CN101411893A - Titanium or titanium alloy material with modified surface as well as preparation method and use thereof - Google Patents

Abstract

The invention discloses a surface modified titanium or titanium alloy material. The surface of the material is provided with at least five layers of bioactive coats consisting of double-layer chitosan-silk fibroin. The material has favorable osteoplast compatibility, can promote multiplication and division of osteoplast, shows potential for further developing a novel medical hard tissue implant material, and has favorable clinical application prospect. The surface modified titanium or titanium alloy material can be prepared by a self-assembly method layer by layer; alternate layers of polycation electrolyte chitosan and polyanion electrolyte gelatin are laminated and assembled on the surface of the titanium or titanium alloy material through electrostatic adsorption action; and the preparation method is simple, can control coating thickness, is not affected by size and shape of the material, is suitable for the materials with complex appearance, has moderate preparation conditions, needs no special equipment, and has low production cost, strong practicability, and better popularization and application values.

Description

Surface-modified titanium or titanium alloy material and its preparation method and application

technical field

The invention relates to a metal material, in particular to a surface-modified titanium or titanium alloy material, and to a preparation method and application of the surface-modified titanium or titanium alloy material.

Background technique

At present, bone defects caused by trauma, fractures, osteoporosis and other diseases are common clinical problems. Every year, millions of hard tissue implants are used for bone fixation or functional tissue replacement around the world. Due to their excellent biocompatibility and comprehensive mechanical properties, titanium and titanium alloys have become the most widely used hard tissue implant materials in clinical practice, and are used to make artificial joints, internal fixation devices and bone reconstruction materials. However, titanium and titanium alloy materials themselves lack biological activity and have no ability to induce osseointegration. Implants made of them cannot form a firm and long-term stable combination with surrounding bone tissue after implantation, and loosening and dislocation often occur . Therefore, the integration and long-term stability between titanium and titanium alloy implants and bone tissue are major clinical challenges. An ideal bone tissue implant should have the ability to "actively" rather than "passively" interact with the surrounding tissues of the host, such as promoting the proliferation and differentiation of osteoblasts.

In view of the shortcomings of titanium and titanium alloy materials, surface modification by surface engineering methods is expected to greatly improve their comprehensive performance, so that they are more suitable for medical applications. In recent years, scholars at home and abroad have carried out a lot of work on the surface modification of titanium and titanium alloy materials. The general idea is to generate organic (such as protein, enzyme, etc.) or inorganic (such as hydroxyapatite, Titanium dioxide, etc.) bioactive coatings. Surface modification methods are roughly divided into three categories: physical modification methods, chemical modification methods, and biochemical methods. Physical modification methods such as plasma spraying methods have high production costs, and the prepared hydroxyapatite coating is physically bonded to titanium and titanium alloy materials, which has the disadvantages of weak bonding force and poor stability, and most Part of the spraying is linear spraying, which is easy to cause problems such as uneven coating thickness for titanium and titanium alloy materials with complex shapes. Chemical modification methods such as acid-base treatment are highly corrosive to titanium and titanium alloy materials, and the obtained titanium dioxide coating has a loose structure, and the bonding firmness needs to be improved. The biochemical method is to bind organic polymer substances such as proteins or enzymes to the surface of titanium and titanium alloy materials through physical adsorption, chemical bonding and carrier attachment. Its advantage is that it can directly and effectively improve the biological activity of titanium and titanium alloy materials. , but a lot of research is needed on how to ensure the biological activity of the protein or enzyme after the reaction. So far, no surface-modified titanium and titanium alloy materials have been successfully applied clinically.

Contents of the invention

In view of this, in order to overcome the deficiencies in the prior art, one of the purposes of the present invention is to provide a surface-modified titanium or titanium alloy material, which has good osteoblast compatibility and can promote the proliferation and differentiation.

To achieve this purpose, the surface-modified titanium or titanium alloy material of the present invention has a bioactive coating on the surface of the titanium or titanium alloy material, and the bioactive coating consists of at least 5 layers of chitosan-silk fibroin Composed of two layers.

Further, the bioactive coating is composed of 5 to 10 layers of chitosan-silk fibroin bilayers;

Further, the bioactive coating is composed of 6 layers of chitosan-silk fibroin bilayers.

The second object of the present invention is to provide a method for preparing the surface-modified titanium or titanium alloy material, which is not affected by the size and shape of the material, has simple operation, mild conditions, no need for special equipment, and low production cost.

