A method for preparing composite coating on titanium surface

Abstract

The invention discloses a method for preparing a composite coating on a titanium surface, belongs to the technical field of medical implant materials, and relates to a titanium implant. The steps are: preparation of a regenerated silk protein solution; pretreatment of medical pure titanium to obtain an acid-pretreated titanium sheet pTi, placing it in a Piranha solution, oxidation treatment to construct a hydroxylated titanium Ti-OH with an oxide layer rich in hydroxyl groups, and reacting it in a hydrochloric acid dopamine solution; preparation of a silk protein-derived peptide; adding Cs powder to an SF solution, then dropping it onto a polydopamine-modified titanium surface, drying it for electrodeposition, and then washing it with deionized water to obtain. The composite coating of the present invention has good compatibility with mesenchymal stem cells, can improve the biological activity and osteogenic activity of the pure titanium surface, has a wide range of clinical application potential, a relatively simple preparation process, a low production cost, and a good value for promotion and application.

Description

Technical Field

[0001] The invention belongs to the technical field of medical implant materials, relates to a titanium implant, and specifically relates to a method for preparing a composite coating on a titanium surface. Background Art

[0002] Titanium and its alloys have low density, high strength, good toughness, non-toxicity, low elastic modulus, good corrosion resistance, low X-ray absorption rate, etc., so they play an increasingly important role in medical metal materials and have been widely used in clinical practice. However, due to the lack of sufficient biological activity of titanium implants, the interaction with the host bone is still mainly mechanical interlocking, and the initial stability and long-term success rate are still unsatisfactory.

[0003] According to the theory of osseointegration, the key to successful implantation is to form a good osseointegration between the implant and the host tissue. How to quickly achieve good osseointegration is very important for achieving the implant success rate and long-term stability. The latest research shows that the physical morphology and chemical properties of the surface of medical biomaterials play a decisive role in the performance of biomaterials. Appropriate modification of the implant surface is conducive to the attachment and growth of tissue cells, especially osteoblasts, on the implant surface, promoting osseointegration.

[0004] The purpose of the present invention is to solve the above-mentioned problems existing in titanium implants, and to modify the titanium surface, and to develop a method for preparing a silk fibroin / silk fibroin-derived peptide / hydroxyapatite composite coating on the titanium surface. Summary of the invention

[0005] The purpose of the invention is to provide a method for preparing a composite coating on a titanium surface.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for preparing a composite coating on a titanium surface, comprising the following steps:

[0008] (1) Preparation of regenerated silk fibroin solution: Silk fibroin fibers were mixed with CaCl 2 / water / ethanol ternary solution, stirring and dissolving into a silk fibroin mixed solution, dialyzing with deionized water in a dialysis bag for 3-5 days, removing impurities and then centrifuging to remove insoluble particles, and storing the supernatant at 4°C to obtain a regenerated silk fibroin solution;

[0009] (2) Pretreatment of medical pure titanium: The medical pure titanium sheet was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water, and then with HF and HNO 3 The titanium sheet is immersed in the mixed solution to remove the titanium oxide film of different components on the surface, and then ultrasonically cleaned with deionized water, dried with nitrogen, and placed in a dryer for standby use to obtain the acid-pretreated titanium sheet pTi;

[0010] (3) Surface activation of medical titanium: The titanium sheet pretreated with acid is placed in a Piranha solution and reacted at 55-65°C for 25-40 min to form a hydroxylated titanium Ti-OH with a hydroxyl-rich oxide layer. The titanium sheet is then ultrasonically cleaned with deionized water, dried with nitrogen and placed in a dryer.

