Method for the production of a one-step coated titanium material with bone-coating abalone shell water-soluble matrix

Abstract

The application discloses a preparation method of a one-step coated abalone shell water-soluble matrix promoting bone-coated titanium material, which can be used in the field of bone repair. The method is as follows: dopamine is oxidized into polydopamine, the abalone shell water-soluble matrix is coated and co-deposited on the surface of titanium, and the modified titanium with the function of promoting bone is prepared by using the biomimetic technology, so that Ca / P is deposited. The modified titanium has the functions of promoting bone, strong interface bonding property of the modified titanium surface and natural tissue, good mineralization capacity, biocompatibility and the function of promoting bone, and is expected to become a new type of bone repair material.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a preparation method and application of a titanium material coated with an abalone shell water-soluble matrix to promote a bone coating in one step. BACKGROUND

[0002] Titanium has unique properties, such as the highest biocompatibility (compared with other metals), high mechanical properties (such as low Young's modulus, high yield strength, high tensile strength and high elongation), excellent corrosion resistance and relatively light weight. It is widely used in the field of bone repair. However, titanium has low bioactivity, poor osteogenesis and bone conduction performance, and poor integration with the body after implantation, and even requires secondary implantation. In order to improve the bioactivity of titanium, the surface of titanium needs to be modified to improve the bone-promoting performance of the surface of titanium.

[0003] The nacre layer of abalone shell is composed of 1-5 wt% of organic matter and 95 wt% of aragonite calcium carbonate crystals, and is a biocompatible, biodegradable and bone-conducting material. The abalone shell water-soluble matrix (WSM) is mainly composed of glycoprotein, protein sugar and soluble protein matrix, and is an acidic substance. It can control the deposition of aragonite calcium carbonate crystals and control the size, number and structure of calcium carbonate crystals. In the present application, dopamine is oxidized to generate polydopamine, which not only plays an adhesive role, but also reacts with the abalone shell water-soluble matrix to form a Schiff base, and the two are co-deposited on the surface of titanium to enhance the mineralization and bone formation ability of the surface of titanium. SUMMARY

[0004] In order to solve the problems of the existing materials, the present application aims to provide a preparation method of a titanium material coated with an abalone shell water-soluble matrix to promote a bone coating in one step, which can uniformly load biological active factors on the surface of titanium-based materials in situ simply and efficiently. The titanium-based material prepared has excellent biocompatibility and bone-promoting performance, and the raw materials are widely available and easy to operate.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] A titanium-based material coated with an abalone shell water-soluble matrix to promote a bone coating in one step, which utilizes dopamine to oxidize and generate macromolecular polydopamine to coat the abalone shell water-soluble matrix, and co-deposits and adheres to the surface of titanium corroded by sodium hydroxide, which can effectively improve the Ca / P element deposition capacity of the surface of titanium and the bone-promoting performance of the titanium-based material, and is applied in the field of bone repair.

[0007] The preparation method of the titanium-based material coated with an abalone shell water-soluble matrix to promote a bone coating in one step specifically comprises the following steps:

[0008] (1) Soak the titanium sheet in ultrapure water, acetone and ethanol in sequence, ultrasonic for 10 min, and dry overnight to obtain clean cp-Ti.

[0009] (2) Take 2 g of sodium hydroxide and add 10 ml of pure water, immerse the cp-Ti obtained in step (1) into the sodium hydroxide solution, and heat in an oven at 60 DEG C for 24 h.

[0010] (3) Take out the titanium sheet etched by alkali in step (2) and immerse it in pure water for cleaning three times, and dry overnight to obtain TiOH.

[0011] (4) Prepare 100 ml of 10 mmol / L tris (hydroxymethyl) aminomethane solution, and adjust the pH to 8.5, then add 0.2 g of dopamine hydrochloride, and then add different amounts of water-soluble matrix of abalone shell, stir at room temperature for 30 min, and immerse the TiOH obtained in step (3) therein.

[0012] (5) Take out the titanium sheet immersed in step (4) and rinse it with water three times, and dry at 40 DEG C overnight to obtain TiOH / PDA / WSM.

[0013] (5) Take out the titanium sheet immersed in step (4) and rinse it with water three times, and dry at 40 DEG C overnight to obtain TiOH / PDA / WSM.

[0014] The ultrasonic power in step (1) is 40 kHz, and the overnight drying temperature is 40 DEG C.

[0015] The immersion cleaning time in step (3) is 10 min, and the overnight drying temperature is 40 DEG C.

[0016] The amount of water-soluble matrix of abalone shell in step (4) is 0.4 g-0.6 g.

[0017] The present application takes pure titanium as the main material, generates polydopamine by self-polymerization of dopamine hydrochloride under alkaline conditions, and at the same time can coat and adhere the water-soluble matrix of abalone shell to the titanium surface, improve the Ca / P element deposition capacity of the titanium surface, improve the bone promoting performance of titanium, and enhance the combination ability with bone tissue.

