, as shown in Figure 8B.
    Osteoblast cell extension assays
    The degree of cell extension and the morphology of the osteoblasts were observed by 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. Dehydration of
    The samples were made by slowly replacing the medium, using ethanol series with increasing concentration (30%, 50%, 70%, 90%) for 30 min with a final dehydration in absolute ethanol for 60 min, which allowed the samples to dry at room temperature in vacuum. The Ti6AI4V and nano-Ti6Al4V samples were mounted on slides and coated with gold for visualization in the SEM.
    20 Figure 9 shows the surface after one day of HOS cell culture on the surface of the coating. The surface appears completely covered by the cells showing good adhesion, proliferation and extent of extension. The micrographs obtained at higher magnifications show the anchoring elements formed by the cells.
    25 Figure 9d shows a detail of the nanocolumns that remain below the osteoblast layer.
    Bacterial cultures with S. aureus
    Preliminary studies of in vitro bacterial adhesion have been performed using a
    30 strain ATCC 29213 of Slaphylococcus aureus (S. aureus) as a model of bacteria under the static conditions commonly recorded in the literature (Montanaro L, al, Future Microbiology 2011, 6 (11): 1329-49.) The samples were sterilized by dry heat at 150 oC for 12 h. S. aureus bacteria grew to their middle logarithmic phase in growth medium Todd Hewitt (THB) Sigma-Aldrich, USA) at 37 oC under agitation
    35 magnetic at 100 rpm, until the optical density measured at 600 nm reached 1.0. At this point the bacteria from the culture were collected by centrifugation at 1500 rpm for 10 min at room temperature. It was washed 3 times with sterile PBS maintaining pH 7.4 and resuspended in PBS at a concentration of 6x108 cells mr1. Subsequently it was incubated at 37 oC under magnetic stirring at 100 rpm for different incubation times, in
    40 presence of the biomaterials under study.
    Adhesion assays of S. aureus The incubation time with the suspended bacteria was 90 minutes. Subsequently, the samples were aseptically removed from the bacterial suspension and three were clarified.
    45 times in PBS to eliminate free bacteria. The quantification of the bacteria bound to the surface of the nanostructured material was carried out by the following method, each piece was placed in an Eppendorf tube containing 1 ml of sterile PBS. Then

    it was sonicated for 30 s assuming that 99.9% of the remaining bacteria were separated from the surface. Then 100 ml of each product obtained after sonication was taken and grown on Tryptic Soy Agar (TSA) discs (Sigma Aldrich, USA), followed by overnight incubation at 370 C. The determination of colony forming units (CFU) resulting from the sum of the three sonication processes allowed to determine the number of original bacteria adhered to the pieces. Bacterial cultures of S. aureus performed on Ti6AI4V surfaces (Figure 10) do not show significant differences with respect to the control after 90 minutes of exposure. However, in the case of nano-Ti6AI4V, the adhesion of S. aureus is three times lower compared to Ti6AI4V.
    S. aureus canfaeal microscopy After 90 minutes of incubation in PBS, the samples were stained for 15 minutes with the Invitrogen Live / Oead backlight bacterial viability kit. Confocal microscopy studies have been performed with a Biorad MC1025 microscope. SYTO 9 fluorescence (live bacteria, green) is excited at a wavelength of 480/500 nm, emitting fluorescence at 500 nm. The fluorescence of propidium iodide (dead / red bacteria) was excited at 490/635 nm and the fluorescence emitted at 618 nm was measured. The images obtained with confocal microscopy after 90 minutes of culture are shown in Figure 11. The images show a lower bacterial adhesion on nano-Ti6AI4V, in total agreement with the count shown in Figure 11. No significant differences in the live / dead ratio were observed when the surfaces of Ti6AI4V and nano-Ti6AI4V were compared. This fact indicates that the antibacterial activity of the coatings is exerted thanks to the non-stick properties thereof, with no bactericidal effects against S.
    aureus
    SEM microscopy of S. aureus The SEM study was carried out by preparing the samples analogously to that described for osteoblast studies. Figure 12 shows the surfaces of Ti6AI4V and nano-Ti6Al4V after 24 hours of culture with S. aureus. Figure 12A corresponds to the surface of the Ti6AI4V sample and bacteria can be seen surrounded by an extracellular matrix identified as bacterial biofilm and covering the polished surface of the substrate. In order to highlight the presence of the bacterial biofilm, Figure 12 contains an insel in which the clean surface of biofilm before the bacterial culture is observed while, on the contrary, the surface of the nano-Ti6Al4V sample shows a micrograph in which the bacteria present have not been able to form biofilm, allowing to see the nanostructure of the nano-Ti6AI4V sample.