[0011] (4) Polydopamine modification and activation of titanium surface: Ti-OH was immersed in dopamine hydrochloride solution for 20-30 hours. After the reaction was completed, it was ultrasonically cleaned in acetone, anhydrous ethanol and deionized water respectively, dried with nitrogen and placed in a dryer;

[0012] (5) Preparation of silk fibroin-derived peptides: α-chymotrypsin was dissolved in an aqueous solution of SF to form a gel-like precipitate Cp component, which was centrifuged, washed with water, and freeze-dried. The supernatant was freeze-dried to recover the soluble polypeptide Cs powder;

[0013] (6) Preparation of composite coating: Cs powder was added to the obtained SF solution, and then evenly dropped onto the polydopamine-modified titanium surface. After drying, electrodeposition was performed. The electrolyte was Ca(NO 3 ) 2 and (NH 4 )H 2 PO 4 The sample acts as a cathode and is immersed in the electrolyte for deposition at room temperature. After drying, it is immersed in Tris buffer and then washed with deionized water to obtain the obtained product.

[0014] Furthermore, the ternary solution is composed of CaCl 2 : A mixed solution of water and ethanol in a molar ratio of 1:8:2.

[0015] The molecular weight cut-off of the dialysis bag is 8000-14000 mW. After removing impurities, the insoluble particles are removed by centrifugation at 3000 r / min for 5 minutes.

[0016] The concentration of HF solution is 0.54 mol / L, HNO 3 The concentration of the titanium sheet was 0.29 mol / L, the volume ratio of the two was 1:1, and the titanium sheet was immersed in stirring conditions for 5 minutes.

[0017] The volume ratio of the Piranha solution is H 2 SO 4 :H 2 O 2 =7:3.

[0018] The pH value of the dopamine hydrochloride solution is 8.5, and the concentration of Tris is 10 Mm.

[0019] The ratio of α-chymotrypsin to substrate is 1:100.

[0020] The Cs powder was added to 3 wt% SF solution at a ratio of 5 mg / ml and then heated at 15 μL / cm 2 The ratio of was evenly added dropwise onto the polydopamine-modified titanium surface and dried at 65°C.

[0021] The electrolyte has a pH of 4 and consists of 0.042 M Ca(NO 3 ) 2 and 0.025 M (NH 4 )H 2 PO 4 The mixture was made with an electrode spacing of 2 cm and a deposition time of 15 min.

[0022] The present invention treats the medical pure titanium sheet to remove the titanium dioxide film on the surface, obtains the acid pretreated titanium sheet pTi, and then treats it with Piranha solution, and oxidizes it to construct a hydroxylated titanium Ti-OH with an oxide layer rich in hydroxyl groups. Dopamine is reacted with TiO 2 The layer directly provides active sites for the oxidative self-polymerization of dopamine to generate a uniform polydopamine layer. Then, the quinone structure of the polydopamine layer converted from catechol in an alkaline environment can undergo Michael addition or Schiff base reaction with the amino groups of silk fibroin and its derivative peptides to generate a strong covalent bond. In the subsequent electrodeposition process, the deposited calcium ions and phosphate ions combine with the protein polypeptide molecules to form a silk fibroin / silk fibroin-derived peptide / hydroxyapatite composite coating in the subsequent alkaline bath process. The morphology of the formed composite coating is mainly flaky and gradually connected into a mesh-like porous structure.

[0023] By comparing the silk fibroin / hydroxyapatite composite coating, the silk fibroin / 2% silk fibroin-derived peptide / hydroxyapatite composite coating, the silk fibroin / 5% silk fibroin-derived peptide / hydroxyapatite composite coating, and the 5% silk fibroin-derived peptide / hydroxyapatite composite coating, it can be found that the silk fibroin / hydroxyapatite coating is deposited in a spherical shape, the silk fibroin / 2% silk fibroin-derived peptide / hydroxyapatite coating is deposited in a narrow flake shape, the silk fibroin / 5% silk fibroin-derived peptide / hydroxyapatite coating is deposited in a flake shape with smaller pores in the middle, and the 5% silk fibroin-derived peptide / hydroxyapatite coating is deposited in a flake shape with larger pores in the middle. Figure 1 , 2 shown.