[0018] The titanium coated with the water-soluble matrix of abalone shell bone promoting coating has the following advantages compared with other titanium surface modification methods:

[0019] (1) Under alkaline conditions, dopamine can self-polymerize to generate polydopamine, which can coat and connect the water-soluble matrix of abalone shell to the titanium surface, and make it uniformly distributed, thereby improving the biocompatibility of titanium;

[0020] (2) Polydopamine and water-soluble matrix of abalone shell can effectively regulate Ca / P element deposition, accelerate mineralization, promote the conversion of calcium phosphate to hydroxyapatite, and improve the bone promoting performance of the material;

[0021] (3) The PDA surface contains a large number of active functional groups such as hydroxyl and amino groups, which can provide the required active groups for secondary graft modification, and can also promote cell adhesion, proliferation and anchoring of active substances;

[0022] (4) The surface modification method has a wide material source, low price and simple and easy manufacturing process. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the infrared spectrum of the reaction of the material used in examples 3 and 4;

[0024] Figure 2 is the surface contact angle of the titanium sheet obtained in examples 1, 2, 3 and 4;

[0025] Figure 3 is the SEM image of the titanium sheet obtained in examples 1, 2, 3 and 4 (a, b, c and d are magnified 2400 X, e, f, g and h are magnified 100000 X);

[0026] Figure 4 is the SEM image of the titanium sheet obtained in examples 1, 2, 3 and 4 after mineralization for three days (a, b, c and d are magnified 100000 X);

[0027] Figure 5 is the result of cck-8 staining of the titanium sheet obtained in examples 1, 3 and 4 co-cultured with MC3T3 cells (the scale is 100 μm);

[0028] Figure 6 is the result of AO / EB staining of the titanium sheet obtained in examples 1, 3 and 4 co-cultured with MC3T3 cells (the scale is 100 μm);

[0029] Figure 7 is the result of alizarin red staining of the titanium sheet obtained in examples 1, 3 and 4 co-cultured with BMSCs cells (the scale is 25 μm);

[0030] Figure 8 is the result of alkaline phosphatase staining of the titanium sheet obtained in examples 1, 3 and 4 co-cultured with BMSCs cells (the scale is 25 μm). DETAILED DESCRIPTION

[0031] In order to make the content of the application more convenient to understand, the technical solutions of the application will be further described below in combination with specific embodiments, but the application is not limited to this.

[0032] The abalone shell water-soluble matrix is prepared according to the preparation method in the patent (CN104940025A Abalone Shell Water-soluble Matrix and Preparation Method and Application Thereof).

[0033] Example 1 (pure titanium)

[0034] Titanium pieces were sequentially immersed in 10 ml ultrapure water, acetone, ethanol, ultrasonic for 10 min, and dried at 40°C overnight to obtain clean cp-Ti.

[0035] Example 2

[0036] (1) Titanium pieces were sequentially immersed in 10 ml ultrapure water, acetone, ethanol, ultrasonic for 10 min, and dried overnight to obtain clean cp-Ti.

[0037] (2) 2 g of sodium hydroxide was weighed and added to 10 ml of pure water, and the cp-Ti obtained in step (1) was immersed in the sodium hydroxide solution and heated in an oven at 60°C for 24 h.

[0038] (3) The titanium pieces etched by alkali in step (2) were taken out and washed with pure water three times, and dried overnight to obtain TiOH.

[0039] Example 3

[0040] (1) Titanium pieces were sequentially immersed in 10 ml ultrapure water, acetone, ethanol, ultrasonic for 10 min, and dried overnight to obtain clean cp-Ti.

[0041] (2) 2 g of sodium hydroxide was weighed and added to 10 ml of pure water, and the cp-Ti obtained in step (1) was immersed in the sodium hydroxide solution and heated in an oven at 60°C for 24 h.

[0042] (3) The titanium pieces etched by alkali in step (2) were taken out and washed with pure water three times, and dried overnight to obtain TiOH.

[0043] (4) A solution of 100 ml of 10 mmol / L tris(hydroxymethyl) aminomethane was prepared, and the pH was adjusted to 8.5, 0.2 g of dopamine hydrochloride was added, and 0.4 g of abalone shell water-soluble matrix was added, and stirred at room temperature for 30 min, and the TiOH obtained in step (3) was immersed therein.

[0044] (5) The titanium pieces immersed in step (4) were taken out, washed with water three times, and dried at 40°C overnight to obtain TiOH / PDA / WSM-L.

[0045] (5) The titanium pieces immersed in step (4) were taken out, washed with water three times, and dried at 40°C overnight to obtain TiOH / PDA / WSM-L.

[0046] Example 4

[0047] (1) Titanium pieces were sequentially immersed in 10 ml ultrapure water, acetone, ethanol, ultrasonic for 10 min, and dried overnight to obtain clean cp-Ti.

[0048] (2) Weigh 2 g of sodium hydroxide, add it to 10 ml of pure water, immerse the cp-Ti obtained in step (1) in the sodium hydroxide solution, and heat it in an oven at 60°C for 24 h.

[0049] (3) Take out the titanium sheet after alkaline etching in step (2), soak and wash it three times with pure water, and dry it overnight to obtain TiOH.

[0050] (4) Prepare 100 ml of 10 mmol / L tris(hydroxymethyl)aminomethane solution and adjust the pH to 8.5, then add...

