CN112064090B

CN112064090B - Microstructure surface preparation method with high blood compatibility and microstructure

Info

Publication number: CN112064090B
Application number: CN202010974036.5A
Authority: CN
Inventors: 张松; 张静; 李东海; 满佳; 李剑峰
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-09-16
Filing date: 2020-09-16
Publication date: 2022-02-08
Anticipated expiration: 2040-09-16
Also published as: CN112064090A

Abstract

The invention discloses a microstructure surface preparation method with high blood compatibility and a microstructure, and the technical scheme is as follows: polishing and cleaning a pure titanium sample; carrying out acid etching treatment on the polished sample, and carrying out ultrasonic cleaning and high-temperature drying on the sample subjected to the acid etching treatment; carrying out primary anodic oxidation treatment on the dried sample to generate TiO on the surface of the sample₂A nanotube layer; putting the sample obtained by the primary anodic oxidation into deionized water for ultrasonic treatment until the TiO on the surface of the sample₂The nanotube layer is completely peeled off; cleaning and drying the sample after the oxide layer falls off; and (3) carrying out secondary anodic oxidation treatment on the sample by adopting electrolyte different from the primary anodic oxidation, and cleaning and drying the sample after the oxidation treatment to obtain the surface of the honeycomb-shaped nano-pore structure. The method can accurately prepare the regular microstructure based on the protein scale on the surface of the pure titanium, and realizes the modification of the medical pure titanium surface with high blood compatibility.

Description

Microstructure surface preparation method with high blood compatibility and microstructure

Technical Field

The invention relates to the field of medical pure titanium processing, in particular to a microstructure surface preparation method with high blood compatibility and a microstructure.

Background

Titanium and titanium alloy are widely applied to some implanted/interventional medical devices, such as cardiovascular stents, guide wires, artificial heart pumps and the like, as a blood compatible material. However, in the case of implant materials, a series of adverse clinical reactions, such as thrombosis, hemolysis, tissue inflammation and other clinical phenomena, occur when blood comes into contact with the implant material. Among these clinical symptoms, thrombosis is one of the most dangerous clinical conditions. Once the thrombus formed on the surface of the implant falls off and enters the blood circulation system, it will block the blood flow and cause some serious clinical reactions such as tissue ischemia, infarction and even death. Thrombosis is a complex multiple cascade of reactions, in which the first time the implant material comes into contact with blood is the adhesion of blood proteins, such as fibrin, globulin and other procoagulant proteins. Then, the protein adhered to the surface of the material undergoes a conformational change, such as the conversion of fibrinogen to fibrin filaments by the action of thrombin. These conformationally altered blood proteins will provide a large number of blood cell binding targets, which in turn will cause activation and adhesion of blood cells (platelets) in the blood, eventually resulting in a thrombus on the surface of the implant.

In recent years, in order to improve the blood compatibility of titanium and titanium alloys, some researchers have studied different surface modification techniques, such as chemical treatment, sol-gel, anodic oxidation, chemical vapor deposition, biomacromolecule grafting, thermal spraying, physical vapor deposition, and ion implantation. The purpose of these modification techniques is to prepare micro-nano composite structures on the surface of titanium and titanium alloy and introduce biomolecules and elements to realize the change of physical and chemical properties. Among them, the microstructure of the material surface is an important factor affecting the blood compatibility of the material. Depending on the coagulation mechanism, at different microscopic scales, factors such as the shape and size of these surface microstructures will exhibit different results in platelet adhesion, aggregation, deformation, and adsorption of blood proteins. However, considering that blood protein is used as a trigger factor of blood coagulation reaction, how to precisely regulate and control the size of the surface microstructure based on the protein scale, further influencing the adhesion property of the material surface to protein, and reducing the adhesion and activation of platelets on the material surface, so that the preparation of titanium and titanium alloy surfaces with high blood compatibility still has great challenges.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a microstructure surface preparation method and a microstructure with high blood compatibility, which can accurately prepare a regular microstructure based on protein scale on the surface of pure titanium and realize the modification of the surface of medical pure titanium with high blood compatibility.