To achieve this purpose, the method for preparing the surface-modified titanium or titanium alloy material of the present invention comprises the following steps:

a, take chitosan, add the acetic acid solution that concentration is 10～30mL/L, stir to dissolve, make the chitosan solution that mass percent concentration is 1%～4%;

b. Weigh the silk fibroin, add deionized water to dissolve it, and make a silk fibroin solution with a concentration of 2% to 10% by mass;

c. Weigh 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and morpholineethanesulfonic acid (MES), add deionized water to dissolve, and make A mixed solution containing EDC with a concentration of 0.05% to 2% by mass and MES with a concentration of 0.05 to 0.3mol/L;

d, put the titanium or titanium alloy material in the chitosan solution prepared in step a, soak for 10-20 minutes, and take it out;

e. Fully washing the titanium or titanium alloy material treated in step d with deionized water;

f. Put the titanium or titanium alloy material cleaned in step e into the silk fibroin solution prepared in step b, soak for 10-20 minutes, and take it out;

g, fully washing the titanium or titanium alloy material treated in step f with deionized water;

h, repeat steps d to g at least 4 times, make the chitosan-silk fibroin bilayer of at least 5 layers self-assembled on the surface of titanium or titanium alloy material layer by layer;

i. Put the titanium or titanium alloy material treated in step h into the EDC-MES mixed solution prepared in step c, and react at a temperature of 4-20°C for 20-60 minutes to obtain surface-modified titanium or titanium Alloy materials.

Further, the mass percent concentration of the chitosan solution in the step a is 2%, and the soaking time in the step d is 20 minutes;

Further, the mass percent concentration of the silk fibroin solution in the step b is 5%, and the soaking time in the step f is 10 minutes;

Further, the mixed solution in step c contains EDC with a concentration of 0.1% by mass and MES with a concentration of 0.1 mol/L, and the reaction temperature in step i is 4° C. and the reaction time is 30 minutes.

Further, the step h is to repeat steps d to g 4 to 9 times, so that the surface of the titanium or titanium alloy material is self-assembled layer by layer with 5 to 10 layers of chitosan-silk fibroin bilayer;

Further, the step h is to repeat steps d to g 5 times, so that the surface of the titanium or titanium alloy material is self-assembled layer by layer with 6 layers of chitosan-silk fibroin bilayers.

The third object of the present invention is to provide the application of the surface-modified titanium or titanium alloy material in the preparation of medical hard tissue implant materials.

The beneficial effects of the present invention are: the present invention discloses a surface-modified titanium or titanium alloy material, which has a bioactive coating consisting of at least five layers of chitosan-silk fibroin double layers on the surface of the material. It has good osteoblast compatibility, can promote the proliferation and differentiation of osteoblasts, and has shown the potential to be further developed into a new type of medical hard tissue implant material, and has good clinical application prospects; the surface-modified titanium of the present invention Or titanium alloy materials can be prepared by layer-by-layer self-assembly method, and polycationic electrolyte chitosan and polyanionic electrolyte silk fibroin are alternately stacked and assembled on the surface of titanium or titanium alloy materials through electrostatic adsorption. The preparation method is simple and the coating The thickness is controllable, not affected by the size and shape of the material, suitable for materials with complex shapes, and the preparation conditions are mild, no special equipment is required, the production cost is low, the practicability is strong, and it has good promotion and application value.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with the accompanying drawings, wherein:

Fig. 1 is a schematic diagram of the proliferation of osteoblasts over time on the surface-modified titanium film of the present invention;

Figure 2 is a schematic diagram of the differentiation of osteoblasts over time on the surface-modified titanium membrane of the present invention.

