CN111945207A

CN111945207A - Sr/Ag-doped micro-arc oxidation coating and preparation method and application thereof

Info

Publication number: CN111945207A
Application number: CN201910415412.4A
Authority: CN
Inventors: 戚孟春; 王艺睿; 张阳阳; 王欢; 李瑛�; 冯晓洁; 董伟; 刘梦楠
Original assignee: North China University of Science and Technology
Current assignee: North China University of Science and Technology
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2020-11-17
Anticipated expiration: 2039-05-17
Also published as: CN111945207B

Abstract

The invention discloses a Sr/Ag-doped micro-arc oxidation coating and a preparation method and application thereof, and the preparation method of the Sr/Ag-doped micro-arc oxidation coating comprises the following steps: treating the titanium substrate for 10-15 min by adopting a micro-arc oxidation method to form a Sr/Ag-doped micro-arc oxidation coating on the titanium substrate, wherein the micro-arc oxidation method for treating the titanium substrate comprises the following steps: electrolyte is placed in the electrolytic cell, the titanium matrix is placed in the electrolyte for electrolysis, the titanium matrix is used as an anode, the electrolytic cell is used as a cathode, and the electrolyte is formed by mixing strontium salt, silver salt, chloroplatinic acid and water. The Sr/Ag-doped micro-arc oxidation coating is prepared in one step by a micro-arc oxidation method, has good osteogenic activity and antibacterial activity, can promote the proliferation and differentiation of osteoblasts, is beneficial to the early adhesion of cells, has antibacterial capability, and particularly has obvious antibacterial effect on staphylococcus aureus.

Description

Sr/Ag-doped micro-arc oxidation coating and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medical instruments, and particularly relates to a Sr/Ag-doped micro-arc oxidation coating as well as a preparation method and application thereof.

Background

Medical pure titanium and titanium alloy are the most common medical in-vivo implantation materials for human bodies; the application of the method in the fields of orthopedics and oral implantation mainly faces two problems: firstly, poor binding with bone tissue, especially in the case of osteoporosis and bone tissue defects, delayed bone healing rate (delayed bone healing) and bone union (osseointegration) failure are easy to occur; secondly, bacterial infection can cause osseointegration failure in a short term and peri-implantitis (periimplentitis) in a long term, and the implant loosens and falls off. Therefore, how to improve the biological performance of titanium and titanium alloy, so that the titanium and titanium alloy have good osteogenesis activity, promote implant osseointegration, have good short-term/long-term antibacterial activity and prevent peri-implant inflammation caused by peri-implant bacterial infection is a main problem of clinical exploration.

The medical titanium and titanium alloy material is a biological inert material, and the biological performance in vivo is poor; in order to improve the in vivo biological performance, a common method is to change the surface micro-morphology and the chemical composition of the material by a surface coating/modification technology, and introduce some chemical or biological components to make the material show specific biological performance, such as good biocompatibility, osteogenic activity, antibacterial activity, and the like.

Strontium (Sr) element is one of the trace elements in the body, and has been confirmed to have good osteogenic activity and to be able to inhibit bone resorption; the metal silver (Ag) has good antibacterial activity, can achieve the purpose of bacteriostasis and antibiosis within a certain concentration range, and can avoid the negative effects caused by cytotoxicity, so the silver (Ag) is widely applied to the surfaces of a plurality of medical devices. Many previous researches attempt to improve the surface osteogenesis activity and antibacterial activity of titanium and titanium alloy materials by applying coating technologies such as magnetron sputtering (magnetron sputtering), Plasma spraying (Plasma spraying), electrochemical deposition (electrochemical deposition), and alkali and thermal treatment (alkali and hydrothermal treatment) and by using Hydroxyapatite (HA), calcium/phosphorus compounds, chitosan, beta-tricalcium phosphate, sodium alginate and the like as carriers and applying Sr and Ag to the surface coating of titanium and titanium alloy materials singly or jointly.

However, the materials prepared by the coating have the following defects: (1) the coating on the surface part of the material lacks the micro-morphology beneficial to the growth of osteoblasts; (2) the combined application of Sr and Ag in the coating usually requires two or more preparation technologies, and the required technologies are complex, high in cost and not easy to popularize; (3) the prepared Hydroxyapatite (HA), chitosan, beta-tricalcium phosphate and other coatings have lower bonding strength with titanium and titanium alloy matrixes, generally less than 20MPa, and are easy to strip in vivo, particularly in bone tissues, so that the biological performance of the coatings is influenced; (4) the coating is easy to dissolve in vivo, so that Sr and Ag in the coating are mainly released in 1-2 weeks, the time for exerting the biological effect is short, and the maximum time can only be maintained for 4 weeks; the implant material is often used for years and even decades in vivo, so that the required long-term antibacterial activity is difficult to guarantee, and the infection caused by bacteria in long-term use, such as peri-implantitis, is difficult to resist.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a preparation method of the Sr/Ag-doped micro-arc oxidation coating, the preparation method adopts a one-step method to prepare the Sr/Ag-doped micro-arc oxidation coating, the surface of the Sr/Ag-doped micro-arc oxidation coating is doped with Sr and Ag at the same time, and the preparation method is simple and economic and is suitable for batch production; the Sr/Ag-doped micro-arc oxidation coating has a micro-morphology suitable for osteoblast growth, high bonding strength and difficult stripping in vivo application; has proper roughness, hydrophilicity, biocompatibility and long-term antibacterial activity, and Ag stably exists in the coating for a long time (more than 3 months), so that the coating can play a role for a long time.

The invention also aims to provide the Sr/Ag-doped micro-arc oxidation coating obtained by the preparation method.

The purpose of the invention is realized by the following technical scheme.

