Bone-targeting in-situ bioactive coating implanted into titanium surface as well as preparation method and application thereof

Abstract

The invention discloses a bone targeting in-situ bioactive coating implanted into a titanium surface as well as a preparation method and application of the bone targeting in-situ bioactive coating, and belongs to the technical field of biomedical materials. The coating takes an implanted titanium metal material as a matrix, and sequentially comprises a titanium dioxide nanotube loaded with bone targeting ions, a polydopamine middle layer and a polyanion amino acid molecule targeting layer from inside to outside, the preparation method comprises the following steps: (1) constructing a titanium dioxide nanotube; (2) carrying bone targeting ions by adopting a hydrothermal technology; (3) spin-coating a self-polymerization dopamine middle layer; and (4) in-situ grafting of a polyanion amino acid molecular targeting layer. The coating improves the biological activity of the implanted titanium alloy surface, controls the release of Ga < 3 + >, promotes the early adhesion of cells, accelerates the induction of the formation of hydroxyapatite, realizes the early in-situ rapid formation of bone tissues on the implanted titanium interface, and endows the surface with broad-spectrum antibacterial property. The invention provides a new thought for surface modification and biological functionalization of implanted titanium and alloy materials thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to an implant titanium surface bone-targeting in-situ bioactive coating and a preparation method and application thereof. BACKGROUND

[0002] Titanium alloys are widely used as orthopedic and oral implant materials due to their reliable mechanical properties, stable chemical properties and excellent biocompatibility. However, the titanium alloy surface has poor bioactivity, and the naturally formed inert oxide layer cannot provide adhesion sites for cell growth, affecting the integration of the surrounding tissue and the implant material interface. The titanium surface is severely deficient in active groups, which cannot induce the in-situ spontaneous rapid formation of hydroxyapatite, resulting in implant loosening and even failure. In addition, bacterial adhesion during the implant surgery process is another major cause of surrounding tissue infection, inflammation and implant failure. Therefore, it is very urgent to endow the implant titanium material surface with bioactivity and antibacterial properties.

[0003] Gallium is a semi-metallic element in the 13th main group, and Ga 3+ has similar ionic radius, physical and chemical properties to Fe 3+ . It has multiple biological functions such as immunosuppression, tumor imaging, anticancer, antibacterial, anti-inflammatory and inhibition of osteoclast generation, and is widely used in the field of biological medicine. Ga 3+ has obvious targeting to bone tissue, significantly accelerates the formation of bone tissue, and inhibits bone resorption, thereby hindering the loss of Ca. Ga 3+ has broad-spectrum antibacterial properties and can penetrate into the bacterial system by simulating "Fe 3+ " through the "Trojan horse" strategy, thereby disturbing iron metabolism, inhibiting DNA and protein synthesis, hindering mitochondrial function, inhibiting bacterial activity and even killing bacteria. However, the biological function of Ga 3+ is also highly dependent on concentration, and excessive Ga 3+ has obvious toxicity to cells and surrounding tissues.

[0004] Gamma-polyglutamic acid is a natural polypeptide polymer, which is first found in the cell wall of Bacillus anthracis and can also be fermented by microbial metabolites such as Bacillus subtilis. Gamma-polyglutamic acid is an anionic polymer formed by the combination of D-glutamic acid or L-glutamic acid through alpha-amino and gamma-carboxyl gamma-amide bond, which can be degraded into endogenous glutamic acid in the human body. Glutamic acid is widely distributed in the human body and has no toxic side effects, and can be used as a collagen component to promote hydroxyapatite nucleation. The side chain of gamma-polyglutamic acid is rich in free carboxyl groups with high activity, which can induce Ca-P deposition and accelerate the formation of hydroxyapatite; it is also easy to react with some substances with amino, thiol, hydroxyl and other functional groups to realize modification, grafting, crosslinking, chelation and other modification effects, which can be used for the preparation of medical hydrogel, drug modification, drug targeted delivery and controlled release, and also can be used as potential biomaterials for wound repair, tissue filling, engineering scaffold, drug carrier, antibacterial agent, vaccine adjuvant and cancer treatment.

[0005] However, the current bone targeting strategy is to load and encapsulate the targeted ions, molecules, polypeptides and other substances into hydrogels, liposomes, mesoporous / porous materials, transport into the body, and release the target to the bone mineral area to play a function by means of body fluid environment acidification, temperature change, enzyme degradation and the like. But this strategy is difficult to achieve at the interface / surface of the implanted material. The biological interaction between the implanted titanium surface and the surrounding tissue interface is the key to early in-situ bone integration. The existing research improves the titanium surface morphology and composition by physical and chemical methods to realize the disordered growth of cells and related tissues, and the research report of the reverse targeting of the surrounding bone tissue to the interface of the implanted material is very limited. SUMMARY

[0006] The purpose of the application: In view of the problems of poor biological activity of orthopedic implant titanium alloy surface, insufficient bone integration with surrounding tissue, and serious bacterial infection after operation, the first purpose of the application is to provide a bone targeting in-situ bioactive coating of implanted titanium surface with bone targeting ions / gamma-polyamino acid synergistic functionalized titanium dioxide nanotubes for improving the biological activity of the implanted titanium alloy surface, promoting the targeted aggregation and ingrowth of surrounding osteoblasts and Ca-P apatite, improving the bone integration ability, and inhibiting bacterial adhesion. The second purpose of the application is to provide a preparation method of the coating, and the third purpose of the application is to provide the application of the coating.

