Biocompatible multilayer-thin-film-type coating as a surface treatment for biomedical substrates, and method for the production thereof

Abstract

The present invention discloses a process for the manufacture of a thin-film multilayered coating used in treating biomedical substrates and a coating in multilayered thin-film form (S/TiN/Ti/TiZr) to treat biomedical substrates used in surgical implants.

Claims

1. A process to manufacture a thin-film multilayered coating for the treatment of biomedical substrates, comprising: polishing the substrate surface to a granulometry of between 700 and 2500 to generate a mirror surface finish; washing the substrate with acetone in ultrasound for 10 to 20 min; submerging the substrate in a PVD-DC magnetron sputtering reactor at a temperature of between 20 and 25 C., vacuum of 5.110-6 torr, and 50 to 60% relative humidity; depositing a TiN coating layer with thickness of between 550 and 590 nm from a Ti precursor target (99.99%) at a pressure of 2 to 4 mtorr, argon flow between 8 and 12 sccm, nitrogen flow between 0.1 and 0.2 sccm, precursor target potential between 80 and 120 W, polarization voltage from 50 to 120 V, and interelectrode distance between 8 and 12 cm; depositing the Ti coating layer with thickness between 550 and 590 nm from a Ti precursor target (99.99%) at a pressure from 2 to 4 mtorr, argon flow between 8 and 12 sccm, precursor target potential between 80 and 120 W, polarization voltage from 50 to 120 V, and interelectrode distance between 8 and 12 cm; and depositing the TiZr coating layer with thickness between 550 and 590 rim from a TiZr precursor target (30-70%) at a pressure from 2 ; to 4 mtorr, argon flow between 8 and 12 sccm, precursor target potential between 60 and 100 W, polarization voltage from 50 to 120 V, and interelectrode distance between 8 and 12 cm.

2. A coated biomedical substrate, comprising: a multilayered thin-film coat comprising a TiN coating layer having a thickness of between 550 and 590 nm over the substrate; an intermediate layer of Ti having a thickness of between 550 and 590 nm; and an external TiZr layer having a thickness between 550 and 590 nm.

3. A surgical implant comprising the coated biomedical substrate of claim 2, wherein the substrate is selected from the group consisting of 316L stainless steel, CoCr alloy, and Ti alloy.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) In a first aspect, the invention reveals a process to manufacture a thin-film multilayered coating for treatment of biomedical substrates used in surgical implants, such as but not limited to 316L stainless steel SS, CoCr alloy, Ti alloylike Ti 64 among other materials, which comprises: a) Polishing the substrate surface to a granulometry of between 700 and 2500 to generate a mirror surface finish b) Washing with acetone in ultrasound for 10 to 20 min c) Submerging the substrate from stage b) into a PVD-DC magnetron sputtering reactor at a temperature between 20 and 25 C., vacuum of 5.110.sup.6 torr, and 50 to 60% relative humidity d) Depositing the TiN coating layer with thickness between 550 and 590 nm from a Ti precursor target (99.99%) at a pressure from 2 to 4 mtorr, argon flow between 8 and 12 sccm, nitrogen flow between 0.1 and 0.2 sccm, precursor target power between 80 and 120 W, polarization voltage from 50 to 120 V, and interelectrode distance between 8 and 12 cm. e) Depositing the Ti coating layer with thickness between 550 and 590 nm from a Ti precursor target (99.99%) at a pressure from 2 to 4 mtorr, argon flow between 8 and 12 sccm, precursor target potential between 80 and 120 W, polarization voltage from 50 to 120 V, and interelectrode distance between 8 and 12 cm. f) Depositing the TiZr coating layer with thickness between 550 and 590 nm from a TiZr precursor target (30-70%) at a pressure from 2 to 4 mtorr, argon flow between 8 and 12 sccm, precursor target potential between 60 and 100 W, polarization voltage from 50 to 120 V, and interelectrode distance between 8 and 12 cm.

(2) In a second aspect, the invention provides a coating in multilayered thin-film form (S/TiN/Ti/TiZr) for a substrate used in surgical materials obtained through the procedure already described, which comprises a TiN coating layer with thickness between 550 and 590 nm directly over the substrate, an intermediate Ti coating layer with thickness between 550 and 590 nm, and an external TiZr layer with thickness between 550 and 590 nm.

(3) This thin-film multilayered coating (S/TiN/Ti/TiZr) has all the mechanical properties (high corrosion and wear resistance) and biocompatibility (high proliferation of cells, low genotoxicity and cytotoxicity, and high osseointegration) required for biomedical applications requiring high growth and cellular proliferation, as shown in the following examples.