A preparation method of a Sr/Ag-doped micro-arc oxidation coating comprises the following steps:

treating the titanium substrate for 10-15 min by adopting a micro-arc oxidation method to form a Sr/Ag-doped micro-arc oxidation coating on the titanium substrate, wherein the micro-arc oxidation method for treating the titanium substrate comprises the following steps: electrolyte is placed in the electrolytic cell, the titanium matrix is placed in the electrolyte for electrolysis, the titanium matrix is used as an anode, the electrolytic cell is used as a cathode, and the electrolyte is formed by mixing strontium salt, silver salt, chloroplatinic acid and water.

In the technical scheme, in the process of treating the titanium substrate by adopting the micro-arc oxidation method, the electrolyte is kept stirred, and the temperature of the electrolyte is kept below 38 ℃, preferably 30-37 ℃.

In the technical scheme, the parameters for processing by adopting the micro-arc oxidation method are as follows: the power supply of the micro-arc oxidation method is provided with a constant voltage mode, the forward voltage is 300V, the frequency is 800Hz, and the duty ratio is 80%.

In the technical scheme, after the titanium substrate is treated by the micro-arc oxidation method, the Sr/Ag-doped micro-arc oxidation coating is washed by deionized water and dried.

In the technical scheme, the concentration of strontium salt in the electrolyte is 90-120 g/L, the concentration of silver salt in the electrolyte is 0.08-0.17 g/L, and the concentration of chloroplatinic acid in the electrolyte is 0.021g/L as a catalyst of the silver salt.

The Sr/Ag-doped micro-arc oxidation coating obtained by the preparation method.

In the technical scheme, in the Sr/Ag-doped micro-arc oxidation coating, the strontium accounts for 4-22 wt% and the silver accounts for 0.2-2.5 wt% in percentage by mass.

In the technical scheme, in the Sr/Ag-doped micro-arc oxidation coating, the strontium accounts for 18.23 +/-0.34 wt% and the silver accounts for 0.58 +/-0.19 wt% in percentage by mass.

In the technical scheme, micropores are formed on the Sr/Ag-doped micro-arc oxidation coating, the porosity of each micropore is 15-23%, and the diameter of each micropore is 0.5-3 mu m.

In the technical scheme, the roughness R of the Sr/Ag-doped micro-arc oxidation coating_aThe value is 1 to 1.2 μm, the contact angle of hydrophilic water is 74.7 + -1.77 DEG, and the bonding strength is 40.19 + -2.51 MPa.

The titanium substrate is covered with the Sr/Ag-doped micro-arc oxidation coating.

The application of the preparation method in promoting osteogenic differentiation is provided.

The preparation method is applied to improving antibacterial property.

The preparation method can be used for improving osteoblast proliferation activity and promoting osteoblast early adhesion.

The Sr/Ag-doped micro-arc oxidation coating is prepared in one step by a micro-arc oxidation method, has good osteogenic activity and antibacterial activity, can promote the proliferation and differentiation of osteoblasts, is beneficial to the early adhesion of cells, has antibacterial capability, and particularly has obvious antibacterial effect on staphylococcus aureus. The preparation method provided by the invention is simple and convenient in process, and provides technical support for preparing the composite coating or drug loading on the surface of the titanium metal implant (titanium matrix).

Drawings

FIG. 1 is a schematic structural view of an apparatus used in the micro-arc oxidation treatment according to the present invention;

FIG. 2 is a titanium substrate;

FIG. 3 is a photograph of a titanium substrate;

FIG. 4 is a titanium substrate covered with a Sr/Ag doped micro-arc oxidation coating;

FIG. 5 is a photograph of samples obtained in examples 1 to 4;

FIG. 6 is an SEM of samples obtained in examples 1-4;

FIG. 7 is a graph of elemental distribution of Ti, O, Sr, and Ag in the EDS for a sample of the M-SrAg group (example 4);

FIG. 8 is an XRD of samples obtained in examples 1 to 4;

FIG. 9 shows the surface roughness (R) of the samples obtained in examples 1 to 4_aA value);

FIG. 10 shows surface hydrophilicity tests of samples obtained in examples 1 to 4;

FIG. 11 shows the bonding strength of the samples obtained in examples 2 to 4;

FIG. 12 shows the proliferation potency of osteoblast cultures obtained in examples 1 to 4 in terms of

numbers

1, 4 and 7 d;

FIG. 13 shows the adhesion of preosteoblasts to the surfaces of samples obtained in examples 1 to 4 at 1 hour and 2 hours after inoculation;

FIG. 14 is a graph showing the number of adherent preosteoblasts on the surface of samples obtained in examples 1 to 4 at 1 hour and 2 hours after the inoculation;

FIG. 15 shows the alkaline phosphatase activity of preosteoblasts cultured on the surface of samples obtained in examples 1 to 4 at 7 d;

FIG. 16 is a graph showing the detection of the surface antibacterial activity of the samples obtained in examples 1 to 4 by a film-sticking method;

FIG. 17 shows the strontium content in the Sr/Ag-doped micro-arc oxide coating obtained in examples 5 to 12;

FIG. 18 shows the proliferative capacity of osteoblast cultures on

samples

1, 3, 5, 7 d;

FIG. 19 shows osteoblasts cultured alkaline phosphatase activity at 7d on the surfaces of five groups of samples;

FIG. 20 shows the proliferative capacity of osteoblast cultures at 1 st, 4 th and 7 th d on five groups of samples with different silver contents;

FIG. 21 shows the short-term antibacterial activity detected by the bacteriostatic ring test;

FIG. 22 shows the short-term and long-term antibacterial activity measured by the plate coating method.

Detailed Description

The detection method and apparatus in the following examples are as follows:

1. analysis by scanning electron microscope

The micro-topography of the surface of the test piece after the micro-arc oxidation treatment is detected by a Scanning Electron Microscope (SEM). High-energy electron beams are incident to the surface of the test piece, secondary electrons are generated by surface excitation after electron bombardment, and the microscopic morphology of the surface of the test piece is displayed through an imaging system. The invention adopts an S-4800 scanning electron microscope of Hitachi, Japan to observe the surface micro-topography of the test piece, and analyzes the surface topography of the test piece under different magnification factors.