[0007] Technical scheme: The implanted titanium surface bone targeting in-situ bioactive coating according to the application is based on an implanted titanium metal material, which comprises, from inside to outside, a titanium dioxide nanotube loaded with bone targeting ions, a polydopamine intermediate layer and a polyanion amino acid molecular targeting layer.

[0008] Further, the implanted titanium metal material is TA2 and Ti6Al4V titanium alloy; the bone targeting ion is Ga 3+The polyanionic amino acid molecule is one or more of γ-polyglutamic acid or γ-polyglutamate salt, with a molecular weight of 10-900 kDa, and the inner diameter of the titanium dioxide nanotube is 30-150 nm.

[0009] The present invention firstly uses anodization technology to construct a titanium dioxide nanotube carrier on the surface of a biomedical titanium alloy, and then uses a hydrothermal method to carry bioactive Ga 3+ ions, deposited from the intermediate layer of polymerized dopamine, and further grafted with γ-polyglutamic acid molecules to construct a bone-targeted bioactive coating. The coating exhibits excellent hydrophilicity, and γ-polyglutamic acid molecules and Ga 3+ Both can induce the aggregation and deposition of Ca-P apatite in situ, while Ga 3+ The ion / γ-polyglutamic acid molecule / nanotube topology synergistically promotes the early adhesion of osteoblasts, improves proliferation activity, upregulates the expression of functional factors, and slowly and continuously releases Ga 3+ The ions inhibit the early adhesion of Gram-positive and Gram-negative bacteria, demonstrating broad-spectrum antibacterial properties. The in situ bone-targeted bioactive coating and its preparation method provided by the present invention provide new ideas for surface modification and biofunctionalization of implanted titanium materials.

[0010] The method for preparing the above-mentioned bone-targeted in-situ bioactive coating implanted on the titanium surface comprises the following steps:

[0011] (1) Titanium dioxide nanotubes are constructed on the surface of implanted titanium metal materials through anodization technology;

[0012] (2) Using hydrothermal technology to load bone-targeting ions into titanium dioxide nanotubes to obtain titanium dioxide nanotubes loaded with bone-targeting ions;

[0013] (3) evenly spin-coating the dopamine-Tris solution on the surface of the titanium dioxide nanotubes loaded with bone-targeting ions to form a polydopamine intermediate layer through self-polymerization deposition;

[0014] (4) A polyanionic amino acid molecule solution is prepared, and after activation treatment, the resulting mixed solution is dripped onto the polydopamine intermediate layer. After amide reaction, a polyanionic amino acid molecule targeting layer is obtained, and finally a bone-targeted in situ bioactive coating is obtained.

[0015] Furthermore, the anodic oxidation in step (1) adopts a constant voltage mode, with the voltage set to 20-100 V, preferably 35-55 V; the time is 10 min-180 min, preferably 40-60 min; the electrolyte is a fluorine-containing organic solution, preferably 0.1-10 wt.% ammonium fluoride, 0.1-10 vol% deionized water and glycerol.

[0016] Further, in step (2), the process parameters of the hydrothermal technique are as follows: the reaction temperature is 20-90℃, the reaction time is 12-72h, and the concentration of the solution containing the bone-targeting ion salt is 1-200mM. Preferably, the reaction temperature is 30-50℃, the reaction time is 20-36h, and the concentration of the solution containing the bone-targeting ion salt is 5-50mM.

[0017] Further, the bone-targeting ion salt is one of the following: a gallium salt of gallium nitrate, gallium sulfate, gallium citrate, gallium maltate, and tris(8-hydroxyquinoline) gallium, and is preferably gallium nitrate.

[0018] Further, in step (3), the spin coating method is as follows: the dopamine-Tris solution is first dropped onto the surface of the titanium dioxide nanotube loaded with the bone-targeting ion, then spin coating is started, and finally the reaction is allowed to stand; the concentration of the dopamine-Tris solution is 1-100mM, preferably 10-50mM, the dopamine used is dopamine hydrochloride, the solvent is a 0.01M aqueous solution of tris(hydroxymethyl)aminomethane hydrochloride, and the pH of the dopamine solution is adjusted to 8.2-8.7 repeatedly using 0.1M dilute hydrochloric acid and NaOH solution during the preparation of the dopamine solution; the parameters of the dropping process are as follows: the unit volume of the solution is 10-1000μL / cm 2 , preferably 100-300μL / cm 2 , the number of spin coating is 1-20 times, preferably 3-6 times, and the spin coating sequence is standing first, then low speed, and finally high speed; the standing time is 1-20min, preferably 5-10min, the low speed is 100-1000rpm for 1-50s, preferably 400-600rpm for 10-20s, the high speed is 1000-5000rpm for 1-50s, preferably 1200-2000rpm for 10-30s; and the standing reaction time is 12-36h, preferably 24-36h.

[0019] Further, in step (4), the concentration of the polyanionic amino acid molecule solution is 1-20mg / mL, preferably 10mg / mL; and the parameters of the activation process are as follows: the carboxyl activation reagent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, the molar ratio of the two is 1:1-1.5, preferably 1:1, the concentration of N-hydroxysuccinimide in the polyanionic amino acid molecule solution is 10-100mM, preferably 40-60mM; and the activation time is 1-24h, preferably 4-8h.

