阅读说明：本技术 一种具有抗凝血涂层的钛合金支架的制备方法及支架 (Preparation method of titanium alloy stent with anticoagulant coating and stent ) 是由 高飞 徐兰馨 于 2021-09-26 设计创作，主要内容包括：本发明涉及血管支架,提供了一种具有抗凝血涂层的钛合金支架的制备方法,包括如下步骤,(1)将钛合金片样品和钛合金支架样品进行清洗、干燥；(2)对步骤(1)中的钛合金和钛合金支架进行活化处理,使其表面产生羟基基团；(3)将步骤(2)中的样品置于具有抗凝血分子的溶液中,浸泡一段时间后取出,清洗并干燥。本发明还提供了由上述具有抗凝血涂层的钛合金支架的制备方法所制备得到的支架。本发明的具有抗凝血涂层的钛合金支架的制备方法,不仅方法简单,成本低,而且抗凝血涂层具有显著的抗凝血性能。(The invention relates to a vascular stent, and provides a preparation method of a titanium alloy stent with an anticoagulant coating, which comprises the following steps of (1) cleaning and drying a titanium alloy sheet sample and a titanium alloy stent sample; (2) activating the titanium alloy and the titanium alloy bracket in the step (1) to generate hydroxyl groups on the surface; (3) and (3) putting the sample in the step (2) into a solution with anticoagulant molecules, taking out after soaking for a period of time, washing and drying. The invention also provides a stent prepared by the preparation method of the titanium alloy stent with the anticoagulant coating. The preparation method of the titanium alloy stent with the anticoagulation coating is simple in method and low in cost, and the anticoagulation coating has obvious anticoagulation performance.)

1. A preparation method of a titanium alloy stent with an anticoagulant coating is characterized by comprising the following steps: comprises the following steps of (a) carrying out,

(1) cleaning and drying a titanium alloy sheet sample and a titanium alloy bracket sample;

(2) activating the titanium alloy and the titanium alloy bracket in the step (1) to generate hydroxyl groups on the surface;

(3) and (3) putting the sample in the step (2) into a solution with anticoagulant molecules, taking out after soaking for a period of time, washing and drying.

2. The method for preparing a titanium alloy stent with an anticoagulant coating according to claim 1, wherein the method comprises the following steps: the titanium alloy in the step (1) is one of nickel titanium, nickel iron titanium and nickel niobium titanium.

3. The method for preparing a titanium alloy stent with an anticoagulant coating according to claim 1, wherein the method comprises the following steps: the activation treatment in the step (2) is to place the sample in a hydrogen peroxide solution and irradiate the sample under ultraviolet light.

4. The method for preparing a titanium alloy stent with an anticoagulant coating according to claim 3, wherein the method comprises the following steps: the mass fraction of the hydrogen peroxide in the step (2) is 5%, the wavelength of the ultraviolet light is 254nm, and the treatment time is 5-7 h.

5. The method for preparing a titanium alloy stent with an anticoagulant coating according to claim 1, wherein the method comprises the following steps: the activation treatment in the step (2) is to place the sample in an alkaline solution and heat-treat.

6. The method for preparing a titanium alloy stent with an anticoagulant coating according to claim 5, wherein the method comprises the following steps: the alkaline solution in the step (2) is a sodium hydroxide solution; the mass fraction of the alkaline solution is 0.01-0.1 g/mL; the heating method is water bath at 50-60 deg.C for 1.5-2.5 h.

7. The method for preparing a titanium alloy stent with an anticoagulant coating according to claim 1, wherein the method comprises the following steps: the anticoagulant molecules in the step (3) are one or more of tannic acid, coumarin, citric acid and gallic acid.

8. The method for preparing a titanium alloy stent with an anticoagulant coating according to claim 7, wherein the method comprises the following steps: the concentration of the anticoagulant molecules in the step (3) is 0.01-0.05mg/mL, the soaking temperature is 35-45 ℃, and the soaking time is 1.5-2.5 h.

9. The stent prepared by the method for preparing the titanium alloy stent with the anticoagulant coating according to any one of claims 1 to 8.

Technical Field

The invention relates to the technical field of intravascular stents, in particular to a preparation method of a titanium alloy stent with an anticoagulant coating and the stent.