2. Energy spectrum analysis

The principle of the X-ray spectrometer is as follows: different chemical elements emit characteristic X-rays, the emitted rays having different frequencies. By detecting the frequencies of the different photons, the class of the element can be determined. It is a very reliable technical method and has wide application. The invention adopts an S-4800 scanning electron microscope to be matched with Energy spectrum analysis (EDS) to analyze the surface of the film layer, mainly measures the content of strontium and silver, and qualitatively and visually observes the distribution of the strontium and the silver.

3. X-ray diffraction analysis

Qualitative phase analysis of the test pieces was performed using an X-ray diffractometer (XRD). XRD phase analysis can distinguish object images according to different diffraction effects of X-rays on different crystals. The analysis of the invention adopts a D/MAX-rBX ray diffractometer of Japan science company to analyze the chemical components of the surface of the test piece. The invention adopts Cu target Kalpha radiation, the working voltage and the working current are respectively 40KV and 100mA, and the characteristic wavelength

The scanning range is 10-80 degrees, the step length is 0.02 degree, and the scanning speed is 2 degrees/min.

4. (1) roughness measurement

And testing the surface roughness of the test piece by using a roughness measuring instrument. During testing, a sensor of the roughness measuring instrument vertically contacts the surface of a tested piece, the sensor slides along the surface of the tested piece, the sensor detects the roughness of the surface of the tested piece through an internal contact pin, the roughness of the surface of the tested piece causes the contact pin to generate displacement, the displacement enables the inductance quantity of an inductance coil of the internal sensor to change, a signal proportional to the roughness of the surface of the tested piece is generated at the output end of a rectifier, the signal enters a data acquisition system after being amplified and converted through a corresponding electric screen, and an internal chip carries out data filtering and parameter calculation on the acquired data and finally displays a corresponding measuring result on a display. The roughness measuring instrument adopted by the invention is Germany DMG expert3D, the roughness of the surface of the test piece is precisely measured, each group of three test pieces is repeated for three times, and finally, the average value is obtained.

(2) Contact Angle analysis

The surface contact angle measuring instrument is mainly used for measuring the contact angle of liquid and the surface of a sample so as to show the hydrophilic effect of the surface of the sample. The instrument is very sensitive and can measure the contact angles of different liquids to the surfaces of different solid materials. The invention adopts a hanging drop contact angle measuring instrument Germany Dataphysics OCA40Micro to measure the liquid contact angle, each group of three test pieces and each test piece is repeatedly measured for three times.

(3) Coating adhesion scratch tester for measuring coating bonding strength

The coating adhesion scratch tester applies the combination of acoustic emission detection technology detection, friction force detection technology and microcomputer automatic control technology, continuously applies load to a scriber (diamond pressure head) through an automatic loading mechanism, and simultaneously moves a sample to enable the scriber to scribe the surface of a film layer. Acoustic emission signals, load variation and friction variation during scratching are obtained through the sensors, input into a computer for corresponding data conversion, and measured results are drawn into a graph to finally obtain the bonding strength of the film layer and the substrate. During testing, three test pieces are measured in each group, and each test piece needs to be repeatedly measured three times. And finally an average is taken.

5. Measurement of osteoblast proliferation Capacity

Dispersing third generation MC3T3-E1 cells (cell bank of Shanghai Life sciences, China) in complete culture medium to form cell suspension, wherein the density of MC3T3-E1 cells in the cell suspension is 1 × 10⁴Each group of samples contains 12 pieces per mlEach group of 3 duplicate wells was prepared by placing the samples in 24-well plates (Guangzhou Jet Biofil Co., Ltd.), inoculating 1mL of cell suspension on the surface of each test piece in each well, adding complete medium to each well, and adding CO₂And culturing in a constant temperature incubator at 37 ℃ for 1d, 4d and 7d respectively. And then detecting the influence of the test piece on the cell proliferation capacity by adopting an MTT method. The specific operation steps are as follows: after 1d, taking the 12-well plate for culturing the MC3T3-E1 cells out of the cell culture box, sucking the original complete culture medium by using a gun head, washing the surface of the test piece for 2-3 times by using a PBS solution, and removing the cells which are not adhered to the surface. Mu.l of fresh complete medium and 100. mu.l of thiazole salt (MTT, purchased from Sigma, USA) reagent were added to each well, shaken gently, and placed in CO₂Culturing in constant temperature incubator with saturated humidity of 37 deg.C and CO content of 5%₂(the residual 95% volume fraction is air), taking out after 4h, adding 600 mul of dimethyl sulfoxide (DMSO, purchased from Shanghai chemical reagent factory) into each well, placing on a shaking table, shaking for 20min, sucking out 200 mul of solution from each well, injecting into a 96-well enzyme label plate, reading by an enzyme label detector under the condition of 492nm wavelength, recording the absorbance (OD) value detected by each group of MTT, wherein the OD value is in direct proportion to the proliferation level of osteoblasts, and comparing the OD value can compare the proliferation level of each group of cells. The 4d and 7d processes were performed as described above. Complete medium was changed every 2-3 d. The OD values of 1d, 4d and 7d of the cultured cells were recorded for statistical analysis, and the averaging was repeated 3 times.