[0020] Further, in step (4), the parameters of the dropping process of the mixed solution to the polydopamine intermediate layer are as follows: 200-1000μL / cm 2, amide reaction 12-36h, repeat 3-6 times, preferably, 500-800ul / cm 2 , reaction 24-36h; after reaction, rinse sample surface with deionized water 3-5 times, air dry.

[0021] The application of the implant titanium surface bone targeting in-situ bioactive coating as an implant material in the field of orthopedics or dentistry.

[0022] Invention principle: titanium dioxide nanotube has a large specific surface area and strong hydrophilicity, which can provide sites for early cell adhesion and provide nutrient storage space for cell growth; the hollow structure can also provide space for active substance loading. 3+ Ga ions have obvious targeting effect on inorganic minerals of bone tissue, and have positive promoting effect on growth behavior of osteoblasts, while significantly inhibiting bone resorption, thus accelerating bone tissue formation and hindering loss of Ca and other minerals. 3+ It has broad-spectrum antibacterial properties and can simulate Fe 3+ Penetrate into the bacterial system, disturb iron metabolism, inhibit DNA and protein synthesis, hinder mitochondrial function, inhibit bacterial activity, and kill bacteria. Therefore Ga 3+ ions are ideal ions for bone mineral targeting and antibacterial properties. As an anionic polymer amino acid, gamma-polyglutamic acid has a structure similar to the extracellular matrix, and the side chain is rich in free carboxyl groups with high activity, which can induce in-situ deposition of Ca-P and accelerate formation of hydroxyapatite; it is also easy to react with some substances with amino, thiol, hydroxyl and other functional groups to achieve grafting. The carboxyl groups of gamma-polyglutamic acid have ideal ion complexing ability, which can delay the release of metal ions and avoid cell and tissue toxicity. A large number of carboxyl groups can also be grafted on the material surface through amide reaction, hydrogen bonding and electrostatic adsorption.

[0023] The present application improves the activity of the implant titanium surface by titanium dioxide nanotube array, provides a large number of sites for osteoblast adhesion, and also provides space for Ga 3+ ion loading; through the polydopamine intermediate layer, the nanotube surface is endowed with high-density amino groups, and then through amide reaction with the carboxyl groups of gamma-polyglutamic acid, gamma-polyglutamic acid is grafted on the material surface, accelerating the induced formation of hydroxyapatite, and relying on the chelating effect of carboxyl groups, controlling the slow release of Ga 3+ ions, guiding early cell behavior, improving cell proliferation activity, promoting early in-situ bone tissue formation, and improving bone integration; at the same time, relying on the slow release of Ga 3+ resist bacterial adhesion and infection during the operation process.

[0024] Beneficial effects: compared with the prior art, the present application has the following remarkable advantages: (1) the bone-targeting bioactive coating provided by the present application can induce rapid deposition of Ca-P and formation of an apatite layer in situ, promoting early adhesion, growth and functional expression of osteoblasts; (2) the coating provided by the present application can effectively delay the release of Ga 3+ ions, avoiding the cell and tissue toxicity caused by burst release; (3) the Ga 3+ ions, gamma polyglutamic acid or gamma polyglutamate, polydopamine and titanium dioxide nanotubes carried by the present application are widely available and low in cost; (4) the technical solution of the present application is feasible and controllable, and the preparation process is simple and the experimental conditions are mild, without the need for special equipment. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 SEM morphology diagram of the surface of the coating prepared for Comparative Example 2 (left) and Example 1 (right);

[0026] Figure 2 Main element distribution diagram of the surface of the coating prepared for Example 1;

[0027] Figure 3 Fourier infrared spectrum diagram of the surface of the coating prepared for Example 1;

[0028] Figure 4 High-resolution diagram of the surface of the coating prepared for Example 1 by X-ray photoelectron spectroscopy;

[0029] Figure 5 Release curve diagram of Ga 3+ ions in the coating prepared for Examples 1-4 and Comparative Examples 3-4 over time;

[0030] Figure 6 Surface hydroxyapatite deposition morphology diagram (14 days) of the coating prepared for Comparative Example 1 (left) and Comparative Example 2 (right);

[0031] Figure 7 Surface morphology diagram (14 days) of the surface of the coating prepared for Example 1 to deposit hydroxyapatite: low magnification (left) and high magnification (right);

[0032] Figure 8 Surface osteoblast adhesion morphology diagram (1 day) of the coating prepared for Comparative Example 1 (left) and Comparative Example 2 (right);

[0033] Figure 9 Surface osteoblast adhesion morphology diagram (1 day) of the coating prepared for Example 1 (left) and Example 4 (right);

[0034] Figure 10Antibacterial property evaluation chart (E. coli) of the coating prepared for Comparative Example 1 (left) and Comparative Example 2 (right);

[0035] Figure 11 Antibacterial property evaluation chart (E. coli) of the coating prepared for Example 1 (left) and Example 4 (right). DETAILED DESCRIPTION

[0036] The present application will be further described below in conjunction with specific examples and drawings.

[0037] Example 1: The implant titanium surface bone-targeting in-situ bioactive coating described in this example has TA2 alloy as the substrate, and comprises, from inside to outside, Ga 3+ ion functionalized titanium dioxide nanotubes, a polydopamine intermediate layer and a γ-polyglutamic acid targeting layer.