[0024] By analyzing and comparing the cross-sectional EDS spectra of silk fibroin / 5% silk fibroin-derived peptide / hydroxyapatite composite coating and the cross-sectional EDS spectra of 5% silk fibroin-derived peptide / hydroxyapatite composite coating, it can be found that the cross-sectional EDS spectra of silk fibroin / 5% silk fibroin-derived peptide / hydroxyapatite composite coating contain a small amount of N element, proving that the protein peptide is complexed with hydroxyapatite. Figure 3 ,4 shown.

[0025] In summary, the present invention first constructs a silk fibroin and its derivative peptide coating on the polydopamine coating on the activated titanium surface, covalently fixes the silk fibroin and its derivative peptide through the polydopamine layer, and then electrochemically deposits an apatite structure and mixes it with a protein polypeptide layer to construct a bionic bone-like structure on the titanium surface.

[0026] Compared with the prior art, the composite coating of the present invention has good compatibility with mesenchymal stem cells, can induce the osteogenic differentiation ability of stem cells, can improve the biological activity and osteogenic activity of the pure titanium surface, has broad clinical application potential, has a relatively simple preparation process, and has a low production cost, and has good promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 They are silk fibroin / hydroxyapatite composite coating; silk fibroin / 2% silk fibroin-derived peptide / hydroxyapatite composite coating; silk fibroin / 5% silk fibroin-derived peptide / hydroxyapatite composite coating; and 5% silk fibroin-derived peptide / hydroxyapatite composite coating.

[0028] Figure 2 They are respectively the cross-sectional SEM images of silk fibroin / 5% silk fibroin-derived peptide / hydroxyapatite composite coating and the cross-sectional SEM images of 5% silk fibroin-derived peptide / hydroxyapatite composite coating;

[0029] Figure 3 This is the cross-sectional EDS spectrum of the silk fibroin / 5% silk fibroin-derived peptide / hydroxyapatite composite coating;

[0030] Figure 4 This is the EDS spectrum of the cross section of 5% silk fibroin-derived peptide / hydroxyapatite composite coating. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with embodiments and drawings:

[0032] Example 1

[0033] 1) Preparation of regenerated silk fibroin solution: 75 g of silkworm raw silk was placed in 3 L of 0.05% Na 2 CO 3 The silk was degummed in boiling water at 100 °C for 30 min, and the process was repeated 3 times. Pure silk fiber was obtained after thorough washing and drying. 2The obtained silk fibroin mixed solution was placed in a dialysis bag (8000-14000 mW), dialyzed with deionized water for 4 days to remove impurities, and then centrifuged at 3000 r / min for 5 min to remove insoluble particles, and the supernatant was stored at 4°C.

[0034] (2) Pretreatment of medical pure titanium: The medical pure titanium sheet was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 30 min each to remove impurities and dirt on the titanium surface; HF (0.54 mol / L) and HNO 3 The titanium sheet was immersed in a mixed solution (volume ratio of 1:1) prepared by (0.29 mol / L) and stirred continuously for 5 min to remove the titanium oxide film of different components on the surface. It was then ultrasonically cleaned with deionized water for 10 min, dried with nitrogen and placed in a dryer for later use to obtain an acid-pretreated titanium sheet.

[0035] (3) Activation of medical titanium surface: Place the titanium sheet pretreated with acid in step 2) in 100 ml of Piranha solution (volume ratio H 2 SO 4 :H 2 O 2 =7:3), react at 60°C for 30 min, oxidize to construct hydroxyl-rich oxide layer of titanium hydroxylate (Ti-OH), i.e., activate titanium, clean with deionized water ultrasonically for 10 min, dry with nitrogen and place in a desiccator for later use.

[0036] (4) Polydopamine modification of activated titanium surface: Prepare a 2 mg / mL dopamine hydrochloride solution (pH = 8.5, Tris = 10 Mm), and immerse the activated titanium in step 3) in the dopamine hydrochloride solution and shake at 37 ° C for 24 h. After the reaction is completed, the polydopamine-modified titanium (Ti-PDA) is ultrasonically cleaned in acetone, anhydrous ethanol and deionized water for 10 min each, blown dry with nitrogen, and placed in a desiccator for later use.