Complete medium: fetal bovine serum (PAN, Germany) with a volume fraction of 10%, penicillin + streptomycin with a volume fraction of 1% and DMEM medium (BI, Israel) with a volume fraction of 89%. Among these, "penicillin + streptomycin" was purchased from israel BI corporation. DMEM medium was purchased from israel BI corporation and has the following composition as shown in the table:

DMEM (H) cell culture Medium (powder type) composition

Serial number	Name of Compound	Content (mg/L)	Serial number	Name of Compound	Content (mg/L)
						1	Anhydrous calcium chloride	200.00	17	L-serine	42.00
2	Ferric nitrate 9H 20	0.10	18	L-threonine	95.00
						3	Potassium chloride	400.00	19	L-tryptophan	16.00
4	Anhydrous magnesium sulfate	97.67	20	L-tyrosine sodium salt	104.00
						5	Sodium chloride	6400.00	21	L-valine	94.00
6	Anhydrous sodium dihydrogen phosphate	125.00	22	D-calcium pantothenate	4.00
						7	L-arginine hydrochloride	84.00	23	Choline chloride	4.00
8	L-cystine hydrochloride	63.00	24	Folic acid	4.00
						9	L-Glutamine	584.00	25	Inositol	7.20
10	Glycine	30.00	26	Nicotinamide	4.00
						11	L-histidine hydrochloride	42.00	27	Riboflavin	0.40
12	L-isoleucine	105.00	28	Thiamine hydrochloride	4.00
						13	L-leucine	105.00	29	Pyridoxine hydrochloride	4.00
14	L-lysine hydrochloride	146.00	30	Glucose	4500.00
						15	L-methionine	30.00	31	Pyruvic acid sodium salt	110.00
16	L-phenylalanine	66.00	32	Phenol Red	15.00

6. Measurement of osteoblast adhesion Capacity

Dispersing third generation MC3T3-E1 cells (cell bank of Shanghai Life sciences, China) in complete culture medium to form cell suspension, wherein the density of MC3T3-E1 cells in the cell suspension is 1 × 10⁴Each group of samples is 12 pieces, each group has 3 multiple holes, the samples are put into a 24-hole culture plate (Jet Biofil Co., Ltd., Guangzhou), 100 mu l of cell suspension is respectively inoculated on the surface of a test piece of each hole, complete culture medium is added, after 1h and 2h, cell nucleuses adhered to the surface of the test piece are dyed, and the number of the cells adhered to the surface of the test piece is observed by a fluorescence microscope. The specific operation steps are as follows: observing the state of the MC3T3-E1 cells under an inverted microscope, and repeating the following steps for 2-3 times when the MC3T3-E1 cells grow to 80-90% fusion: the complete medium was removed from the cell culture plate, 5ml of PBS solution (Sigma, usa) was added and removed. Repeating for 2-3 times, adding trypsin (Gibco, USA) for digestion for 3-5min, observing cell morphology under inverted phase contrast microscope, adding 6ml of complete culture medium to stop digestion when cell changes from long fusiform adherent state to spherical state, blowing cell growth surface with pipette to make cell suspend in complete culture medium, transferring into 50ml centrifuge tube for centrifugation, centrifuging, and collecting supernatantDiscarding the supernatant; adding new complete culture medium to make MC3T3-E1 cells re-suspended to be evenly dispersed, extracting 100 μ l, inoculating on the surface of each group of test pieces in a 12-well plate, placing in an incubator at 37 ℃ for culture, removing the complete culture medium after 1h and 2h respectively, and gently rinsing with PBS buffer solution for 2-3 times, 10min each time, and removing cells which are not adhered on the surface. Fixing with 4% paraformaldehyde (4% paraformaldehyde from Shanghai chemical reagent factory) for 1 hr, washing with PBS buffer solution for 10min for 2-3 times, and removing the fixing solution, 4% paraformaldehyde. Discarding PBS buffer, breaking the wells with 0.5 wt% Triton-X (a breaker, available from Shanghai Sorbo Biotech Co., Ltd.) for 15min, rinsing with PBS buffer for 2-3 times, each for 10min, and removing the breaker. The cell nucleus is stained with Propidium Iodide (PI) for 3min, and rinsed 2-3 times with PBS buffer, 10min each time, to remove PI. The red nuclei were observed by immunofluorescence microscopy and 5 fields were randomly selected for cell counting. The averaging was repeated 3 times.

7. Measurement of ALP Activity of osteoblast

Dispersing third generation MC3T3-E1 cells (cell bank of Shanghai Life sciences, China) in complete culture medium to form cell suspension, wherein the density of MC3T3-E1 cells in the cell suspension is 1 × 10⁴And (2) inoculating 1mL of cell suspension on the surface of each test piece, wherein the total number of the test pieces is 12, each group comprises 3 multiple wells, and after culturing for 7 days, detecting the activity of alkaline phosphatase. The specific operation steps are as follows: placing the test piece inoculated with the MC3T3-E1 osteoblasts into CO₂Culturing in a constant-temperature incubator, and replacing the complete culture medium every two days. After incubation for 7d, the complete medium was removed, gently rinsed with PBS buffer, the non-adherent cells were removed from the surface, and the PBS buffer was blotted. Adding 30 μ l of lysis solution (purchased from WU HAN doctor Ded Biotech Co., Ltd.) into each well, lysing for 1h on ice, transferring the lysed cells into a centrifuge tube, centrifuging at 14000rpm for 15min at 4 deg.C, quickly sucking the supernatant, adding into a4 deg.C pre-cooled centrifuge tube, and centrifuging again. A portion of the supernatant was quickly aspirated and added to a 96-well microplate (purchased from Jet Biofil, Guangzhou, Ltd.), and the reactant in the BCA kit (Biyun day) was added thereto, and the absorbance OD value was measured with an microplate reader at a wavelength of 520 nm. From the OD values, the protein concentration was calculated. Suction partAdding the supernatant into the reactant in ALP kit (Nanjing institute of bioengineering), measuring absorbance at 520nm, and calculating according to the formula: alkaline phosphatase activity was calculated as absorbance value/protein concentration of ALP kit. The averaging was repeated 3 times.