Background

The current main measure for treating intracranial aneurysm is to implant a dense mesh stent, which is a blood flow guiding device and is mainly woven by nickel-titanium alloy or cobalt-chromium alloy wires, after the dense mesh stent is implanted into a parent artery, the blood flow rate and flow of an affected part can be changed, thrombus is gradually formed in the aneurysm, the re-endothelialization of the blood vessel at the aneurysm is promoted, and the normal function of the blood vessel is remodeled. Is currently the "second choice" for treating intracranial aneurysms. However, with the development of clinical practice, activation and adhesion of blood platelets, fibrinogen and other substances are likely to be induced in the early stage of stent implantation, and thrombus formation and even vascular restenosis occur on the stent. Resulting in insufficient blood supply to the brain of the patient, even cerebral infarction, etc. Because double-antibody is needed to be taken for anticoagulation treatment after implantation, the risk of bleeding caused by unknown reasons is increased, and the double-antibody is the main reason for death after implantation of the dense mesh stent.

Therefore, surface modification of dense mesh stents to improve their anticoagulation, reduce the risk of restenosis and reduce the probability of late bleeding is the hot spot of current modification studies on dense mesh stents. The construction of an anticoagulant micromolecule functional coating on a dense net bracket is reported at home and abroad; constructing hydrophilic polymer coating, etc. However, the problems of less research, insignificant modification effect and the like still exist.

Disclosure of Invention

The invention aims to provide a preparation method of a titanium alloy stent with an anticoagulation coating, which is simple and low in cost, and the anticoagulation coating has obvious anticoagulation performance.

The embodiment of the invention is realized by the following technical scheme: the preparation method of the titanium alloy stent with the anticoagulation coating comprises the following steps,

(1) cleaning and drying a titanium alloy sheet sample and a titanium alloy bracket sample;

(2) activating the titanium alloy and the titanium alloy bracket in the step (1) to generate hydroxyl groups on the surface;

(3) and (3) putting the sample in the step (2) into a solution with anticoagulant molecules, taking out after soaking for a period of time, washing and drying.

Further, the titanium alloy in the step (1) is one of nickel titanium, nickel iron titanium and nickel niobium titanium.

Further, the activation treatment in step (2) is to place the sample in a hydrogen peroxide solution and irradiate under ultraviolet light. More specifically, the mass fraction of the hydrogen peroxide is 5%, the wavelength of the ultraviolet light is 254nm, and the treatment time is 5-7 h.

Further, the activation treatment in the step (2) is to place the sample in an alkaline solution and heat-treat. More specifically, the alkaline solution is a sodium hydroxide solution; the mass fraction of the alkaline solution is 0.01-0.1 g/mL; the heating method is water bath at 50-60 deg.C for 1.5-2.5 h.

Further, the anticoagulant molecules in the step (3) are one or more of tannic acid, coumarin, citric acid and gallic acid. More specifically, the concentration of anticoagulant molecules is 0.01-0.05mg/mL, the soaking temperature is 35-45 ℃, and the soaking time is 1.5-2.5 h.

The invention also provides a stent prepared by the preparation method of the titanium alloy stent with the anticoagulant coating.

The technical scheme of the embodiment of the invention at least has the following advantages and beneficial effects: the preparation method of the titanium alloy stent with the anticoagulant coating comprises the steps of exposing a large number of active groups such as hydroxyl on the surface of a metal substrate by carrying out alkali activation treatment or ultraviolet irradiation on the surface of nickel-titanium metal, and inducing the generation of titanium dioxide micromolecules of a substrate, wherein the anticoagulant molecules are combined with the substrate to form a stable functional coating based on the principles of self-assembly and covalent grafting, and the stable functional coating and the titanium dioxide micromolecules formed by the substrate exert the anticoagulant function together.

Drawings

FIG. 1 is a result of demonstrating hydrophilicity before and after modification of a dense mesh scaffold in test example 1 of the present invention;

FIG. 2 is a graph showing the fluorescent staining effect and morphology of the modified nickel-titanium alloy sheet of test example 2 before and after platelet adhesion;

FIG. 3 is a morphology chart of an experimental optical mirror and an electron microscope of the modified front half body and the rear half body of the dense mesh support in experimental example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

The preparation method of the titanium alloy stent with the anticoagulant coating comprises the following steps,

(1) cleaning and drying a nickel-titanium alloy sheet sample and a nickel-titanium alloy bracket sample;

(2) putting the sample in the step (1) in 5% hydrogen peroxide, and irradiating for 5 hours under 254nm ultraviolet light;

(3) and (3) putting the sample in the step (2) into 0.01mg/mL tannic acid solution, soaking at 35 ℃, taking out after soaking for 1.5h, cleaning and drying.

Example 2

The preparation method of the titanium alloy stent with the anticoagulant coating comprises the following steps,

(1) cleaning and drying a nickel-titanium alloy sheet sample and a nickel-titanium alloy bracket sample;

(2) putting the sample in the step (1) in 5% hydrogen peroxide, and irradiating for 7h under 254nm ultraviolet light;

(3) and (3) putting the sample in the step (2) into 0.1mg/mL tannic acid solution, soaking at the temperature of 45 ℃, taking out after soaking for 2.5h, washing and drying.