[0037] (5) Preparation of silk fibroin-derived peptides: α-chymotrypsin (ratio of enzyme to substrate: 1:100) was dissolved in an aqueous solution of SF. After 24 h at 37°C, a gel-like precipitate (Cp component) was formed, which was centrifuged, washed twice with water, and then freeze-dried. The supernatant was freeze-dried and the soluble polypeptide (Cs component) was recovered.

[0038] (6) Preparation of silk fibroin / hydroxyapatite composite coating: The obtained 3 wt% SF solution was diluted with water at 15 μl / cm 2 The obtained sample was directly subjected to electrodeposition, and the electrolyte (pH = 4) was composed of 0.042 M Ca(NO3 ) 2 and 0.025 M (NH 4 )H 2 PO 4 composition. The electrode spacing was 2 cm and the deposition time was 15 min. The sample acted as a cathode and was immersed in the electrolyte for deposition at room temperature, then taken out and dried in a drying oven. In order to obtain hydroxyapatite, the deposited titanium sheet was immersed in 10 mM Tris buffer (pH = 8.5) at 60 ° C for 2 hours and then washed with deionized water.

[0039] Example 2

[0040] (1) Preparation of regenerated silk fibroin solution: 75 g of silkworm raw silk was placed in 3 L of 0.05% Na 2 CO 3 The silk was degummed in boiling water at 100 °C for 30 min, and the process was repeated 3 times. Pure silk fiber was obtained after thorough washing and drying. 2 The obtained silk fibroin mixed solution was placed in a dialysis bag (8000-14000 mW), dialyzed with deionized water for 4 days to remove impurities, and then centrifuged at 3000 r / min for 5 min to remove insoluble particles, and the supernatant was stored at 4°C.

[0041] (2) Pretreatment of medical pure titanium: The medical pure titanium sheet was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 30 min each to remove impurities and dirt on the titanium surface; HF (0.54 mol / L) and HNO 3 The titanium sheet was immersed in a mixed solution (volume ratio of 1:1) prepared by (0.29 mol / L) and stirred continuously for 5 minutes to remove the titanium oxide film of different components on the surface. It was then ultrasonically cleaned with deionized water for 10 minutes, dried with nitrogen and placed in a dryer for use to obtain an acid-pretreated titanium sheet (pTi).

[0042] (3) Activation of medical titanium surface: Place the acid-pretreated titanium sheet in 100 ml of Piranha solution (volume ratio H 2 SO 4 :H 2 O 2 =7:3), react at 60°C for 30 min, oxidize to construct hydroxyl-rich titanium hydroxylate (Ti-OH) oxide layer, clean with deionized water ultrasonically for 10 min, dry with nitrogen and place in a desiccator for later use.

[0043] (4) Polydopamine modification and activation of titanium surface: Prepare a 2 mg / mL dopamine hydrochloride solution (pH = 8.5, Tris = 10 Mm), soak Ti-OH in the dopamine hydrochloride solution and shake at 37 ° C for 24 h. After the reaction is completed, the polydopamine-modified titanium (Ti-PDA) is ultrasonically cleaned in acetone, anhydrous ethanol and deionized water for 10 min each, blown dry with nitrogen and placed in a desiccator for later use.

[0044] (5) Preparation of silk fibroin-derived peptides: α-chymotrypsin (ratio of enzyme to substrate: 1:100) was dissolved in an aqueous solution of SF. After 24 h at 37°C, a gel-like precipitate (Cp component) was formed, which was centrifuged, washed twice with water, and then freeze-dried. The supernatant was freeze-dried and the soluble polypeptide (Cs component) was recovered.