In order to achieve the purpose, the invention is realized by the following technical scheme:

in a first aspect, embodiments of the present invention provide a method for preparing a microstructure surface with high blood compatibility, including:

polishing and cleaning a pure titanium sample;

carrying out acid etching treatment on the polished sample, and carrying out ultrasonic cleaning and high-temperature drying on the sample subjected to the acid etching treatment;

carrying out primary anodic oxidation treatment on the dried sample to generate TiO on the surface of the sample₂A nanotube layer;

putting the sample obtained by the primary anodic oxidation into deionized water for ultrasonic treatment until the TiO on the surface of the sample₂The nanotube layer is completely peeled off; cleaning and drying the sample after the oxide layer falls off;

and (3) carrying out secondary anodic oxidation treatment on the sample by adopting electrolyte different from the primary anodic oxidation, and cleaning and drying the sample after the oxidation treatment to obtain the surface of the honeycomb-shaped nano-pore structure.

As a further implementation mode, the polishing and cleaning comprises the steps of carrying out hot inlaying, physical polishing and ultrasonic cleaning on a pure titanium sample, wherein the hot inlaying temperature is 120-130 ℃, and the inlaying time is 6-8 minutes; physically polishing until the surface roughness of the sample reaches Ra0.2-Ra0.3 mu m; and during ultrasonic cleaning, taking the sample out of the mosaic block, and respectively putting the sample into acetone, absolute ethyl alcohol and deionized water for cleaning for a set time.

As a further implementation manner, during the acid etching treatment, the polished sample is placed in the mixed acid solution with the set concentration, and the mixed acid solution containing the sample is placed in the ultrasonic cleaning equipment, and the acid etching treatment is combined with the ultrasonic oscillation treatment for the set time, so that the removal of the surface oxidation layer is realized.

As a further implementation, the mixed acid solution comprises the following components: 0.2-0.8wt% of HF, 0.6-1.4wt% of HNO₃And the balance of deionized water.

As a further implementation mode, the primary anodic oxidation treatment process comprises the following steps: putting the dried sample after acid etching treatment into a reaction chamber containing NH₄F. Deionized water andcarrying out anodic oxidation treatment in the electrolyte prepared by ethylene glycol to generate TiO₂A nanotube layer; wherein, the sample is used as an anode, and the graphite sheet is used as a cathode; and continuously stirring the electrolyte at a constant speed in the anodic oxidation process.

As a further implementation mode, the electrolyte for the secondary anodic oxidation treatment is a mixed solution of phosphoric acid and ethylene glycol, and the electrolyte is continuously stirred at a constant speed in the anodic oxidation process.

As a further implementation, the electrolyte of the primary anodic oxidation treatment comprises the following components: 0.2-0.8wt% NH₄F, 1.0-5.0vol% of deionized water, and the balance of ethylene glycol; the electrolyte for secondary oxidation treatment comprises the following components: 4.0-6.0wt% of H₃PO₄And the balance being ethylene glycol.

As a further implementation mode, the anodic oxidation power supply is a direct-current stabilized power supply, and the specified oxidation voltage is 20-100V in consideration of uniform and controllable growth of the primary anodic oxidation nano-tubes and the secondary anodic oxidation honeycomb-shaped nano-holes; the primary oxidation time is 0.5-1.5h, and the secondary oxidation time is 1-10 min.

As a further implementation mode, the surface of the obtained honeycomb-shaped nano-pore structure is subjected to annealing treatment and then is cooled along with the furnace.

In a second aspect, the embodiment of the present invention further provides a microstructure with high blood compatibility, and the microstructure is prepared by the preparation method.