8. Study of antibacterial Activity

The invention adopts the antibacterial ring method to detect the antibacterial performance, and the prepared test piece (sample) is vibrated and sterilized at high temperature for standby use to detect the antibacterial performance. Dispersing Staphylococcus aureus in bacterial culture solution to form bacterial suspension, wherein the total number of staphylococcus aureus colonies in each 1mL of bacterial suspension is 1 × 10⁶The CFU, pipette 20 μ l of a bacterial suspension of staphylococcus aureus (bacterial broth consisting of bacteria, peptone, sodium chloride and beef extract powder, purchased from tokyo obozoocin biotechnology ltd.) to a solid medium consisting of peptone, sodium chloride, beef extract powder and agar, purchased from tokyo oboocin biotechnology ltd), wherein the bacterial suspension was uniformly coated on the test piece with a triangular coating bar, and the test piece coating surface was placed in the central position of a petri dish (purchased from Corning, usa) containing the solid medium. The culture was carried out in an aerobic environment for 24 hours. The solid media were photographed and counted.

The invention uses a flat plate coating method to detect the short-term and long-term antibacterial rates of five groups of test pieces, and the test piece (short-term) prepared immediately by the invention and the test piece (long-term) of the invention after being soaked in PBS solution for 30 days are respectively used for detecting the short-term and long-term antibacterial rates: placing the test piece in a sterile 24-hole culture plate, adding 3 multiple holes per group, and adding bacterial suspension (Staphylococcus aureus is dispersed in bacterial culture solution to form bacterial suspension) into the culture plate by a pipette at a rate of 50 μ L/hole, wherein the total number of bacterial colonies of Staphylococcus aureus in each 1mL of bacterial suspension is 1 × 10⁵CFU) was applied to the coated surface of the test pieces, and sufficient sterile PBS solution was added to the perimeter of the blank wells and the well plate to prevent evaporation of the bacterial suspension, and the samples were incubated in a 37 ℃ incubator. After 24h of culture, adding 1mL of sterile PBS solution into each hole, fully blowing and beating, eluting non-adhered bacteria, continuously diluting each group by 10 times with the PBS solution, sucking 50 mu L of bacterial suspension, dripping the bacterial suspension into a solid culture medium, and adding the solid culture medium into the solid culture medium100 mu LPBS solution is added into each solid culture medium to facilitate the sterilization of a triangular glass coating rod to evenly coat the bacteria, and the bacteria are cultured in a constant temperature incubator at 37 ℃. After 24h, the solid media were photographed and counted according to R ═ N₀–N_t)/N₀And multiplying by 100% formula to calculate the antibacterial rate of each group. Wherein N is₀And Nt represents the M-Sr group (N)₀) And the average viable bacterial count for the silver-containing M-Sr/Ag group (Nt).

In the following embodiments, the titanium alloy is polished by 1000-mesh, 1500-mesh and 2000-mesh abrasive paper step by step to remove oil stains and oxide layers on the surface of the titanium alloy, and then ultrasonic vibration cleaning is respectively carried out by acetone, alcohol and deionized water for 15min, and the titanium alloy is placed in a drying oven for drying and standby.

The apparatus used for performing the micro-arc oxidation treatment in the following examples is shown in fig. 1, and includes: the device comprises an electrolytic cell, a WH-1A micro-arc oxidation power supply, a stirrer and a heat insulation plate, wherein the heat insulation plate is arranged at the bottom end of the electrolytic cell, the electrolytic cell is made of stainless steel and is filled with electrolyte, and a blade of the stirrer extends into the electrolyte and is used for stirring the electrolyte; the thermometer is extended into the electrolyte and used for detecting the temperature of the electrolyte. The titanium substrate is a pure titanium sheet, the pure titanium sheet is used as an anode, the electrolytic cell is used as a cathode, and the anode and the cathode are respectively and electrically connected with the WH-1A micro-arc oxidation power supply.

The titanium substrate can be a screw, as shown in fig. 2 and 3, and after being covered with the Sr/Ag-doped micro-arc oxidation coating of the invention, as shown in fig. 4, in the embodiment of the invention, for the sake of simple operation, a pure titanium sheet is used as the titanium substrate, and the pure titanium sheet is a TA2 disk with the diameter of 14.5mm × 1 mm. The technical scheme of the invention is further explained by combining specific examples.

Example 1 (comparative)

The pure titanium sheet is not treated after being polished by sand paper and is named as Ti group.

Example 2 (comparative)

A method of preparing a coating comprising the steps of:

after the pure titanium sheet is polished by sand paper, micro-arc oxidation treatment is carried out, and the pure titanium sheet is named as M group. The micro-arc oxidation treatment method comprises the following steps: the method comprises the steps of placing electrolyte in an electrolytic cell made of stainless steel by adopting voltage provided by a WH-1A micro-arc oxidation power supply, placing a pure titanium sheet in the electrolyte to serve as an anode, using the electrolytic cell made of stainless steel as a cathode, and arranging a stirrer in the middle to stir the electrolyte so as to ensure that electrolyte in the electrolyte is uniformly distributed. The experiment adopts a constant voltage mode, the forward voltage is 300V, the frequency is 800Hz, the duty ratio is 80%, the treatment time is 10min, the sodium silicate aqueous solution is used as electrolyte, and the concentration of the sodium silicate in the electrolyte is 0.6 mol/L. Enough electrolyte is in the electrolytic cell, and the temperature of the electrolyte is controlled at 37 ℃ under the control of a cooling system. After micro-arc oxidation treatment, the sample is pressurized and washed for 10min by flowing water and then dried.

Example 3 (comparative)

A method of preparing a coating comprising the steps of:

the pure titanium sheet is polished by sand paper and then treated by micro-arc oxidation strontium-doped treatment, wherein the micro-arc oxidation strontium-doped treatment is 20.15 wt%, and is named as M-Sr group. The micro-arc oxidation treatment method comprises the following steps: the method comprises the steps of adopting a voltage provided by a WH-1A micro-arc oxidation power supply, taking a pretreated sample as an anode, taking a stainless steel electrolytic cell as a cathode, arranging a stirrer in the middle to stir an electrolyte, adopting a constant voltage mode in an experiment, taking a forward voltage of 300V, a frequency of 800Hz, a duty ratio of 80%, a treatment time of 10min, taking a strontium acetate aqueous solution as the electrolyte, and pressurizing and washing the sample for 10min by flowing water after the micro-arc oxidation treatment, and then drying the sample.