(4) The following examples are presented for the purpose of describing the preferred aspects of the invention, but do not constitute a limitation to its scope.

EXAMPLE 1

(5) A bar of commercial grade biocompatible 316L Steel, 1.25 cm in diameter, was used as substrate. It was fractioned into cylinders 4 mm thick and their surface was polished by using silicon carbide sandpaper with granulometry between 700 and 2500 to generate a mirror surface finish. Prior to being submerged in the PVD-sputtering reactor, the cylinders were subjected to ultrasound washes for 15 min in acetone fluid, eliminating foreign agents like grease and dust, contamination due to manipulation. For synthesis of thin-film coatings from different materials (Ti, TiN,Ti/TiN, TiZr, TiZr/Ti/TiN), a DC magnetron sputtering system was used, consisting of a multisource PVD magnetron sputtering system (AJA ATC1500 INTERNATIONAL) with deposition reactor (15 internal diameter and 17 height), vacuum system and valves, and substrate holder.

(6) Said system is housed in a class 1000 clean room for the areas where the coatings are deposited and class 10,000 for the room in general; this means that there are 1000 and 10,000 particles per cubic foot of air in these zones, respectively; the clean room is evaluated and certified by ISO 9001 standard C4Contamination Control.

(7) Conditions were adjusted to prepare the magnetron sputtering system to produce coatings, such as: clean room temperature at 22 C., relative humidity 55%, and initial atmospheric pressure of 762 torr. FIG. 1 shows the logarithmic pressure (log P) ratio versus time of particle evacuation from the reactor, defining a base vacuum value of 5.110.sup.6 torr as sufficient condition for the synthesis process of the materials, taking an approximate time value of 2 h 30 min minimum to reach the base pressure and deposit the coatings.

(8) For the synthesis of the different protective layers on the biocompatible 316L steel substrate, high-purity Ti and TiZr precursor targets were used. For all the coatings deposited, polarization voltage at 100V, base pressure of 5.110.sup.6 mtorr, and 10-cm interelectrode distance were established as fixed variables in the process. The synthesis conditions of each of the coating layers with 570-nm thickness (measured via profilometry) are presented in Table 1.

(9) TABLE-US-00001 TABLE 1 Synthesis conditions of Ti, TiN, Ti/TiN, TiZr, and TiZr/Ti/TiN thin layer materials Working Precursor pressure Ar flow N.sub.2 flow Target power Material target (mtorr) (sccm) (Sccm) (W) Ti Ti (99.99%) 3 10 100 TiZr TiZr (30%70%) 3 10 80 TIN Ti (99.99%) 3 10 0.15 100

EXAMPLE 2

(10) The following presents the comparison of the analysis of surface, mechanical, tribological, and corrosion properties of the TiN/Ti/TiZr multilayer coating of the invention compared to Ti, TiN/Ti, TiZr, and TiN thin protective layers.

(11) Analysis of Mechanical Properties

(12) Hardness and modulus of elasticity measurements were made by using a nanoindenter (NANOVEA module IBISTechnology), using the traditional Oliver and Pharr method, to fit the discharge curve. Nanoindentations were carried out in the following ranges: low (L), medium (M), and high (H) loads (L: 0.01-0.4 mN; M: 0.41-1 mN, and H: 1.1-10 mN) obtaining hardness and modulus of elasticity profiles in function of depth, determining a 1-mN ideal load that covers 10% of the thickness for the coatings. Nanoindentation tests were conducted by using a Berkovich pyramidal indenter coupled to the IBIS nanoindentation head by Fischer-Cripps Labs and a displacement control frame with compliance of 0.00035 um/mN, IBIS SOFWARE was used to control indentation, correction, and analysis of results. Hardness and modulus of elasticity were calculated recurring to the Oliver and Pharr model.

(13) Analysis of Surface and Tribological Properties

(14) To study the friction coefficient and wear of the coatings, a ball-on-disk system (CSEMTribometer) was used. The experimental conditions to measure the tribological properties were constant for the set of thin layers studied; the experimental conditions are shown in Table 2.