    权利要求:
    Claims (1)
    [1]
    1 Implant comprising a titanium coating deposited on a substrate characterized in that
    -the substrate comprises a biomaterial with a mean quadratic roughness lower than5 nm in an area of 4 ~ m2,-The coating has a purity greater than 95% and comprises titaniumnanostructured formed by metallic titanium and a layer of titanium oxide,
    10 -The nanostructured titanium has a nanocolumnar shape where the diameter of the nanocolumns is 30 to 100 nm and the height varies between 100 and 300 nanometers, the space between nanocolumns varies between 50 and 150 nanometers with an inclination of the nanocolumn with respect to the vertical of the substrate between 0 ° and 30 °.
    Implant according to claim 1 wherein the substrate biomaterial comprises at least one of the following materials
    - commercially pure titanium with purity greater than 99% of medical grade for periodontal implants, - medical grade metal alloys such as CrCo, stainless steel, Ti6AI4V for 20 orthopedic, cranial and maxillofacial applications,
    3 Implant according to claim 2 wherein the substrate biomaterial comprises Ti6AI4V.
    4 Implant according to any of claims 2 to 3 wherein the biomaterial of the
    The substrate is formed in structures comprising discs, screws, nails, stems, osteosynthesis plates and other fracture fixation devices, generally made of stainless steel.
    Method of obtaining the implant of claims 1 to 4, which comprises depositing the coating on the substrate using oblique incidence techniques (GLAD).
    The method according to claim 5, wherein the deposition is carried out in a sputtering system.
    Method according to claim 6, wherein the sputtering system comprises a magnetron.
    8 Method according to claim 7 wherein the deposit comprises the following 35 steps:
    a) introduction of the substrate into the cathode spray system chamber,b) chamber closure and vacuum completion,c) introduction of gas into the chamber,d) electromagnetic excitation of the gas particles present in the chamber
    40 by means of a source, e) collision of the particles present in the chamber against a titanium target, f) deposit of the material released from the target on the substrate, characterized in that the product of the operating pressure (P,) by distance white substrate (L), fulfills the condition of ballistic regime for the pulverization of Ti, given by pgL <12 Pa cm; and the substrate forms an inclination angle greater than 60 ° with the target.
    Method according to claim 8, characterized in that the vacuum reached is less than 104 Pa, in the chamber it is satisfied that the ratio LId is greater than 3.5, d being the diameter of the blank and L the white-substrate distance
    Use of the implant defined in claims 1 to 4 in bone implantology.
    Use according to claim 10 in temporary or permanent use implants.
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    同族专利:
    公开号 | 公开日
    US20170224458A1|2017-08-10|
    EP3135242A1|2017-03-01|
    EP3135242A4|2018-01-03|
    ES2552278B1|2016-09-14|
    WO2015162329A1|2015-10-29|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2006116752A2|2005-04-28|2006-11-02|The Regents Of The University Of California|Compositions comprising nanostructures for cell, tissue and artificial organ growth, and methods for making and using same|
    RU2281122C1|2005-06-30|2006-08-10|Евгений Александрович Левашов|Medicine-destination biocompatible multicomponent nanostructural coatings|
    ITRM20070033A1|2007-01-23|2008-07-24|Romana Film Sottili S R L|PROCESS FOR OSSEOINTEGRATION OF SURGICAL PROSTHESIS|KR101993391B1|2018-01-10|2019-06-26|단국대학교 천안캠퍼스 산학협력단|Method for manufacturing surface-modified dental implant materials|
    CN113403619A|2021-06-04|2021-09-17|浙江大学|near-infrared/pH dual-response iodine-loaded titanium alloy implant and preparation method thereof|
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    ES201430616A|ES2552278B1|2014-04-25|2014-04-25|BIOCOMPATIBLE IMPLANTS OF NANOESTRUCTURED TITANIUM WITH ANTIBACTERIAL PROPERTIES|ES201430616A| ES2552278B1|2014-04-25|2014-04-25|BIOCOMPATIBLE IMPLANTS OF NANOESTRUCTURED TITANIUM WITH ANTIBACTERIAL PROPERTIES|
    PCT/ES2015/070345| WO2015162329A1|2014-04-25|2015-04-24|Biocompatible implants made of nanostructured titanium with antibacterial properties|
    EP15782740.3A| EP3135242A4|2014-04-25|2015-04-24|Biocompatible implants made of nanostructured titanium with antibacterial properties|
    US15/306,751| US20170224458A1|2014-04-25|2015-04-24|Biocompatible implants made of nanostructured titanium with antibacterial properties|
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