The beneficial effects of the above-mentioned embodiment of the present invention are as follows:

(1) one or more embodiments of the invention can accurately regulate and control the size of a surface microstructure based on protein scale, and reduce the adhesion and activation of platelets on the surface of a material, thereby preparing titanium and titanium alloy surfaces with high blood compatibility;

(2) one or more embodiments of the invention adopt a low-cost, easy-to-operate anodic oxidation technology, and realize the precise regulation and preparation of a regular honeycomb structure on a medical pure titanium matrix by adopting different electrolyte components; the size of the prepared regular structure can be accurately controlled within the blood protein scale, and feasibility is provided for researching the material microstructure and the blood protein adhesion mechanism.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a flow diagram in accordance with one or more embodiments of the invention;

FIG. 2 is a schematic illustration of anodization according to one or more embodiments of the present invention;

FIG. 3 is a schematic illustration of nanotube generation in accordance with one or more embodiments of the invention;

FIG. 4 is a diagram of a cellular nanostructure without annealing treatment according to one or more embodiments of the invention;

FIG. 5 is a honeycomb nanostructured surface after a 450 ℃ annealing process according to one or more embodiments of the present invention;

FIG. 6 is a graph of platelet adhesion results for a titanium substrate surface according to one or more embodiments of the present invention;

FIG. 7 is an enlarged view of platelet adhesion results for a titanium substrate surface according to one or more embodiments of the present invention;

FIG. 8 is a graph of cellular nanostructure surface platelet adhesion results after 450 ℃ annealing treatment according to one or more embodiments of the invention;

FIG. 9 is an enlarged view of cellular nanostructure surface platelet adhesion results after 450 ℃ annealing treatment according to one or more embodiments of the invention;

wherein, 1, anode, 2, cathode, 3, electrolyte, 4, magnetic stirrer.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an", and/or "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof;

the first embodiment is as follows:

the present embodiment provides a method for preparing a microstructure surface with high blood compatibility, as shown in fig. 1, comprising the following steps:

cleaning and polishing a sample in the step (1):

firstly, a pure titanium (BT1-00, purity not less than 99.8%) sample is prepared, in this example, the sample size is 12mm multiplied by 0.2mm, of course, in other examples, the test size can be other values, and can be selected according to actual requirements. And carrying out thermal inlaying, physical polishing and ultrasonic cleaning on the sample, wherein the thermal inlaying temperature is 120-130 ℃ and the inlaying time is 6-8 minutes in consideration of the firmness of the inlaying block. Preferably, the mosaic temperature is 130 ℃.

The physical polishing process comprises the following steps: and sequentially polishing the test piece by using 1200-mesh and 2000-mesh sandpaper, and then respectively polishing by using diamond polishing agents with the particle sizes of 3.5 mu m and 1.5 mu m until the surface roughness of the test piece reaches Ra0.2-0.3 mu m. The ultrasonic cleaning process comprises the following steps: and taking out the sample from the mosaic block, and respectively putting the sample into acetone, absolute ethyl alcohol and deionized water for cleaning for 10-15 min.

And (2) acid etching treatment:

and (3) putting the polished sample into a mixed acid solution with a specific concentration, putting the mixed acid solution containing the sample into ultrasonic cleaning equipment, and combining the acid etching treatment process with ultrasonic oscillation treatment for 3-5min to remove the surface oxide layer.

Wherein, the parameters of the ultrasonic oscillation are as follows: the power is 240W, and the ultrasonic frequency is 40 KHz. After acid etching treatment, the sample is respectively put into acetone, absolute ethyl alcohol and deionized water for ultrasonic cleaning for 8-12 minutes, and then dried for 3-5 minutes at 50-60 ℃. Preferably, the ultrasonic cleaning time is 10 min.

The mixed acid solution comprises the following components: 0.2-0.8wt% of HF, 0.6-1.4wt% of HNO₃And the balance of deionized water. Preferably, the mixed acid solution component comprises 0.66 wt% HF, 0.8wt% HNO₃And the balance being deionized water.