Example 4

after the pure titanium sheet is polished by sand paper, the micro-arc oxidation strontium-doped silver treatment is carried out, wherein the micro-arc oxidation strontium-doped 21.41 wt% is doped with silver and 0.69 wt% is named as M-SrAg group. The micro-arc oxidation treatment method comprises the following steps: the voltage provided by a WH-1A micro-arc oxidation power supply is adopted, a pretreated sample is taken as an anode, a stainless steel electrolytic tank is taken as a cathode, a constant voltage mode is adopted in an experiment, the forward voltage is 300V, the frequency is 800Hz, the duty ratio is 80%, the treatment time is 10min, a mixed solution of strontium acetate, silver nitrate, chloroplatinic acid (the chloroplatinic acid is taken as a silver catalyst) and water is taken as an electrolyte, and the concentrations of the strontium acetate, the silver nitrate and the chloroplatinic acid in the electrolyte are respectively 0.6mol/L (strontium acetate), 0.001mol/L (silver nitrate) and 0.00005mol/L (chloroplatinic acid) in sequence. After micro-arc oxidation treatment, the sample is pressurized and washed for 10min by flowing water, and then is stored in a dry environment.

The photographs of the samples obtained in examples 1 to 4 are shown in fig. 5, and the coating/Sr/Ag-doped micro-arc oxidation coating of the samples obtained in examples 1 to 4 is shown in fig. 6 when observed under a Scanning Electron Microscope (SEM), as can be seen from the figure, microscopic holes with different sizes are formed on the surface of the coating or the Sr/Ag-doped micro-arc oxidation coating, and the shape of the micro-holes is approximately round or like a crater; the porosity is between 15 percent and 23 percent, and the diameter of each micropore is 0.5 to 3 mu m; the micropores are distributed more uniformly.

The energy spectrum analysis (EDS) of the sample obtained in example 4 enables the detection of simultaneous incorporation of Sr, Ag into the coating (table 1) and uniform distribution in the coating, as shown in fig. 7.

Table 1 surface chemical element content (wt%) of samples obtained in examples 1 to 4 obtained by EDS

As can be seen from Table 1, the Ti group consists of four elements of Ti, O, Al (aluminum) and V (vanadium), wherein the main component is Ti, and O, Al and V are less in content; after the micro-arc oxidation treatment is carried out on the group M, because the electrolyte is sodium silicate, the contents of O and Si on the surface of the film layer are increased and become main components, and Ti is used as the secondary component, but Al and V are less; the M-Sr and M-SrAg groups, the main components of which are Sr (20.15%, 21.41%) in addition to Ti and O due to the addition of strontium acetate in the electrolyte, further confirmed the presence of strontium titanate (SrTiO) in the film layer₃) (ii) a In the M-SrAg group, the film contains Ag element due to the addition of silver nitrate, but the relative content of the Ag element is not high and is 0.69 wt%. EDS shows that Ti, O, Sr and Ag are uniformly distributed in the coating as shown in figure 7.

The X-ray diffraction (XRD) analysis of the samples obtained in examples 1-4 is shown in FIG. 8, in which the titanium in the coating/Sr/Ag-doped micro-arc oxidation coating is mainly TiO₂The Sr element is mainly present in the form of SrTiO₃As shown in the figure, Ti, M,The main phases of the surfaces of the four groups of film layers of M-Sr and M-SrAg are basically consistent, and diffraction peaks of titanium (a) and rutile phase (delta) titanium dioxide are mainly present. In addition to the diffraction peaks, the M group also has an anatase phase (■) titanium dioxide diffraction peak; besides the diffraction peak of anatase phase titanium dioxide on the surface of the M-Sr and M-SrAg group film layer, strontium titanate (SrTiO) is also added due to the addition of strontium acetate₃) Diffraction peak of (. diamond-solid.).

As shown in FIGS. 9 to 11, the roughness R of the Sr/Ag-doped micro-arc oxidation coating obtained in example 4 was measured_aThe value is 1-1.2 μm (figure 9), the contact angle of hydrophilic water is 74.7 +/-1.77 degrees (figure 10), and the values are all superior to medical pure titanium or titanium alloy; the bonding strength of the Sr/Ag doped micro-arc oxidation coating obtained in example 4 was 40.19 + -2.51 MPa (FIG. 11), which is as good as the Sr/Ag undoped micro-arc oxidation coating.

As shown in FIG. 9, the roughness of the M-, M-Sr-, and M-SrAg groups was significantly higher than that of the Ti group, and the M-, M-Sr-, and M-SrAg groups were compared, and the Sr-doped/Ag micro-arc oxidized coating obtained by the M-SrAg group (example 4) was the roughest; the rougher the surface is, the more favorable the growth of osteoblasts is;

as shown in FIG. 10, after the micro-arc oxidation treatment, the hydrophilicity of the surface of the test piece is significantly increased compared with the surface of the test piece without the micro-arc oxidation treatment, and the hydrophilicity of the M group, the M-Sr group and the M-SrAg group is superior to that of the Ti group, and the M-SrAg group has proper hydrophilicity.

The bonding strength of the coating/Sr/Ag doped micro-arc oxidation coating was tested by a coating adhesion scratch tester, and the results are shown in FIG. 11. The binding strength of the M group, the M-Sr group and the M-SrAg group is 40.17 + -2.11 MPa, 40.13 + -2.05 MPa and 40.19 + -2.51 MPa, respectively. The bonding strength of the M-SrAg group is about 40MPa and is better than that of the existing coatings such as Hydroxyapatite (HA), chitosan, beta-tricalcium phosphate and the like (the bonding strength of the existing coatings is mostly less than 20MP), so that compared with the bonding strength of other existing coatings, the Sr-doped/Ag micro-arc oxidation coating prepared by the micro-arc oxidation method HAs the advantages that the bonding strength prepared by the micro-arc oxidation method is not influenced by doping strontium and silver, and the bonding strength is still good.