Example 3

The preparation method of the titanium alloy stent with the anticoagulant coating in the embodiment is basically the same as that of the embodiment 1, except that the nickel-titanium alloy in the step (1) of the embodiment 1 is replaced by the nickel-iron-titanium alloy.

Example 4

The preparation method of the titanium alloy stent with the anticoagulant coating in the embodiment is basically the same as that of the embodiment 1, except that the nickel-titanium alloy in the step (1) of the embodiment 1 is replaced by the nickel-niobium-titanium alloy.

Example 5

The preparation method of the titanium alloy stent with the anticoagulant coating comprises the following steps,

(1) cleaning and drying a nickel-titanium alloy sheet sample and a nickel-titanium alloy bracket sample;

(2) putting the sample in the step (1) into 0.01mg/mL sodium hydroxide solution, and heating for 1.5h in a water bath at 50 ℃;

(3) and (3) putting the sample in the step (2) into 0.01mg/mL tannic acid solution, soaking at 35 ℃, taking out after soaking for 1.5h, cleaning and drying.

Example 6

The preparation method of the titanium alloy stent with the anticoagulant coating comprises the following steps,

(1) cleaning and drying a nickel-titanium alloy sheet sample and a nickel-titanium alloy bracket sample;

(2) putting the sample in the step (1) into 0.1mg/mL sodium hydroxide solution, and heating for 1.5h in a water bath at 60 ℃;

(3) and (3) putting the sample in the step (2) into 0.01mg/mL tannic acid solution, soaking at 35 ℃, taking out after soaking for 1.5h, cleaning and drying.

Example 7

The preparation method of the titanium alloy stent with the anticoagulant coating comprises the following steps,

(1) cleaning and drying a nickel-titanium alloy sheet sample and a nickel-titanium alloy bracket sample;

(2) putting the sample in the step (1) into 0.1mg/mL sodium hydroxide solution, and heating for 1.5h in a water bath at 60 ℃;

(3) and (3) putting the sample in the step (2) into 0.05mg/mL tannic acid solution, soaking at the temperature of 45 ℃, taking out after soaking for 2.5h, cleaning and drying.

Example 8

The preparation method of the titanium alloy stent with the anticoagulant coating in the embodiment is basically the same as that in the embodiment 7, except that the tannic acid in the step (3) is replaced by coumarin.

Example 9

The preparation method of the titanium alloy stent with the anticoagulant coating in the embodiment is basically the same as that of the embodiment 7, except that the tannic acid in the step (3) is replaced by citric acid.

Example 10

The preparation method of the titanium alloy stent with the anticoagulation coating in the embodiment is basically the same as that in the embodiment 7, except that the gallic acid is replaced by the tannic acid in the step (3).

Test examples 1 to 3

Experimental examples 1-3 the samples prepared in example 1 were used as experimental samples and were designated NiTi @ TA (where TA represents tannic acid). A sample of bare, untreated nickel titanium alloy was used as a control and was designated NiTi.

Test example 1

The UP water drop experiments are respectively carried out on the experimental sample and the reference sample, the results are shown in the attached drawing 1, and it can be seen from the attached drawing 1 that the nickel-titanium alloy dense-mesh stent after modification shows better hydrophilicity, water drops on the surface of the sample after modification can be uniformly spread, which fully shows that the prepared anticoagulation coating can improve the hydrophilicity of the stent.

Test example 2

And (3) verifying the in vitro platelet adhesion and activation condition of the anticoagulant functional coating. The experimental procedure was as follows,

(1) the blood used in this example was donated to an unknown volunteer. Mixing fresh blood with 3.8% sodium citrate at a ratio of 10:1, centrifuging in a centrifuge at 1500rpm for 15min, and collecting supernatant to obtain Rich plasma (PRP);

(2) adding the prepared samples into a 24-well plate, dropwise adding 100 mu L of enriched plasma on the surface of each sample, and incubating for 1h in a constant-temperature incubator at 37 ℃;

(3) taking out the incubated sample, washing the incubated sample for three times by using 0.9% sodium chloride, and fixing the incubated sample for 4 hours by using 2.5% glutaraldehyde;

in this embodiment, the adhesion morphology and activation condition of platelets are observed by immunofluorescence staining and scanning electron microscopy, and the immunofluorescence staining comprises the following steps:

(1) washing the fixed sample for 3 times by using 0.9% sodium chloride;

(2) dripping 70 μ L of Rhodamine (Rhodamine-pharaoidin) solution on the surface of each sample, and standing for 15min in a dark condition;

(3) the stained sample was washed 3 times with 0.9% sodium chloride and blown dry and observed under a fluorescence microscope.