Detailed ways

Chitosan is a product of deacetylation of chitin in the shell of crustaceans, insects and other invertebrates. It is a natural polycation polysaccharide (pKa=6.3) with good film-forming properties and biocompatibility. , biodegradability, and a variety of biological activities such as broad-spectrum antibacterial, promoting epithelial cell growth, inhibiting smooth muscle hyperplasia, etc., are widely used in tissue engineering, drug release and other fields. Silk fibroin is a natural high-molecular protein derived from silk (pI=4.22). It contains 18 kinds of amino acids, 11 of which are essential amino acids for human body. It has excellent biocompatibility and mechanical properties and can be slowly degraded. , with the in-depth study of its unique amino acid composition and crystal structure and other physical and chemical properties, the application of silk fibroin in the field of biomedical materials is becoming more and more extensive and in-depth. The present invention selects chitosan and silk fibroin as polycation and polyanion electrolyte respectively, utilizes the electrostatic interaction between polycation/anion, adopts the method for layer-by-layer self-assembly to carry out the surface modification of titanium or titanium alloy material, and to The prepared surface-modified titanium or titanium alloy materials were identified for biological activity.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Raw materials: chitosan (deacetylation degree 95%, molecular weight 200,000, Qingdao Haihui Biological Products Co., Ltd.); flat titanium film (deposited by electron beam evaporation, surface roughness about 2.5nm) and Ti-6Al-4V titanium alloy (Northwest Institute of Nonferrous Metals); silk fibroin is self-made, and the preparation method is as follows: take natural silk by weighing, under the condition of temperature 100 ℃, degumming 2 times with the sodium bicarbonate solution that concentration is 5mL/L, every time 30 minutes, use After fully washing with deionized water, add calcium chloride-ethanol-water mixed solution with a molar ratio of 1:2:8, stir for 6 hours at a temperature of 70°C to dissolve, then dialyze with deionized water for 3 days, freeze-dry , to produce silk fibroin.

Embodiment one

This embodiment is a surface-modified titanium film, and its preparation method comprises the following steps:

A, take by weighing chitosan 0.5g, adding concentration is 50mL of acetic acid solution of 10mL/L, magnetic stirring makes dissolving for 20 minutes, makes the chitosan solution that mass percent concentration is 1%;

b. Weigh 1.0 g of silk fibroin, add 50 mL of deionized water to dissolve it, and make a silk fibroin solution with a concentration of 2% by mass;

c. Weigh 0.025g of EDC and 0.533g of MES, add 50mL of deionized water to dissolve, and make a mixed solution containing EDC with a concentration of 0.05% by mass and MES with a concentration of 0.05mol/L;

d, the titanium film is placed in the chitosan solution prepared in step a, soaked for 10 minutes, and taken out;

e. Washing the titanium membrane processed in step d twice with deionized water, soaking for 2 minutes each time;

f. Put the titanium film cleaned in step e into the silk fibroin solution prepared in step b, soak for 20 minutes, and take it out;

g, cleaning the titanium film processed by step f with deionized water twice, soaking for 2 minutes each time;

h, repeat steps d to g4 times, make the chitosan-silk fibroin bilayer of 5 layers on the surface of titanium film self-assembled layer by layer;

i. Put the titanium film treated in step h into the EDC-MES mixed solution prepared in step c, and react at a temperature of 10° C. for 60 minutes to prepare a surface-modified titanium film.

Embodiment two

This embodiment is a surface-modified titanium film, and its preparation method comprises the following steps:

A, take chitosan 1.0g, add concentration and be 50mL of acetic acid solution of 10mL/L, magnetic stirring makes dissolving for 20 minutes, makes the chitosan solution that mass percent concentration is 2%;

b. Weigh 2.5 g of silk fibroin, add 50 mL of deionized water to dissolve, and make a silk fibroin solution with a concentration of 5% by mass;

c. Weigh 0.050g of EDC and 1.066g of MES, add 50mL of deionized water to dissolve, and make a mixed solution containing EDC with a mass percentage concentration of 0.1% and MES with a concentration of 0.1mol/L;

d, the titanium film is placed in the chitosan solution prepared in step a, soaked for 20 minutes, and taken out;

e. Washing the titanium film treated in step d with deionized water 3 times, soaking for 1 minute each time;

f. Put the titanium film cleaned in step e into the silk fibroin solution prepared in step b, soak for 10 minutes, and take it out;

g, cleaning the titanium film processed by step f with deionized water 3 times, soaking for 1 minute each time;

h, repeat step c to f5 times, make the chitosan-silk fibroin bilayer of 6 layers on the surface of titanium film self-assembled layer by layer;

i. Put the titanium film treated in step h into the EDC-MES mixed solution prepared in step c, and react at a temperature of 4°C for 30 minutes to prepare a surface-modified titanium film.