[0045] (6) Preparation of silk fibroin / silk fibroin-derived peptide / hydroxyapatite composite coating: Cs powder was added to the obtained 3 wt% SF solution at a ratio of 2 mg / ml to obtain a mixed solution of SF solution containing 2% Cs. The obtained mixed solution was heated at 15 μl / cm 2 The obtained sample was directly subjected to electrodeposition, and the electrolyte (pH = 4) was composed of 0.042 M Ca(NO 3 )2 and 0.025 M (NH4) H2PO4. The electrode spacing was 2 cm and the deposition time was 15 min. The sample acted as a cathode and was immersed in the electrolyte for deposition at room temperature, then taken out and dried in a drying oven. In order to obtain hydroxyapatite, the deposited titanium sheet was immersed in 10 mM Tris buffer (pH = 8.5) at 60 ° C for 2 hours and then washed with deionized water.

[0046] Example 3

[0047] (1) Preparation of regenerated silk fibroin solution: 75 g of silkworm raw silk was placed in 3 L of 0.05% Na 2 CO 3 The silk was degummed in boiling water at 100 °C for 30 min, and the process was repeated 3 times. Pure silk fiber was obtained after thorough washing and drying. 2 The obtained silk fibroin mixed solution was placed in a dialysis bag (8000-14000 mW), dialyzed with deionized water for 4 days to remove impurities, and then centrifuged at 3000 r / min for 5 min to remove insoluble particles, and the supernatant was stored at 4°C.

[0048] (2) Pretreatment of medical pure titanium: The medical pure titanium sheet was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 30 min each to remove impurities and dirt on the titanium surface; HF (0.54 mol / L) and HNO 3 The titanium sheet was immersed in a mixed solution (volume ratio of 1:1) prepared by (0.29 mol / L) and stirred continuously for 5 minutes to remove the titanium oxide film of different components on the surface. It was then ultrasonically cleaned with deionized water for 10 minutes, dried with nitrogen and placed in a dryer for use to obtain an acid-pretreated titanium sheet (pTi).

[0049] (3) Activation of medical titanium surface: Place the acid-pretreated titanium sheet in 100 ml of Piranha solution (volume ratio H 2 SO 4 :H 2 O 2 =7:3), react at 60°C for 30 min, oxidize to construct hydroxyl-rich titanium hydroxylate (Ti-OH) oxide layer, clean with deionized water ultrasonically for 10 min, dry with nitrogen and place in a desiccator for later use.

[0050] (4) Polydopamine modification and activation of titanium surface: Prepare a 2 mg / mL dopamine hydrochloride solution (pH = 8.5, Tris = 10 Mm), soak Ti-OH in the dopamine hydrochloride solution and shake at 37 ° C for 24 h. After the reaction is completed, the polydopamine-modified titanium (Ti-PDA) is ultrasonically cleaned in acetone, anhydrous ethanol and deionized water for 10 min each, blown dry with nitrogen and placed in a desiccator for later use.

[0051] (5) Preparation of silk fibroin-derived peptides: α-chymotrypsin (ratio of enzyme to substrate: 1:100) was dissolved in an aqueous solution of SF. After 24 h at 37°C, a gel-like precipitate (Cp component) was formed, which was centrifuged, washed twice with water, and then freeze-dried. The supernatant was freeze-dried and the soluble polypeptide (Cs component) was recovered.

[0052] (6) Preparation of silk fibroin / silk fibroin-derived peptide / hydroxyapatite composite coating: Cs powder was added to the obtained 3 wt% SF solution at a ratio of 5 mg / ml to obtain a mixed solution of SF solution containing 5% Cs. The obtained mixed solution was heated at 15 μl / cm 2 The obtained sample was directly subjected to electrodeposition, and the electrolyte (pH = 4) was composed of 0.042 M Ca(NO 3 ) 2 and 0.025 M (NH 4 )H 2 PO 4The electrode spacing was 2 cm and the deposition time was 15 min. The sample acted as a cathode and was immersed in the electrolyte for deposition at room temperature, then taken out and dried in a drying oven. In order to obtain hydroxyapatite, the deposited titanium sheet was immersed in 10 mM Tris buffer (pH = 8.5) at 60 ° C for 2 hours and then washed with deionized water.