[0038] The preparation process of the above coating is as follows:

[0039] (1) TA2 alloy was made into a sample of 50 mm x 50 mm, and was polished layer by layer through #320, 400, 600, 800, 1000, etc. sandpaper, and then was ultrasonically cleaned with ethanol, deionized water, and then was blown dry, and was immersed in 200 mL of an electrolyte containing 0.5 wt. % NH4F + 2 vol. % deionized water + glycerol, the voltage was kept at 45 V, and anodization was performed for 1 h, and then the sample was cleaned with glycerol, ethanol, and deionized water for 5 min, respectively, and was blown dry, and a titanium dioxide nanotube carrier was obtained on the surface.

[0040] (2) The nanotube material prepared in step (1) was immersed in 250 mL of a 50 mM aqueous gallium nitrate solution, and was hydrothermally reacted at 40°C for 24 h, and was cleaned with deionized water and then was blown dry, and Ga 3+ ion functionalized titanium dioxide nanotubes were obtained.

[0041] (3) A 10 mM dopamine-Tris solution was prepared (in the preparation of the dopamine-Tris solution, dopamine hydrochloride was selected as the dopamine, and a 0.01 M aqueous solution of tris(hydroxymethyl)aminomethane hydrochloride was selected as the solvent, and during the preparation process, 0.1 M dilute hydrochloric acid and NaOH solution were repeatedly used to adjust the pH to 8.5), and the Ga 3+ ion functionalized titanium dioxide nanotubes prepared in step (2) were made into a material of 10 mm x 10 mm and were placed on a spin coater, 100 μL / cm 2 of the dopamine-Tris solution was dropped slowly, and after standing for 5 min, the sample was rotated at a low speed of 400 rpm for 30 s, and then was rotated at a high speed of 1500 rpm for 15 s, and the spin coating was repeated for 5 times, and then the sample was allowed to stand for 24 h, and nanotubes carrying a polydopamine intermediate layer were obtained.

[0042] (4) Configuration of 10 mg / mL aqueous solution of gamma-polyglutamic acid (700 kDa) and addition of 50 mM of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (molar ratio of 1:1), activation of carboxyl groups for 4 h; pipetting the mixed solution, static drop 500 μL / cm 2 Amide reaction on the polydopamine intermediate layer obtained in step (3) for 24 h; this static drop + amide reaction step is repeated 3 times; subsequent washing with deionized water 3-5 times, air drying, to obtain.

[0043] Example 2: The implant titanium surface bone targeting in situ bioactive coating described in this embodiment, Ti6Al4V alloy as the matrix, from inside to outside includes Ga 3+ loaded titanium dioxide nanotubes, a polydopamine intermediate layer and a gamma-polyglutamic acid sodium targeting layer.

[0044] The preparation process of the above coating is as follows:

[0045] (1) Ti6Al4V alloy is made into a sample of 50 mm x 50 mm, and is polished layer by layer through #320, 400, 600, 800, 1000 and other gradient sandpaper, and then is ultrasonically cleaned with ethanol, deionized water and air dried in sequence, immersed in 200 mL of electrolyte containing 0.5 wt.% NH4F + 2 vol.% deionized water + glycerol, the voltage is kept at 50 V, anodized for 1 h, and then cleaned with glycerol, ethanol and deionized water for 5 min respectively, and then dried to obtain a titanium dioxide nanotube carrier on the surface.

[0046] (2) The nanotube material of step (1) is immersed in 250 mL of 5 mM aqueous gallium nitrate solution, hydrothermally reacted at 40°C for 24 h, deionized and dried to obtain Ga 3+ ion functionalized titanium dioxide nanotubes.

[0047] (3) Preparation of 10 mM dopamine-Tris solution (in the preparation of dopamine-Tris solution, dopamine hydrochloride is selected as dopamine, and aqueous solution of 0.01 M tris-hydroxymethyl aminomethane hydrochloride is selected as solvent, and 0.1 M dilute hydrochloric acid and NaOH solution are repeatedly used to adjust pH to 8.5 during the preparation process), Ga 3+ ion functionalized titanium dioxide nanotubes prepared in step (2) are made into a material of 10 mm x 10 mm and placed on a spin coater, static drop 100 μL / cm 2 of dopamine-Tris solution, static for 5 min, low speed 400 rpm rotation for 30 s, then high speed 1500 rpm rotation for 15 s, spin coating is repeated 5 times, static reaction for 24 h, to obtain nanotubes with a polydopamine intermediate layer.

[0048] (4) Configuration of 15 mg / mL aqueous solution of gamma-polyglutamic acid sodium (500 kDa), and adding 50 mM of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (molar ratio of 1:1), activation of carboxyl 4h; suction mixed solution, 500 μL / cm 2 Amide reaction on the polydopamine intermediate layer obtained in step (3) for 24h; the static drop + amide reaction step is repeated 3 times; then washed with deionized water for 3-5 times, air dried, to obtain.

[0049] Example 3: The implant titanium surface bone targeting in situ bioactive coating described in this embodiment, with TA2 alloy as the matrix, includes Ga 3+ loaded titanium dioxide nanotube, polydopamine intermediate layer and gamma-polyglutamic acid sodium targeting layer from inside to outside.