(15) TABLE-US-00002 TABLE 2 Experimental conditions for the analysis of tribological properties Spherical counterpart Alumina (Al.sub.2O.sub.3) Diameter of the Spherical counterpart 6 mm Normal applied load 1N Run distance 15 m Test rate 10 m/s Test radius 3 mm Frequency of data recording 2 Hz

(16) The friction coefficient analysis had statistical software support equipment (CSEMtribotest, XTribo 2.5) and the ORIGIN mathematical package. To calculate thickness, roughness, and wear, a profilometer (XP2 AMBIOS) was used. The rate of wear measurement was made by using the transversal area of the wear track after the BOD test and the Archard model, which proposes that the wear coefficient is directly proportional to the volume worn and inversely proportional to the normal load applied and the glide path.

(17) Adherence Analysis

(18) The scratch test was performed with Micro Test equipment, using a Rockwell C-type indenter with 200 m radius, variable load from 0 to 100 N, rate of load application: 1 N/s, distance: 6 mm, and displacement rate: 4.5 mm/min.

(19) Stereoscopy

(20) Stereoscopies were taken by using Olympus SZ4045 equipment (reference #OCS07) to determine form of wear and cellular morphology, facilitating the calculation of cellular density per square micrometer.

(21) Scanning Electron Microscopy and Energy Dispersive Spectroscopy (SEM/EDS)

(22) For surface morphology description and cellular observation, SEM equipment was used (JEOL series JSM-6460) with EDS probe (model Oxford INCAEnergy EDS system), with possibility for magnification from 5to 300,000, 3-nm resolution, and acceleration potential from 0 to 30 KV.

(23) Through magnifications from 2000 to 5000, surface details were determined inherent to the material synthesis process through magnetron sputtering, in addition to cell growth and morphology. With EDS the elemental chemical composition was determined, discretely detecting the atomic percentage of elements present in the coatings and substrate. For count calculations and cell population, micrographs were carried out on substrates and layers where initially fibroblast cells from series L929were manually deposited for 72 h of cell growth at 37 C.; the magnifications used were 50 and 500. Given that these were non-conducting biological samples, they were subjected to metallization process (evaporation of a nanometric layer of gold, generating electric conduction).

(24) Corrosion Resistance

(25) Potentiodynamic curves were analyzed, using the potentiostat-galvanostat system for Ti, TiN, TiZr, TiN/Ti, and TiN/Ti/TiZr thin-film coatings deposited on 316L biocompatible substrate using Potenciostat/Galvavanostat equipment (model 273 A EG&E PRINCETON APPLIED RESEARCH) with cell (model k47 Corrosion Cell System EG&E INSTRUMEN-PRINCETON APPLIED RESEARCH). Reference electrode Hg/KCI was used and platinum as counterelectrode to analyze the thin-film coatings; using a potentiodynamic scan of 1 mV/s. Coating behavior was observed by using two electrolytic solutions, brine (3.5%p/p NaCI) and simulation of blood fluid (Hank's solution); Hank's electrolyte is an artificial physiological solution with environment rich in chloride and pH 7.4. Hank's balanced saline solution is a standard culture medium used in biomedical research for cellular preservation; it is not toxic, has balanced pH, and its osmolarity is 320 mOsm/Kg. This medium has been studied profoundly, showing that during the first 24 h of storage, the fibroblasts remain vital. Said experiments were conducted at human body temperature (37 C.), using water bath during the process. The composition and concentration of the blood solution are shown in Table 3.

(26) TABLE-US-00003 TABLE 3 Composition and concentration of Hank's physiological solution Reagent Concentration (g/L) NaCl 8 KCl 0.4 NaHCO.sub.3 0.35 C.sub.6H.sub.12O.sub.6 1 NaH.sub.2PO.sub.4H.sub.2O 0.25 MgCl.sub.26H.sub.2O 0.19 Na.sub.2HPO.sub.42H.sub.2O 0.06 MgSO.sub.47H.sub.2O 0.06
Biological Experimentation

(27) To evaluate biocompatibility, cellular proliferation, cell count, cytotoxicity, and genotoxicity were determined.

(28) Cell count of fibroblasts was accomplished by using SEM and stereoscopy. Samples were cultured in the form of mouse fibroblast monolayer from cell line L929, making observations after 72 h in controlled 37 C. environment on the cell capacity to grow on the surfaces of the coatings synthesized via magnetron sputtering, through SEM and stereoscopy, determining cellular density in the optical field area.