[0053] Example 4

[0054] (1) Preparation of regenerated silk fibroin solution: 75 g of silkworm raw silk was placed in 3 L of 0.05% Na 2 CO 3 The aqueous solution was treated in boiling water at 100 °C for 30 minutes, and repeated 3 times to degummed the silk, and then washed and dried to obtain pure silk fiber. The pure silk fiber was added to the ternary solution (formed by CaCl2 / water / ethanol with a molar ratio of 1:8:2) and stirred at 72 °C to dissolve into a mixed solution of silk protein. The obtained mixed solution of silk was placed in a dialysis bag (8000~14000 mW), dialyzed with deionized water for 4 days to remove impurities, and then centrifuged at 3000r / min for 5 minutes to remove insoluble particles. The supernatant was stored at 4 °C.

[0055] (2) Pretreatment of medical pure titanium: The medical pure titanium sheet was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water for 30 min each to remove impurities and dirt on the titanium surface; HF (0.54 mol / L) and HNO 3 The titanium sheet was immersed in a mixed solution (volume ratio of 1:1) prepared by (0.29 mol / L) and stirred continuously for 5 minutes to remove the titanium oxide film of different components on the surface. It was then ultrasonically cleaned with deionized water for 10 minutes, dried with nitrogen and placed in a dryer for use to obtain an acid-pretreated titanium sheet (pTi).

[0056] (3) Activation of medical titanium surface: Place the acid-pretreated titanium sheet in 100 ml of Piranha solution (volume ratio H 2 SO 4 :H 2 O 2 =7:3), react at 60°C for 30 min, oxidize to construct hydroxyl-rich titanium hydroxylate (Ti-OH) oxide layer, clean with deionized water ultrasonically for 10 min, dry with nitrogen and place in a desiccator for later use.

[0057] (4) Polydopamine modification and activation of titanium surface: Prepare a 2 mg / mL dopamine hydrochloride solution (pH = 8.5, Tris = 10 Mm), soak Ti-OH in the dopamine hydrochloride solution and shake at 37 ° C for 24 h. After the reaction is completed, the polydopamine-modified titanium (Ti-PDA) is ultrasonically cleaned in acetone, anhydrous ethanol and deionized water for 10 min each, blown dry with nitrogen and placed in a desiccator for later use.

[0058] (5) Preparation of silk fibroin-derived peptides: α-chymotrypsin (ratio of enzyme to substrate: 1:100) was dissolved in an aqueous solution of SF. After 24 h at 37°C, a gel-like precipitate (Cp component) was formed, which was centrifuged, washed twice with water, and then freeze-dried. The supernatant was freeze-dried and the soluble polypeptide (Cs component) was recovered.

[0059] (6) Preparation of silk fibroin-derived peptide / hydroxyapatite composite coating:

[0060] Cs powder was added to deionized water at a ratio of 5 mg / ml to obtain a 5% Cs aqueous solution. The resulting solution was diluted to 15 μl / cm 2 The obtained sample was directly subjected to electrodeposition, and the electrolyte (pH = 4) was composed of 0.042 M Ca(NO 3 ) 2 and 0.025 M (NH 4 )H 2 PO 4 composition. The electrode spacing was 2 cm and the deposition time was 15 min. The sample acted as a cathode and was immersed in the electrolyte for deposition at room temperature, then taken out and dried in a drying oven. In order to obtain hydroxyapatite, the deposited titanium sheet was immersed in 10 mM Tris buffer (pH = 8.5) at 60 ° C for 2 hours and then washed with deionized water.