Embodiment Three

Present embodiment is the Ti-6Al-4V titanium alloy of surface modification, and its preparation method comprises the following steps:

A, take chitosan 1.5g, add concentration and be 50mL of the acetic acid solution of 20mL/L, magnetic stirring makes dissolving for 20 minutes, makes the chitosan solution that mass percent concentration is 3%;

b. Weigh 4.0 g of silk fibroin, add 50 mL of deionized water to dissolve it, and make a silk fibroin solution with a concentration of 8% by mass;

c. Weigh 0.075g of EDC and 2.133g of MES, add 50mL of deionized water to dissolve, and make a mixed solution containing EDC with a mass percentage concentration of 0.15% and MES with a concentration of 0.2mol/L;

d, put titanium alloy in the chitosan solution that step a makes, soak for 15 minutes, take out;

e, cleaning the titanium alloy processed by step d with deionized water 3 times, soaking for 1 minute each time;

f. Put the titanium alloy cleaned in step e into the silk fibroin solution prepared in step b, soak for 15 minutes, and take it out;

g, cleaning the titanium alloy processed through step f with deionized water 3 times, soaking for 1 minute each time;

h, repeat steps c to f7 times, make the chitosan-silk fibroin bilayer of 8 layers self-assembled layer by layer on the titanium film surface;

i. Put the titanium film treated in step h into the EDC-MES mixed solution prepared in step c, and react at a temperature of 15° C. for 45 minutes to prepare a surface-modified titanium film.

Embodiment four

Present embodiment is the Ti-6Al-4V titanium alloy of surface modification, and its preparation method comprises the following steps:

A, take chitosan 2.0g, add concentration and be 50mL of acetic acid solution of 30mL/L, magnetic stirring makes dissolving for 20 minutes, makes the chitosan solution that mass percent concentration is 4%;

b. Weigh 5.0 g of silk fibroin, add 50 mL of deionized water to dissolve, and make a silk fibroin solution with a concentration of 10% by mass;

c. Weigh 0.100g of EDC and 3.199g of MES, add 50mL of deionized water to dissolve, and make a mixed solution containing EDC with a concentration of 0.2% by mass and MES with a concentration of 0.3mol/L;

d, the titanium film is placed in the chitosan solution prepared in step a, soaked for 20 minutes, and taken out;

e. Washing the titanium film treated in step d with deionized water 4 times, soaking for 1 minute each time;

f. Put the titanium film cleaned in step e into the silk fibroin solution prepared in step b, soak for 20 minutes, and take it out;

g. Washing the titanium film processed by step f with deionized water 4 times, soaking for 1 minute each time;

h, repeat steps c to f9 times, make the chitosan-silk fibroin bilayer of 10 layers self-assembled layer by layer on the titanium film surface;

i. Put the titanium film treated in step h into the EDC-MES mixed solution prepared in step c, and react at a temperature of 20° C. for 20 minutes to prepare a surface-modified titanium film.

Bioactivity identification of the surface-modified titanium or titanium alloy material of the present invention

1. Identification of proliferation ability of osteoblasts

Method: Mouse calvarial primary osteoblasts were inoculated on the surface of unmodified titanium membrane, the surface of the surface-modified titanium membrane of the present invention and polystyrene cell culture well plates (tissue culture polystyrenes, TCPS). Density is 5000/cm ² , with DMEM culture solution containing fetal bovine serum at a concentration of 100mL/L, cultured at a temperature of 37°C and a CO ₂ gas concentration of 50mL/m ³ , and the culture solution is replaced every 3 days; On the 1st, 4th, and 7th day of culture, the cells were digested with a trypsin solution with a concentration of 2.5 mg/mL, and the cells were counted with a Z1-type Coulter Counter (Beckman Coulter GmbH, Germany) to detect cell proliferation. ability;

Results: As shown in Figure 1, compared with the unmodified titanium film and TCPS, the surface-modified titanium film of the present invention has good osteoblast compatibility and can promote the proliferation of osteoblasts.