Step (3), primary anodic oxidation treatment:

putting the dried sample after acid etching treatment into a reaction chamber containing NH₄F. Performing anodic oxidation treatment in electrolyte prepared from deionized water and ethylene glycol to obtain TiO as shown in FIG. 3₂And (4) a nanotube layer.

Specifically, as shown in fig. 2, the sample dried by acid etching was used as an anode 1, a graphite sheet was used as a cathode 2, and the area ratio of the cathode 2 to the anode 1 was 1.5: 1. The anode 1 and the cathode 2 are put into the electrolyte 3, and the cathode 2 and the anode 1 are connected with a power supply through a lead. The magnetic stirrer 4 is arranged at the bottom of the container for containing the electrolyte 3.

The power supply is a direct-current stabilized power supply, the oxidation voltage is 20-100V, and the oxidation time is 0.5-1.5 h. In the process of anodic oxidation, a magnetic stirrer 4 is adopted to continuously stir the electrolyte 3 at a constant speed. The electrolyte 3 comprises the following components: 0.2-0.8wt% NH₄F, 1.0-5.0vol% of deionized water, and the balance of ethylene glycol. Preferably, the electrolyte 3 comprises 0.5 wt% NH₄F (analytical grade), 2 vol% deionized water, and the balance ethylene glycol.

Step (4), stripping an oxide layer:

putting a sample obtained by the primary anodic oxidation into deionized water for ultrasonic treatment, wherein the parameters of the ultrasonic treatment are as follows: the power is 240W, the ultrasonic frequency is 40KHz, and the processing time is 10-20 min; until TiO on the surface of the sample₂The nanotube layer is completely peeled off. Then, the completely peeled sample was put into deionized water to be washed and dried.

And (5) carrying out secondary anodic oxidation treatment:

the electrolyte in the secondary oxidation is a mixed solution of phosphoric acid and ethylene glycol, the oxidation time is 1-10min, and the electricity is oxidizedThe pressure is 20-100V. And in the anodic oxidation process, a magnetic stirrer is adopted to continuously stir the electrolyte at a constant speed. And after the oxidation is finished, putting the sample into deionized water for soaking and cleaning, and then taking out and naturally drying to obtain the surface of the regular hexagon honeycomb nanopore structure shown in fig. 4. Preferably, the electrolyte in the secondary oxidation is 5.0 wt% H₃PO₄The rest is the oxidation time of the ethylene glycol is 3min, and the oxidation voltage is 50V.

And (6) annealing treatment: and annealing the surface of the obtained regular hexagonal honeycomb nano-pore structure at the annealing temperature of 300-800 ℃ for 1-3h, and finally cooling along with the furnace. Preferably, the annealing temperature is 450 ℃, the annealing time is 2h, and finally furnace cooling is carried out, and the microstructure after annealing is shown in FIG. 5. The adhesion results of the platelets on the surface of the honeycomb nanostructure after the annealing treatment at 450 ℃ are shown in fig. 8-9.

And (7) selecting fresh anticoagulated blood to evaluate the anti-platelet adhesion property of the prepared honeycomb nanostructure surface. Fig. 6-7 show the platelet adhesion results of the titanium substrate surface of this example.

First, all samples were sterilized with 75% ethanol, and then after being soaked in Phosphate Buffered Saline (PBS) for 30 minutes, the samples were taken out and dried in the air. Then, the samples were placed in 6-well plates (3 pure titanium substrates, 3 annealed samples), and 400. mu.l of whole blood was added to each sample surface. Then putting the mixture into a constant-temperature incubator for incubation under the culture condition of 37 ℃ and the humidity of 60 percent for 2 hours.

Next, the sample was taken out, and the blood cells not adhering to the surface were removed by washing 3 times with PBS. Subsequently, the samples were placed in new 6-well plates, 2ml of 2.5% glutaraldehyde solution was pipetted by a disposable pipette, and dropped into the 6-well plates to fix the blood cells adhered to the surfaces of the samples, and fixed at 4 ℃ for 4 hours. The fixed samples were then removed and placed in pre-numbered 6-well plates. The samples were dehydrated by standing using gradient ethanol solutions (50%, 70%, 80%, 90% and 100%), respectively, for 15min for each gradient.