(29) Cell Proliferation Ttest (Kit XTT Cell Proliferation ROCHE)

(30) The cells were seeded in 24 of the 96 wells of a 96 well tissue culture plate and were incubated with XTT solution (final concentration 0.3 mg/mL) from 4 to 24 h. After this incubation period, the orange-colored formazan solution is made, which is quantified in spectrophotometically by using an ELISA plate reader. The increased number of living cells yields an increase in the total activity of mitochondrial dehydrogenase in the sample, which permits correlating the response with the amount of formazan formed. This result is used to measure cellular proliferation in response to different factors, like cytokines and growth factors. Line L929 is seeded in cell culture microplates, in 24 of the 96 wells, with flat bottom and a final volume of 200 mL of fresh culture medium; the culture is kept in a moist atmosphere (37 C., 5% CO.sub.2). After the initial incubation period, the medium is removed and the cells are washed with PBS solution three times for 5 min; thereafter, each well is added 50 mL of the mixture of XTT and 100 mL of the growth medium, generating a final XTT concentration of 0.3 mg/mL. The plates are incubated from 24 to 96 h in moist atmosphere (37 C., 5% CO.sub.2). Absorption measurements are performed every 24 h using the microplate reader to verify cellular proliferation of L929 fibroblasts stimulated with magnetic field signals and those not treated with the respective growth controls.

(31) Cytotoxicity Test (Kit LDH ROCHE)

(32) The cells are incubated for 24 h under conditions (37 C., 5% CO.sub.2, 90% humidity) to permit their firmly adhering to the matrix. After the incubation time, the growth medium is removed and the cells are washed three times with PBS for 5 min, and growth medium is added. During the test time, the plates are incubated under the same conditions already described. Cytotoxicity determinations are made at 6 and 24 h, using the ELISA plate reader.

(33) Genotoxicity Test (SOS CHROMOTESTTM)

(34) Preliminary dilution of the bacteria was carried out and density was verified, then, 100 L of the diluted bacterial suspension were added in each well of the columns containing material that will be assayed. The microplates were incubated at 37 C. for 2 h and the relative amount of galactosidase, produced as a result of this interaction, is measured by adding a chromogenic substrate.

(35) Comparative Results

(36) Table 4 summarizes the surface, mechanical, tribological, and corrosion properties of the multilayered coating of the invention (TiN/Ti/TiZr) compared to thin layers of Ti, TiN/Ti, TiZr, and TiN. Table 5 presents the results of the biological tests of the multilayered coating of the invention (TiN/Ti/TiZr) compared to the 316L stainless steel substrate used as material for surgical substrates.

(37) TABLE-US-00004 TABLE 4 Surface, mechanical, tribological, and corrosion properties MATERIAL PROPERTY RESPONSE Ti Cell count 233.33% Cellular proliferation High Cytotoxicity 3.3% Genotoxicity Likely COF 0.8 Hardness 6.76 GPa Modulus of elasticity 179.92 GPa TiN Cell count 160% Cellular proliferation High Cytotoxicity 1.5% Genotoxicity Likely COF 0.28 Hardness 17.34 GPa Modulus of elasticity 235.47 GPa TiN/Ti Cell count 180% Cellular proliferation High Cytotoxicity 2.2% Genotoxicity Unlikely COF 0.71 Hardness 6.68 GPa Modulus of elasticity 254.78 GPa TiZr Cell count 368.8% Cellular proliferation Medium Cytotoxicity 3.8% Genotoxicity Unlikely COF 0.5 Hardness 7 GPa Modulus of elasticity 177.16 GPa TiN/Ti/TiZr (coating Cell count 397.7% of the invention) Cellular proliferation Medium Cytotoxicity 3.9% Genotoxicity Unlikely COF 0.68 Hardness 6 GPa Modulus of elasticity 154.7 GPa

(38) TABLE-US-00005 TABLE 5 Results of biological tests of the invention coating (TiN/Ti/TiZr) against 316L steel PROPERTY TiN/Ti/TiZr 316L Cell count 397.7% 100% Cellular density* 44.8 10.sup.4 11.2 10.sup.4 cells/cm.sup.2 cells/cm.sup.2 Cytotoxicity 3.9% 2.4% Genotoxicity SOSIP Unlikely <0 Likely >0 COF 0.68 0.90 Hardness 6 GPa 4.81 GPa Modulus of Elasticity 154.7 GPa 222.6 GPa Roughness 75 nm 64 nm Wear rate 71 2 10.sup.12 12 2 10.sup.12 mm.sup.3/Nm mm.sup.3/Nm Corrosion potential 167 mV 270 mV NaCl Solution 3.5% Corrosion potential 222 mV 295 mV Hank's Solution *Line L929 after incubation during 72 hours

(39) Although the present invention has been described through the preferred embodiments, nonlimiting of the invention, it is understood that the modifications and variations that conserve its essential and elemental content are within the scope of the claims included.

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