In this example, the steps of scanning and observing platelets are as follows:

(1) and (3) carrying out dehydration treatment on the sample: putting the sample in 50%, 75%, 90% and 100% alcohol solution in sequence, each time for 15 min;

(2) dealcoholizing treatment: placing the sample in 50%, 75%, 90% and 100% isoamyl acetate solutions in sequence, each time for 15 min;

(3) drying at critical point, spraying gold, and observing by scanning electron microscope.

The sample surface platelet adhesion fluorescent staining prepared in this example is shown as part a in fig. 2, and it can be seen that a large number of platelets adhere to the surface of the unmodified nickel-titanium alloy sheet, and the modified sample surface has little or no platelet adhesion, which fully indicates that the modified anticoagulant coating has a good anticoagulant effect.

The scanning electron microscope for platelet adhesion on the surface of the sample prepared in this example is shown in part B of fig. 2, a large amount of platelets adhere to the surface of the unmodified sample, the surface of the modified sample is smooth and flat, and almost no platelets adhere to the surface of the sample, and the result is consistent with fluorescent staining. The result shows that the anticoagulation micromolecule modified coating has good anticoagulation capacity.

Test example 3

And verifying the anticoagulation performance of the prepared anticoagulation coating in an animal model. The model selected in the experiment is a rabbit model, and the experiment is carried out by the following steps:

(1) healthy New Zealand white rabbits were selected as models. Preparing an anesthetic with a certain concentration, injecting the anesthetic with a corresponding dose into the rabbit body through the ear margin vein part according to an equivalent ratio of the volume to the weight of the rabbit of 1mL:1kg, and anesthetizing the rabbit. The hair removal treatment is carried out on the skin of the rabbit where the neck arteriovenous is positioned, so that the following operation is convenient.

(2) Putting the nickel-titanium dense net bracket sample into an assembled PVC four-way catheter, injecting physiological saline into the assembled PVC four-way catheter until the assembled PVC four-way catheter is full of saline, flicking the catheter, removing air bubbles remained in the catheter, and clamping two ends of the catheter by hemostatic clamps for later use.

(3) After the skin of the rabbit neck is cut open, the muscle tissue is stripped layer by layer until the artery and vein blood vessels are exposed. Carefully separating out blood vessels, infiltrating a sterile indwelling needle, penetrating the infiltrated sterile indwelling needle into the stripped and fixed neck artery and vein, and then respectively connecting the indwelling needles at two ends of the arteriovenous with two ends of the assembled four-way catheter to form a closed blood circulation loop with the blood vessels in the white rabbit body.

(4) After taking down the hemostatic forceps at the two ends of the artery and vein, the blood flows into the four-way pipe with the sample from the white rabbit and circulates in a reciprocating way, the time is recorded as an experiment starting node, the artery and vein are fixed by the vascular forceps after the experiment is started for 0.5h, the blood circulation is stopped, the catheter device is taken down, and the physiological saline is used for flushing. The experimental sample is taken out and fixed in glutaraldehyde (2.5%) solution, and then the appearance is observed.

The experimental rear optical mirror and the scanning electron microscope of the half body of the nickel-titanium alloy dense mesh stent manufactured by the embodiment are shown in the attached figure 3, the optical mirror shows that the blockage of the unmodified nickel-titanium alloy dense mesh stent is serious, the modified dense mesh stent is unblocked as before, the electron microscope shows that the surface of the unmodified dense mesh stent has more substance adhesion, the modified dense mesh stent is opposite, and the result shows that the anticoagulation modified coating has good anticoagulation capacity.

In conclusion, the invention constructs the modified micromolecule functional coating with the anticoagulation function, which comprises natural micromolecule tannic acid and coumarin existing in the plant body, commonly used alkali activation and ultraviolet irradiation are adopted for material surface pretreatment, and the anticoagulation micromolecules are fixed on the substrate based on the principles of self-assembly and covalent grafting, so that the coating with the anticoagulation function is formed.

Different from the traditional medicine-carrying coating system, the covalent grafting of the invention ensures that the micromolecules fixed on the surface are more stable and firmer, can realize the long-acting functionalization of the surface, does not relate to the medicine release kinetics, and is simpler and more effective. The invention adopts the anticoagulation micromolecule which can inhibit the adhesion and activation of the blood platelet and has good anticoagulation effect;

the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.