And finally, putting the sample together with the 6-hole plate into a freeze drying box for freeze drying, drying for 13 hours at the cold trap temperature of-50 ℃ under the condition of 20Pa, taking out the sample, spraying gold, and observing by using an SEM.

In the embodiment, regular nano structures based on blood protein size are prepared on the surface of medical pure titanium, and the preparation of nano structures with different sizes can be conveniently realized by regulating and controlling anodic oxidation voltage and time; the regular honeycomb structure prepared on the surface of the medical pure titanium shows excellent anti-platelet adhesion property, and a new idea is provided for improving the blood compatibility of the medical pure titanium.

Example two:

this example provides a microstructure with high blood compatibility, made using the preparation method described in example one.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a microstructure surface having high blood compatibility, comprising:

polishing and cleaning a pure titanium sample;

carrying out acid etching treatment on the polished sample, and carrying out ultrasonic cleaning and high-temperature drying on the sample subjected to the acid etching treatment; the high temperature range is 50-60 ℃;

carrying out secondary anodic oxidation treatment on the sample by adopting electrolyte different from the primary anodic oxidation, and cleaning and drying the sample after the oxidation treatment to obtain the surface of the cellular nanopore structure;

the electrolyte for the secondary anodic oxidation treatment is a mixed solution of phosphoric acid and ethylene glycol, and the anodic oxidation process is carried out

Continuously stirring the electrolyte at a constant speed;

the primary anodic oxidation treatment process comprises the following steps: putting the dried sample after acid etching treatment into a reaction chamber containing NH₄F. Carrying out anodic oxidation treatment in electrolyte prepared by deionized water and ethylene glycol to generate TiO₂A nanotube layer; wherein, the sample is used as an anode, and the graphite sheet is used as a cathode; continuously stirring the electrolyte at a constant speed in the anodic oxidation process;

the electrolyte for primary anodic oxidation treatment comprises the following components: 0.2-0.8wt% NH₄F, 1.0-5.0vol% of deionized water, and the balance of ethylene glycol; the electrolyte for secondary oxidation treatment comprises the following components: 4.0-6.0wt% of H₃PO₄The balance being ethylene glycol;

the anode oxidation power supply is a direct-current stabilized power supply, and the oxidation voltage is 20-100V; the primary oxidation time is 0.5-1.5h, and the secondary oxidation time is 1-10 min;

and annealing the surface of the obtained honeycomb-shaped nano pore structure, and then cooling along with the furnace.

2. The method for preparing the microstructure surface with high blood compatibility according to claim 1, wherein the polishing and cleaning comprises thermal inlaying, physical polishing and ultrasonic cleaning of a pure titanium sample, wherein the thermal inlaying temperature is 120-130 ℃, and the inlaying time is 6-8 minutes; physically polishing until the surface roughness of the sample reaches Ra0.2-Ra0.3 mu m; and during ultrasonic cleaning, taking the sample out of the mosaic block, and respectively putting the sample into acetone, absolute ethyl alcohol and deionized water for cleaning for a set time.

3. The method for preparing a microstructure surface having high blood compatibility according to claim 1, wherein the acid etching treatment is performed by putting the polished sample into a mixed acid solution having a set concentration and putting the mixed acid solution containing the sample into an ultrasonic cleaning device, and the acid etching treatment is combined with the ultrasonic oscillation treatment for a set time to remove the surface oxide layer.

4. The method for preparing a microstructure surface having high hemocompatibility according to claim 3, wherein the mixed acid solution comprises the following components: 0.2-0.8wt% of HF, 0.6-1.4wt% of HNO₃And the balance of deionized water.

5. Microstructure with high hemocompatibility, characterized in that it is produced by the production method according to any one of claims 1 to 4.