[0050] The preparation process of the coating is as follows:

[0051] (1) TA2 alloy is made into a sample of 50 mm x 50 mm, and is polished layer by layer through #320, 400, 600, 800, 1000 and other gradient sandpaper, and then is ultrasonically cleaned with ethanol, deionized water and glycerol in sequence, and is dried by blowing, immersed in 200 mL of electrolyte containing 0.5 wt.% NH4F + 2 vol.% deionized water + glycerol, anodized at a voltage of 45 V for 1 h, and then cleaned with glycerol, ethanol and deionized water for 5 min respectively, and dried by blowing, so that a titanium dioxide nanotube carrier is obtained on the surface.

[0052] (2) The nanotube material of step (1) is immersed in 250 mL of 20 mM aqueous gallium nitrate solution, and hydrothermally reacted at 40°C for 24 h, and then cleaned with deionized water and dried by blowing, so that Ga 3+ ion functionalized titanium dioxide nanotubes are obtained.

[0053] (3) A 20 mM dopamine-Tris solution is prepared (in the preparation of the dopamine-Tris solution, dopamine hydrochloride is selected as dopamine, and an aqueous solution of 0.01 M tris-hydroxymethyl aminomethane hydrochloride is used as solvent, and 0.1 M dilute hydrochloric acid and NaOH solution are repeatedly used to adjust pH to 8.5 during the preparation process), Ga 3+ ion functionalized titanium dioxide nanotubes prepared in step (2) are made into a material of 10 mm x 10 mm and placed on a spin coater, 100 μL / cm 2 of dopamine-Tris solution is dropped statically, and after standing for 5 min, it is rotated at a low speed of 400 rpm for 30 s, and then rotated at a high speed of 1500 rpm for 15 s, and the spin coating is repeated 5 times, and then reacted for 24 h after standing, so that nanotubes carrying a polydopamine intermediate layer are obtained.

[0054] (4) Configuration of 10 mg / mL aqueous solution of gamma-polyglutamic acid sodium (700 kDa), and adding 50 mM of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (molar ratio of 1:1), activation of carboxyl 4h; suction mixed solution, 500 μL / cm 2 The upper amide reaction was carried out on the polydopamine intermediate layer obtained in step (3) for 24h; the static drop + amide reaction step was repeated 3 times; then washed with deionized water for 3-5 times, air dried, and obtained.

[0055] Example 4: The implant titanium surface bone targeting in situ bioactive coating described in this embodiment, with TA2 alloy as the substrate, includes, from inside to outside, Ga 3+ loaded titanium dioxide nanotubes, a polydopamine intermediate layer and a gamma-polyglutamic acid targeting layer.

[0056] The preparation process of the coating is as follows:

[0057] (1) TA2 alloy was made into a sample of 50 mm x 50 mm, and was polished layer by layer through #320, 400, 600, 800, 1000 and other gradient sandpaper, then was ultrasonically cleaned with ethanol, deionized water and glycerol in sequence, and was dried by blowing, immersed in 200 mL of electrolyte containing 0.5 wt.% NH4F + 2 vol.% deionized water + glycerol, anodized at a voltage of 50 V for 1 h, and then cleaned with glycerol, ethanol and deionized water for 5 min, respectively, and dried by blowing, so that a titanium dioxide nanotube carrier was obtained on the surface.

[0058] (2) The nanotube material of step (1) was immersed in 250 mL of 100 mM aqueous gallium nitrate solution, and hydrothermally reacted at 50°C for 24 h, then was cleaned with deionized water and dried by blowing, so that Ga 3+ ion functionalized titanium dioxide nanotubes were obtained.

[0059] (3) A 20 mM dopamine-Tris solution was prepared (in the preparation of the dopamine-Tris solution, dopamine hydrochloride was selected as dopamine, and an aqueous solution of 0.01 M tris-hydroxymethyl aminomethane hydrochloride was used as solvent, and the pH was adjusted to 8.5 repeatedly using 0.1 M dilute hydrochloric acid and NaOH solution during the preparation process), Ga 3+ ion functionalized titanium dioxide nanotubes prepared in step (2) were made into a material of 10 mm x 10 mm and placed on a spin coater, 100 μL / cm 2 of dopamine-Tris solution was dropped statically, and after standing for 5 min, it was rotated at a low speed of 400 rpm for 30 s, and then rotated at a high speed of 1500 rpm for 15 s, the spin coating was repeated 5 times, and then the reaction was carried out for 24 h, so that nanotubes carrying a polydopamine intermediate layer were obtained.

[0060] (4) Configuration of 10 mg / mL aqueous solution of gamma-polyglutamic acid (500 kDa) and addition of 50 mM of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (molar ratio 1 : 1), activation of carboxyl groups for 4 h; pipetting the mixed solution, static drop 500 μί / cm 2 Amide reaction on the polydopamine intermediate layer obtained in step (3) for 24 h; this static drop + amide reaction step is repeated 3 times; subsequent washing with deionized water 3-5 times, air drying, and the like, to obtain.

[0061] Comparative Example 1 : TA2 alloy was made into a sample of 50 mm x 50 mm, and after layer-by-layer polishing with #320, 400, 600, 800, 1000, and the like, sandpaper, ethanol, deionized water were sequentially used for ultrasonic oil removal, cleaning, and blowing dry.

[0062] Comparative Example 2: TA2 alloy was made into a sample of 50 mm x 50 mm, and after layer-by-layer polishing with #320, 400, 600, 800, 1000, and the like, sandpaper, ethanol, deionized water were sequentially used for ultrasonic oil removal, cleaning, and blowing dry, and then immersed in 200 mL of an electrolyte containing 0.5 wt.% NH4F + 2 vol.% deionized water + glycerol, the voltage was kept at 45 V, and after anodizing for 1 h, the surface obtained a titanium dioxide nanotube carrier after cleaning with glycerol, ethanol, deionized water for 5 min, and blowing dry.