The MTT method was used to examine the effect of the surface of the samples obtained in examples 1 to 4 on the cell proliferation ability. The results of cell proliferation experiments (fig. 12) show that after the micro-arc oxidation treatment, the proliferation of osteoblasts is obviously promoted, and the proliferation activity of osteoblasts is stronger with the time. On

days

1 and 4, the proliferative activity of the M-SrAg group was significantly higher than that of the Ti group, but there was no significant difference in the effect on osteoblast proliferation among the M group, the M-Sr group, and the M-SrAg group. The incorporation of Sr has little effect on osteoblasts at 1d and 4 d. Sr does not promote osteoblast proliferation as well. On day 7, MTT results showed significant and statistical differences between groups. With the increase of time, the influence of strontium on osteoblasts is gradually increased, and the advantages are more obvious. Thus, the proliferative activity of the M-SrAg group exceeded that of the M group.

As shown in FIGS. 13 and 14, the osteoblast adhesion test showed that the cell adhesion numbers of the M group, the M-Sr group and the M-SrAg group were greater than those of the Ti group, and the Sr/Ag-doped micro-arc oxidation coating layer promoted the early adhesion of osteoblasts compared with medical titanium. Cell adhesion is a prerequisite for the formation of osseointegration, and the state of the material surface determines the adhesion of osteoblasts. The adhesion may be affected by the roughness and hydrophilicity of the material surface. The nuclei of osteoblasts were stained with Propidium Iodide (PI), and the adhesion effect of osteoblasts on the surfaces of the four groups of test pieces was observed under a fluorescence microscope. The M-SrAg group promotes osteoblast early adhesion compared to the Ti group.

As shown in FIG. 15, the Sr/Ag doped micro-arc oxidation coating promoted the formation of the osteogenic differentiation marker alkaline phosphatase (ALP). Alkaline phosphatase (ALP) is one of the important markers for osteoblast maturation with bone forming ability. The effect of the samples of examples 1-4 on the osteogenic differentiation capacity of cells was examined using alkaline phosphatase (ALP) activity. The results show that the alkaline phosphatase activity of the M-SrAg group is highest, which indicates that the M-SrAg group has the function of remarkably promoting the osteogenic differentiation.

Staphylococcus aureus is one of the important bacteria causing inflammation around implants. The Sr/Ag-doped micro-arc oxidation coating/coating obtained in the example 1-4 is detected to have antibacterial activity against staphylococcus aureus by a film pasting method, the detection result is shown in fig. 16, the Sr/Ag-doped micro-arc oxidation coating obtained in the example 4 inhibits the growth of staphylococcus aureus, and the antibacterial rate reaches 99%. Staphylococcus aureus selectionWith an initial concentration of 1X 10⁵The concentration of the CFU/ml bacterial liquid (components: bacteria, peptone, sodium chloride and beef extract powder; purchased from Beijing Omboxing Biotechnology Limited liability company) is higher than that of staphylococcus aureus bacteria detected in peri-implantitis, so that the reliability of the experimental result is ensured.

Examples 5 to 12

A preparation method of the Sr-doped micro-arc oxidation coating comprises the following steps:

the titanium matrix is treated for 10min by a micro-arc oxidation method, and a Sr/Ag-doped micro-arc oxidation coating is formed on the titanium matrix, wherein the titanium matrix is placed in an electrolyte of an electrolytic cell for electrolysis, the titanium matrix is used as an anode, the electrolytic cell made of stainless steel is used as a cathode, the electrolyte is a strontium acetate aqueous solution, and the concentration of strontium acetate in the strontium acetate aqueous solution is shown in Table 2. During the process of treating the titanium matrix by adopting the micro-arc oxidation method, the electrolyte is kept stirred and the temperature of the electrolyte is kept at 37 ℃. Wherein the parameters for processing by adopting the micro-arc oxidation method are as follows: the WH-1A micro-arc oxidation power supply is provided with a constant voltage mode, the forward voltage is 300V, the frequency is 800Hz, and the duty ratio is 80%.

After the titanium substrate was treated by the micro-arc oxidation method, the Sr-doped micro-arc oxidation coating was washed with deionized water under pressure, and vacuum-dried by a vacuum freeze-dryer (VFD-1000, shanghai biran instruments manufacturing ltd).

TABLE 2

In the micro-arc oxidation process, different strontium acetate concentrations (30g/L, 45g/L, 60g/L, 90g/L, 120g/L, 150g/L, 200g/L and 300g/L) are respectively added into the electrolyte, and the Sr content in the coating is measured through energy spectrum analysis (EDS), as shown in figure 17, when the strontium acetate concentration in the electrolyte is 120g/L, the Sr content in the coating is at most 21.01 +/-0.67%, on the basis, the strontium acetate concentration in the electrolyte is increased or decreased, the Sr content in the coating is lower than 21.01 +/-0.67%, and the strontium doping range determined by the method is 4-22 wt%.

Examples 13 to 17

the titanium matrix is treated for 10min by adopting a micro-arc oxidation method, and a Sr/Ag-doped micro-arc oxidation coating is formed on the titanium matrix, wherein the titanium matrix is placed in an electrolyte of an electrolytic cell for electrolysis, the titanium matrix is used as an anode, the electrolytic cell made of a stainless steel material is used as a cathode, the electrolyte is an aqueous solution formed by mixing strontium salt (strontium acetate), chloroplatinic acid and silver salt (silver nitrate), the concentration of the strontium salt in the electrolyte is shown in Table 3, the concentration of the silver salt in the electrolyte is 0.17g/L, and the concentration of the chloroplatinic acid in the electrolyte is 0.021g/L as a catalyst of the silver salt. During the process of treating the titanium matrix by adopting the micro-arc oxidation method, the electrolyte is kept stirred and the temperature of the electrolyte is kept at 37 ℃. Wherein the parameters for processing by adopting the micro-arc oxidation method are as follows: the WH-1A micro-arc oxidation power supply is provided with a constant voltage mode, the forward voltage is 300V, the frequency is 800Hz, and the duty ratio is 80%.