Claims

1. A method for preparing a composite coating on a titanium surface, It is characterized in that The steps are: (1) Preparation of regenerated silk fibroin solution: Silk fibroin fibers were mixed with CaCl 2 / water / ethanol ternary solution, stirring and dissolving into a silk fibroin mixed solution, dialyzing with deionized water in a dialysis bag for 3-5 days, removing impurities and then centrifuging to remove insoluble particles, and storing the supernatant at 4°C to obtain a regenerated silk fibroin SF solution; (2) Pretreatment of medical pure titanium: The medical pure titanium sheet was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water, and then with HF and HNO 3 The titanium sheet is immersed in the mixed solution to remove the titanium oxide film of different components on the surface, and then ultrasonically cleaned with deionized water, dried with nitrogen, and placed in a dryer for standby use to obtain an acid-pretreated titanium sheet; (3) Surface activation of medical titanium: The titanium sheet pretreated with acid is placed in a Piranha solution and reacted at 55-65°C for 25-40 min to form a hydroxylated titanium Ti-OH with a hydroxyl-rich oxide layer. The titanium sheet is then ultrasonically cleaned with deionized water, dried with nitrogen and placed in a dryer. The volume ratio of the Piranha solution is H 2 SO 4 :H 2 O 2 =7:3; (4) Polydopamine modification and activation of titanium surface: Ti-OH was immersed in dopamine hydrochloride solution for 20-30 hours. After the reaction was completed, it was ultrasonically cleaned in acetone, anhydrous ethanol and deionized water respectively, dried with nitrogen and placed in a dryer; (5) Preparation of silk fibroin-derived peptides: α-chymotrypsin was dissolved in SF solution to form a gel-like precipitate Cp component, which was centrifuged, washed with water, and freeze-dried. The supernatant was freeze-dried to recover the soluble polypeptide Cs powder; (6) Preparation of composite coating: Cs powder was added to the obtained SF solution, and then evenly dropped onto the polydopamine-modified titanium surface. After drying, electrodeposition was performed. The electrolyte was Ca(NO 3 ) 2 and (NH 4 )H 2 PO 4 The sample acts as a cathode and is immersed in the electrolyte for deposition at room temperature. After drying, it is immersed in Tris buffer and then washed with deionized water to obtain the obtained product.

2. The method for preparing a composite coating on a titanium surface according to claim 1, It is characterized in that The ternary solution is composed of CaCl 2 : A mixed solution of water and ethanol in a molar ratio of 1:8:

2.

3. The method for preparing a composite coating on a titanium surface according to claim 1, It is characterized in that The molecular weight cut-off of the dialysis bag is 8000-14000 mW. After removing impurities, the insoluble particles are removed by centrifugation at 3000 r / min for 5 minutes.

4. The method for preparing a composite coating on a titanium surface according to claim 1, It is characterized in that The concentration of HF solution is 0.54 mol / L, HNO 3 The concentration of the titanium sheet was 0.29 mol / L, the volume ratio of the two was 1:1, and the titanium sheet was immersed in stirring conditions for 5 minutes.

5. The method for preparing a composite coating on a titanium surface according to claim 1, It is characterized in that The pH value of the dopamine hydrochloride solution is 8.5, and the concentration of Tris is 10 Mm.

6. The method for preparing a composite coating on a titanium surface according to claim 1, It is characterized in that The ratio of α-chymotrypsin to substrate is 1:100, and the substrate is SF solution.

7. The method for preparing a composite coating on a titanium surface according to claim 1, It is characterized in that The Cs powder was added to 3 wt% SF solution at a ratio of 5 mg / ml and then heated at 15 μL / cm 2 The ratio of was evenly added dropwise onto the polydopamine-modified titanium surface and dried at 65°C.

8. The method for preparing a composite coating on a titanium surface according to claim 1, It is characterized in that The electrolyte has a pH of 4 and consists of 0.042 M Ca(NO 3 ) 2 and 0.025 M (NH 4 )H 2 PO 4 The mixture was made with an electrode spacing of 2 cm and a deposition time of 15 min.

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