[0051] Add 0.2 g of dopamine hydrochloride and 0.6 g of abalone shell water-soluble matrix, stir at room temperature for 30 min, and then soak the TiOH obtained in step (3) in it.

[0052] (5) Take out the titanium sheet soaked in step (4), rinse it three times with water, and dry it overnight at 40°C to obtain TiOH / PDA / WSM-H.

[0053] Figure 1 This is the infrared spectrum of PDA, WSM, and their reaction products, at 1514 cm⁻¹. -1 The characteristic peaks are attributed to the carbon-carbon double bonds in the benzene ring and were not observed in the abalone shell water-soluble matrix, indicating that the product contains dopamine; 1139 cm⁻¹ -1 The characteristic peaks are attributed to carbon-nitrogen single bonds and are not observed in dopamine, indicating that the product contains abalone shell water-soluble matrix.

[0054] Depend on Figure 2 It is known that the Ti plate is hydrophobic and has the largest contact angle. After coating the Ti plate with PDA / WSM, its surface contact angle decreased significantly, indicating that the PDA / WSM coating improved the hydrophilicity of the Ti plate. Compared with the original Ti plate, the hydrophilic PDA / WSM coating facilitates cell adhesion.

[0055] Depend on Figure 3 It can be seen that the PDA / WSM coating adheres uniformly to the surface of the titanium material. The catechol structure in the coating can interact with the Ti surface through the chelation between metal atoms and phenolic hydroxyl groups, so the coating adheres stably to the Ti surface.

[0056] Figure 4 This is a simulated body fluid mineralization result. As shown in the figure, after three days of mineralization, there was almost no Ca / P element deposition on the Ti and TiOH surfaces, and no hydroxyapatite was formed. However, the PDA / WSM coating helps to deposit Ca... 2+ and PO4 3- Aggregation and ion exchange on the surface of titanium materials can promote the nucleation of hydroxyapatite, thereby generating a fully covered mineralized layer.

[0057] Figure 5 and Figure 6 are the cell proliferation and cell morphology after staining by co-culturing the titanium material with MC3T3 cells. The PDA / WSM coating promotes the proliferation of MC3T3 cells, and the promoting effect becomes stronger as the WSM content increases. The cells co-cultured with the modified titanium material have good morphology, and basically no dead cells exist, proving that the modified titanium material has good biocompatibility.

[0058] Figure 7 and Figure 8 are the results of alizarin red staining and alkaline phosphatase staining of BMSCs cells co-cultured with the titanium material, and the number of red calcium nodules shown in Figure 7 and the number of alkaline phosphatase dyed blue in Figure 8 show that the mineralization ability and osteogenesis-promoting ability of BMSCs cells co-cultured with the modified titanium material are stronger than those of BMSCs cells co-cultured with the Ti plate, and the promoting effect becomes stronger as the WSM content increases, indicating that the PDA / WSM coating can improve the mineralization ability and osteogenesis-promoting ability of cells near the wound, thereby enhancing the bonding between the titanium material and the bone tissue and promoting bone repair.

[0059] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

Claims

1. A method for the production of a one-step coated scallop shell water-soluble substrate-bone-promoting coated titanium material, characterized by: The titanium material is prepared by coating the abalone shell water-soluble matrix with dopamine, inducing self-polymerization of hydrochloric acid dopamine in an alkaline environment, having strong adhesion, and uniformly loading the abalone shell water-soluble matrix on the titanium surface to in-situ biomimetic construct the titanium material modified by polydopamine and the abalone shell water-soluble matrix. The method comprises the following steps: (1) titanium pieces are sequentially immersed in ultrapure water, acetone and ethanol, and ultrasonic treatment is performed for 10 min, and then dried overnight; (2) 2 g of sodium hydroxide is weighed and added into 10 ml of pure water, and then the titanium pieces treated in step (1) are immersed in the sodium hydroxide solution, and then placed in an oven for heating at 60℃ for 24 h; (3) the titanium pieces treated by alkali etching in step (2) are taken out and immersed in pure water for cleaning three times, and then dried overnight; (4) 100 ml of 10 mmol / L tris(hydroxymethyl) aminomethane solution is prepared, and the pH is adjusted to 8.5, then 0.2 g of dopamine hydrochloride is added, and then the abalone shell water-soluble matrix is added, and then stirred at room temperature for 30 min, and then the titanium pieces obtained in step (3) are immersed in the solution; (5) the titanium pieces immersed in step (4) are taken out, washed with water for three times, and then dried at 40℃ overnight to obtain the titanium material.

2. The production method according to claim 1, characterized by: In step (1), the ultrasonic power is 40 kHz, and the overnight drying temperature is 40℃.

3. The method of claim 1, wherein: In step (3), the immersion and cleaning time is 10 min, and the overnight drying temperature is 40℃.

4. The production method according to claim 1, characterized by: In step (4), the amount of the abalone shell water-soluble matrix is 0.4 g-0.6 g.

5. The titanium material prepared by the method according to any one of claims 1-4 is applied in the field of bone repair materials.

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