After the titanium substrate was treated by the micro-arc oxidation method, the Sr/Ag-doped micro-arc oxidation coating was washed with deionized water under pressure, and vacuum-dried in a vacuum freeze-dryer (VFD-1000, shanghai belang instruments manufacturing ltd).

TABLE 3

The Sr contents (mass ratio) of the Sr-doped/Ag micro-arc oxidation coating layer were measured to be 0, 4.84 + -0.57%, 15.08 + -0.24%, 18.23 + -0.34%, 20.87 + -0.61% by energy spectrum analysis (EDS), as shown in Table 3.

As shown in FIG. 18, it can be seen that the Sr contents of the MT-Sr60Ag0.17, MT-Sr90Ag0.17 and MT-Sr120Ag0.17 groups promoted osteoblast proliferation compared with the MT-Sr30Ag0.17 groups, but the Sr contents of the groups were not different.

As shown in FIG. 19, it can be seen that MT-Sr90Ag0.17 and MT-Sr120Ag0.17 groups form more bone differentiation promoting marker alkaline phosphatase (ALP). As can be seen from the graph, the more Sr content in the Sr/Ag-doped micro-arc oxidation coating of the MT-Sr30Ag0.17 group, the MT-Sr60Ag0.17 group and the MT-Sr90Ag0.17 group, the more ALP formation is promoted.

Examples 18 to 22

the titanium matrix is treated for 10min by adopting a micro-arc oxidation method, and a Sr/Ag-doped micro-arc oxidation coating is formed on the titanium matrix, wherein the titanium matrix is placed in an electrolyte of an electrolytic cell for electrolysis, the titanium matrix is used as an anode, the electrolytic cell made of stainless steel is used as a cathode, the electrolyte is an aqueous solution formed by mixing strontium salt (strontium acetate), chloroplatinic acid and silver salt (silver nitrate), the concentration of the silver salt in the electrolyte is shown in Table 4, the concentration of the strontium salt in the electrolyte is 90g/L, and the concentration of the chloroplatinic acid in the electrolyte is 0.021g/L as a catalyst of the silver salt. During the process of treating the titanium matrix by adopting the micro-arc oxidation method, the electrolyte is kept stirred and the temperature of the electrolyte is kept at 37 ℃. Wherein the parameters for processing by adopting the micro-arc oxidation method are as follows: the WH-1A micro-arc oxidation power supply is provided with a constant voltage mode, the forward voltage is 300V, the frequency is 800Hz, and the duty ratio is 80%.

TABLE 4

The Ag contents of the Sr-doped/Ag micro-arc oxidation coatings obtained in examples 18 to 22 were measured by Energy Dispersive Spectroscopy (EDS) to be 0, 0.23% + -0.08%, 0.62% + -0.26%, 1.05% + -0.12%, and 1.35% + -0.33%, respectively, as shown in Table 4.

Osteoblast proliferation assays for examples 18-22 are shown in FIG. 20, in which the Sr content in the Ag0, Ag0.04, Ag0.08, Ag0.17 and Ag0.34 groups promoted osteoblast proliferation.

The antibacterial property detection of each group with different silver-doped content (fig. 21, 22) is specifically illustrated by the picture: the more the mass ratio of silver contained in the coating is, the more obvious short-term and long-term antibacterial property is.

As shown in FIG. 21, the Sr/Ag-doped micro-arc oxidation coating layer shows a certain antibacterial property, and the diameter of the antibacterial ring is increased along with the increase of the content of silver.

As shown in fig. 22, short-term antibacterial ratio: the antibacterial rate of the Ag0.04 group is close to 85 percent, the colony number of the Ag0.08 group is obviously reduced, the antibacterial rate is close to 99.98 percent, and the Ag0.17 group and the Ag0.34 group do not see bacterial colonies, and the antibacterial rate reaches 100 percent. Long-term antibacterial rate: the long-term antibacterial rate of the Ag0.17 group and the Ag0.34 group is over 70 percent, and the antibacterial agent shows good short-term and long-term antibacterial properties.

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

Claims

1. The preparation method of the Sr/Ag-doped micro-arc oxidation coating is characterized by comprising the following steps of:

2. The method according to claim 1, characterized in that during the treatment of the titanium substrate by micro-arc oxidation, the electrolyte is kept under agitation and the temperature of the electrolyte is kept below 38 ℃, preferably 30-37 ℃.

3. The preparation method according to claim 2, wherein the parameters for the treatment by the micro-arc oxidation method are as follows: the power supply of the micro-arc oxidation method is provided with a constant voltage mode, the forward voltage is 300V, the frequency is 800Hz, and the duty ratio is 80%.

4. The method for preparing a titanium substrate according to claim 3, wherein after the titanium substrate is treated by the micro-arc oxidation method, the Sr/Ag-doped micro-arc oxidation coating is washed by deionized water and dried.

5. The preparation method according to claim 4, wherein the concentration of strontium salt in the electrolyte is 90-120 g/L, the concentration of silver salt in the electrolyte is 0.08-0.17 g/L, and the concentration of chloroplatinic acid in the electrolyte as a catalyst for silver salt is 0.021 g/L.

6. The Sr/Ag-doped micro-arc oxidation coating obtained by the preparation method according to the claims 1-5.

7. A titanium substrate coated with the Sr/Ag doped micro arc oxidation coating of claim 6.

8. Use of the preparation method according to claims 1 to 5 for promoting osteogenic differentiation.

9. Use of the preparation method according to claims 1 to 5 for improving antibacterial properties.

10. Use of the preparation method according to claims 1 to 5 for increasing osteoblast proliferation activity and promoting osteoblast early adhesion.