[0063] Comparative Example 3: The implant titanium surface bone-targeting in-situ bioactive coating described in this comparative example has TA2 alloy as the substrate, and includes, from inside to outside, Ga 3+ loaded titanium dioxide nanotubes, a polydopamine intermediate layer, and a gamma-polyglutamic acid targeting layer. Compared with Example 1, the molecular weight of the gamma-polyglutamic acid is 1100 kDa, and the solution concentration is adjusted to 100 mg / mL.

[0064] The preparation process of the above coating is as follows:

[0065] (1) TA2 alloy was made into a sample of 50 mm x 50 mm, and after layer-by-layer polishing with #320, 400, 600, 800, 1000, and the like, sandpaper, ethanol, deionized water were sequentially used for ultrasonic oil removal, cleaning, and blowing dry, and then immersed in 200 mL of an electrolyte containing 0.5 wt.% NH4F + 2 vol.% deionized water + glycerol, the voltage was kept at 45 V, and after anodizing for 1 h, the surface obtained a titanium dioxide nanotube carrier after cleaning with glycerol, ethanol, deionized water for 5 min, and blowing dry.

[0066] (2) The nanotube material of step (1) was immersed in 250 mL of 50 mM aqueous gallium nitrate solution, and hydrothermal reaction was carried out at 40°C for 24 h, and then deionized water was used for cleaning and blowing dry, to obtain Ga 3+Ionically functionalized titania nanotubes.

[0067] (3) 10 mM dopamine-Tris solution was prepared (in the preparation of dopamine-Tris solution, dopamine hydrochloride was selected as dopamine, and 0.01 M aqueous solution of tris-hydroxymethyl aminomethane hydrochloride was selected as solvent, and 0.1 M dilute hydrochloric acid and NaOH solution were used to repeatedly adjust pH to 8.5 during the preparation process), and Ga 3+ Ionically functionalized titania nanotubes were prepared into a material with a size of 10 mm x 10 mm and placed on a spin coater, and 100 μL / cm 2 Dopamine-Tris solution was prepared, and after standing for 5 min, low-speed rotation at 400 rpm was performed for 30 s, followed by high-speed rotation at 1500 rpm for 15 s, and the spin coating was repeated 5 times, and the reaction was allowed to stand for 24 h to obtain nanotubes carrying a polydopamine intermediate layer.

[0068] (4) 100 mg / mL aqueous solution of γ-polyglutamic acid (1100 kDa) was prepared, and 50 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (molar ratio of 1:1) were added to activate the carboxyl group for 4 h; the mixed solution was taken, and 500 μL / cm 2 was dropped onto the polydopamine intermediate layer obtained in step (3) to perform amide reaction for 24 h, and the dropping + amide reaction step was repeated 3 times; then, the sample was washed with deionized water for 3-5 times, and air-dried to obtain the sample.

[0069] Comparative Example 4: The implant titanium surface bone-targeting in-situ bioactive coating described in this comparative example has TA2 alloy as a substrate, and comprises, from inside to outside, Ga 3+ loaded titania nanotubes, a polydopamine intermediate layer, and a γ-polyglutamic acid targeting layer. Compared with Example 1, the molecular weight of γ-polyglutamic acid is <100 kDa, and the solution concentration is adjusted to 2 mg / mL.

[0070] The preparation process of the coating is as follows:

[0071] (1) TA2 alloy was prepared into a sample with a size of 50 mm x 50 mm, and was polished layer by layer through #320, 400, 600, 800, 1000, and the like, and then was cleaned with ethanol, deionized water, and the like in sequence, and was dried, and was immersed in 200 mL of an electrolyte containing 0.5 wt. % NH4F + 2 vol. % deionized water + glycerol, and the voltage was kept at 45 V, and anodic oxidation was performed for 1 h, and then the sample was cleaned with glycerol, ethanol, and deionized water for 5 min, and was dried, and the surface obtained titania nanotube carrier.

[0072] (2) The nanotube material prepared in step (1) was immersed in 250 mL of 50 mM aqueous gallium nitrate solution, and hydrothermal reaction was carried out at 40 °C for 24 h. After deionized water washing and air drying, Ga 3+ ionically functionalized TiO2 nanotubes were obtained.

[0073] (3) A 10 mM dopamine-Tris solution was prepared (in the preparation of the dopamine-Tris solution, dopamine hydrochloride was selected as dopamine, and an aqueous solution of 0.01 M tris-hydroxymethyl aminomethane hydrochloride was used as solvent, and 0.1 M dilute hydrochloric acid and NaOH solution were used to repeatedly adjust the pH to 8.5 during the preparation process). The Ga 3+ ionically functionalized TiO2 nanotubes prepared in step (2) were made into a material with a size of 10 mm x 10 mm and placed on a spin coater. 100 μL / cm 2 of the dopamine-Tris solution was dropped slowly, and after standing for 5 min, the material was rotated at a low speed of 400 rpm for 30 s, and then rotated at a high speed of 1500 rpm for 15 s. The spin coating was repeated 5 times, and after standing for 24 h, nanotubes with a polydopamine intermediate layer were obtained.