2. Identification of osteoblast differentiation ability

Method: Primary osteoblasts from the mouse skull were inoculated on the surface of the unmodified titanium membrane, the surface of the surface-modified titanium membrane of the present invention and in TCPS respectively, the cell density was 5000/cm ² , and the concentration was 100mL/ The DMEM culture fluid of L fetal bovine serum was cultivated under the conditions of temperature 37°C and CO ₂ gas concentration of 50mL/m ³ , and the culture fluid was changed every 3 days; After digesting mL of trypsin solution, centrifuge, add Triton X-100 (Triton X-100) solution with a concentration of 10mL/L to the cell pellet, and repeat "freezing-room temperature thawing" for a total of 3 times to obtain a cell lysate First, use the BCA protein quantification kit (Sigma Company, U.S.) to measure the protein content of cells, then add the substrate nitrophenylphosphate to react, and use a microplate counter to measure the synthesis of nitrophenol at a wavelength of 450nm. The number of micromoles of nitrophenol synthesized in minutes "μmol nitrophenol/min/mg protein" indicates the activity of alkaline phosphatase (ALP) and detects the ability of cell differentiation;

Results: As shown in Figure 2, compared with the unmodified titanium membrane and TCPS, the surface-modified titanium membrane of the present invention has good osteoblast compatibility and can promote the differentiation of osteoblasts.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described with reference to the preferred embodiments of the present invention, those skilled in the art should understand that it can be described in the form Various changes may be made in matter and details thereof without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (19)

1, the titanium of surface modification or titanium alloy material, the surperficial biologically active coating at titanium or titanium alloy material is characterized in that: described bioactivity coatings is formed by chitosan-fibroin albumen of at least 5 layers is double-deck.

2, the titanium of surface modification according to claim 1 or titanium alloy material is characterized in that: described bioactivity coatings is formed by chitosan-fibroin albumen of 5～10 layers is double-deck.

3, the titanium of surface modification according to claim 1 or titanium alloy material is characterized in that: described bioactivity coatings is formed by chitosan-fibroin albumen of 6 layers is double-deck.

4, the titanium of the described surface modification of preparation claim 1 or the method for titanium alloy material may further comprise the steps:

A, take by weighing chitosan, adding concentration is the acetic acid solution of 10～30mL/L, stirs and makes dissolving, makes mass percentage concentration and be 1%～4% chitosan solution;

B, take by weighing fibroin albumen, add deionized water and make dissolving, make mass percentage concentration and be 2%～10% silk fibroin protein solution;

C, take by weighing 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) and morpholino b acid

(MES), add deionized water and make dissolving, make contain mass percentage concentration be 0.05%～2% EDC and

Concentration is the mixed solution of the MES of 0.05～0.3mol/L;

D, titanium or titanium alloy material are put in the chitosan solution that step a makes, soaked 10～20 minutes, take out;

E, titanium or the titanium alloy material deionized water thorough washing that will handle through steps d;

F, the titanium that will clean through step e or titanium alloy material are put in the silk fibroin protein solution that step b makes, and soak taking-up 10～20 minutes;

G, titanium or the titanium alloy material deionized water thorough washing that will handle through step f;

H, repeating step d to g at least 4 times make in the surface self assembly successively of titanium or titanium alloy material chitosan-fibroin albumen bilayer of at least 5 layers;

I, the titanium that will handle through step h or titanium alloy material are put in the EDC-MES mixed solution that step c makes, and react 20～60 minutes under 4～20 ℃ of conditions of temperature, promptly make the titanium or the titanium alloy material of surface modification.

5, the method for the titanium of preparation surface modification according to claim 4 or titanium alloy material is characterized in that: the mass percentage concentration of chitosan solution is 2% among the described step a, and soak time is 20 minutes in the described steps d.

6, the method for the titanium of preparation surface modification according to claim 4 or titanium alloy material is characterized in that: the mass percentage concentration of silk fibroin protein solution is 5% among the described step b, and soak time is 10 minutes among the described step f.

7, the method for the titanium of preparation surface modification according to claim 4 or titanium alloy material, it is characterized in that: to contain mass percentage concentration be that 0.1% EDC and concentration are the MES of 0.1mol/L to mixed solution among the described step c, and reaction temperature is that 4 ℃, response time are 30 minutes in the described step I.

8, the method for the titanium of preparation surface modification according to claim 4 or titanium alloy material, it is characterized in that: described step h is for repeating steps d to g4～9 time, makes in the surface self assembly successively of titanium or titanium alloy material chitosan-fibroin albumen bilayer of 5～10 layers.

9, the method for the titanium of preparation surface modification according to claim 8 or titanium alloy material is characterized in that: described step h is for repeating steps d to g5 time, makes in the surface self assembly successively of titanium or titanium alloy material chitosan-fibroin albumen bilayer of 6 layers.

10, the titanium of the described surface modification of claim 1 or the titanium alloy material application in the medical hard tissue implanting material of preparation.

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