[0074] (4) A 2 mg / mL aqueous solution of γ-polyglutamic acid (<100 kDa) was prepared, and 50 mM of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (molar ratio of 1:1) were added to activate the carboxyl group for 4 h. The mixed solution was taken, and 500 μL / cm 2 of the solution was dropped slowly onto the polydopamine intermediate layer obtained in step (3) to carry out amide reaction for 24 h. The step of slow dropping and amide reaction was repeated 3 times. Then, the material was washed with deionized water for 3-5 times, and air dried to obtain the material.

[0075] Figure 1 The surface SEM morphology of the coating prepared for Comparative Example 2 (left) and Example 1 (right). The surface of Comparative Example 2 (left) formed a regular arrangement of uniform porous TiO2 nanotube structure, with an inner diameter of about 70 nm and a wall thickness of about 16 nm. The material prepared in Example 1 (right) still maintained the basic shape and morphology of the nanotube, but the inner diameter was significantly reduced to 38 nm and the wall thickness was significantly increased to 31 nm.

[0076] Figure 2 The surface EDS main element distribution map of the coating prepared for Example 1. The surface elements mainly consisted of Ti, O, C, N, Ga, etc. Ti had components from the titanium matrix and components from the surface TiO2, O mainly existed in the TiO2 nanotube, and N was due to the amino group of the polydopamine layer. Ga came from Ga 3+ ions.

[0077] Figure 3The surface Fourier infrared spectrum of the coating prepared in Example 1 was obtained. The stretching vibration absorption peak of Ti-O-Ti appeared near 650-700 cm -1 A broad peak corresponding to -OH stretching vibration was obvious near 3250 cm -1 The C=C stretching vibration at 1590 cm -1 and the C-H in-plane bending peak at 1490 cm -1 confirmed the benzene ring structure of polydopamine; the C-N vibration absorption peak at 1260 cm -1 corresponded to the amide III band, and the C-N at 2920 cm -1 corresponded to the characteristic peak of polyglutamic acid. The above results showed that the nanotube was TiO2 structure, and was successively covered with polydopamine and polyglutamic acid molecules.

[0078] Figure 4 The surface X-ray photoelectron spectroscopy high-resolution spectrum of the coating prepared in Example 1 was obtained. Ti 2p can be fitted into two peaks of Ti 2p3 / 2 (458.48 eV) and Ti 2p1 / 2 (464.38 eV), corresponding to the doublet structure of TiO2. O 1s was fitted into two peaks, corresponding to titanium oxide at 531.28 eV and hydroxide at 532.98 eV. C 1s was fitted into C-C / C-H at 284.6 eV, C-N / C-O at 286.08 eV, and C=O at 287.98 eV, indicating the oxidative self-polymerization of dopamine. N 1s had two fitted peaks, corresponding to N-C near 399.58 eV and N-H near 401.18 eV, wherein the N-C peak had higher intensity and area, corresponding to the amide bond, indicating that the γ-polyglutamic acid was successfully carried by the amide covalent grafting reaction with the polydopamine layer. Ga 2p had two characteristic peaks near 1145 eV and 1118 eV, corresponding to Ga 2p1 / 2 and Ga 2p3 / 2 , respectively, indicating that gallium existed in the form of trivalent ion in the nanotube.

[0079] Figure 5 The release curve of Ga 3+ ions in the coating prepared in Example 1-Example 4, Comparative Example 3 and Comparative Example 4 over time. The release amount of Example 1 was 8.1 mg / L within 336 h, the release amount of Example 2 was 3.7 mg / L, the release amount of Example 3 was 6.3 mg / L, and the release amount of Example 4 was 9.8 mg / L. The Ga 3+ions, but the total amount of release is closely related to the concentration of the ions carried. The release amount of Comparative Example 3 is 4.1 mg / L, and the total amount and release rate are much lower than those of Example 1; while the release amount of Comparative Example 4 is 9.5 mg / L, and the total amount and release rate are greater than those of Example 1. The Ga 3+ ions carried by Example 1 are the same, while the concentration and molecular weight of the γ-polyglutamic acid solution of Comparative Example 3 are both greater, and the Ga 3+ ions have a significant slow-release effect; while the concentration and molecular weight of the γ-polyglutamic acid solution of Comparative Example 4 are both smaller, and the Ga 3+ ions have no significant slow-release effect.

[0080] Figure 6 The surface hydroxyapatite deposition morphology (14 days) of the coating prepared from Comparative Example 1 (left) and Comparative Example 2 (right). The surface of Comparative Example 1 (left) has spherical protruding particles, which are the apatite particles formed by the nucleation and growth of Ca-P in the simulated body fluid. However, the coverage area of the particles is small, and the surface has no crystalline morphology. The surface of Comparative Example 2 (right) has a significantly increased coverage of particles, and agglomeration occurs, which is because the hydroxide introduced on the surface of the nanotube layer can induce the formation of apatite, and the amount of apatite formed is still small.

[0081] Figure 7 The surface hydroxyapatite deposition morphology (14 days) of the coating prepared from Example 1: low magnification (left) and high magnification (right). The surface of Example 1 is almost covered by hydroxyapatite particles, and the enlarged image shows a clear crystalline petal-like morphology, indicating that the nucleation and growth of hydroxyapatite are significantly induced.

[0082] Figure 8 The surface osteoblast adhesion morphology (1 day) of the coating prepared from Comparative Example 1 (left) and Comparative Example 2 (right). The surface of Comparative Example 1 (left) has a small amount of adhered osteoblasts, and most of the cells are in a round shape, indicating poor cell compatibility; the surface of Comparative Example 2 (right) has a significantly increased number of adhered cells, and filopodia begin to appear. The nanotube surface has a larger surface area and roughness, which can provide more attachment sites for cell adhesion.

[0083] Figure 9 The surface osteoblast adhesion morphology (1 day) of the coating prepared from Example 1 (left) and Example 4 (right). The surface of Example 1 (left) has a large number of adhered osteoblasts, and the cell morphology shows more filopodia, with a predominance of polygonal and dendritic cells. The number of adhered cells and cell activity are significantly increased compared to Comparative Examples 1 and 2. The number of adhered cells of Example 4 (right) begins to decrease significantly, and the proliferation activity also decreases, indicating that the Ga 3+ carrying and release have a certain impact on cells.

[0084] Figure 10 Evaluation of the antibacterial properties (E. coli) of the coatings prepared in Comparative Example 1 (left) and Comparative Example 2 (right). Comparative Example 1 (left) exhibits a high number of viable bacterial colonies, indicating poor antibacterial properties. Comparative Example 2 (right), with its tubular array structure, exhibits a reduced number of bacterial colonies, but the physical antibacterial effect of the surface morphology is very limited, resulting in a high number of bacterial colonies.

[0085] Figure 11 Evaluation of the antibacterial properties of the coatings prepared in Example 1 (left) and Example 4 (right) (Escherichia coli). Example 1 (left) and Example 4 (right) loaded with Ga 3+ The number of colonies on the surface was significantly reduced after the addition of ions. 3+ The concentrations of ions carried and released are positively correlated.

[0086] From the above examples and comparative examples, it can be seen that the present invention combines γ-polyglutamic acid molecules and Ga 3+ The bone-targeting properties of ions and the topological structure of nanotubes guide osteoblast-related cells, apatite and other tissues to grow at the implant interface in the early stage, accelerating bone integration. 3+ Biological functions are highly concentration-dependent, and loading into nanotubes requires further controlled release. γ-polyglutamic acid molecules combined with the middle polydopamine layer effectively release Ga 3+ , avoiding cytotoxicity; the released Ga 3+ The ions have a broad-spectrum antibacterial effect.

Claims

1. A bone-targeted in situ bioactive coating implanted on a titanium surface, characterized in that: The implanted titanium metal material is used as the matrix, and from the inside to the outside it includes titanium dioxide nanotubes loaded with bone-targeting ions, a polydopamine middle layer and a polyanionic amino acid molecular targeting layer.

2. The coating according to claim 1, characterized in that The bone targeting ion is Ga 3+ .

3. The coating according to claim 1, characterized in that The polyanionic amino acid molecule is one or more of gamma-polyglutamic acid or gamma-polyglutamate salt, and has a molecular weight of 10-900 kDa.

4. The coating according to claim 1, characterized in that The inner diameter of the titanium dioxide nanotube is 30-150 nm.

5. A method for preparing a bone-targeted in situ bioactive coating on an implantable titanium surface according to claim 1, characterized in that: The following steps are involved: (1) Titanium dioxide nanotubes are constructed on the surface of implanted titanium metal materials through anodization technology; (2) Using hydrothermal technology to load bone-targeting ions into titanium dioxide nanotubes to obtain titanium dioxide nanotubes loaded with bone-targeting ions; (3) evenly spin-coating the dopamine-Tris solution on the surface of the titanium dioxide nanotubes loaded with bone-targeting ions to form a polydopamine intermediate layer through self-polymerization deposition; (4) A polyanionic amino acid molecule solution is prepared, and after activation treatment, the resulting mixed solution is dripped onto the polydopamine intermediate layer. After amide reaction, a polyanionic amino acid molecule targeting layer is obtained, and finally a bone-targeted in situ bioactive coating is obtained.

6. The preparation method according to claim 5, characterized in that In step (2), the process parameters of the hydrothermal technology are: reaction temperature of 20-90° C., reaction time of 12-72 h, and concentration of the solution containing bone-targeting ion salt of 1-200 mM.

7. The preparation method according to claim 5, characterized in that In step (3), the spin coating method is: first, the dopamine-Tirs solution is dripped onto the surface of the titanium dioxide nanotubes loaded with bone-targeting ions, then spin coating is started, and finally the reaction is allowed to proceed; wherein, the parameters of the static drip are: the unit volume of the solution is 10-1000 μL / cm 2 The number of spin coating is 1-20 times, the spin coating order is first standing, then low speed, and finally high speed, standing for 1-20 minutes, low speed is 100-1000rpm, 1-50s, high speed is 1000-5000rpm, 1-50s; the standing reaction time is 12-36h.

8. The preparation method according to claim 5, characterized in that In step (4), the concentration of the polyanionic amino acid molecule solution is 1-20 mg / mL, and the parameters of the activation treatment are: the activated carboxyl reagent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, the molar ratio of the two is 1:1-1.5, the concentration of N-hydroxysuccinimide in the polyanionic amino acid molecule solution is 10-100 mM, and the activation time is 1-24 h.

9. The preparation method according to claim 5, characterized in that In step (4), the mixed solution is dripped into the polydopamine intermediate layer at a rate of 200-1000 μL / cm 2 , amide reaction 12-36h, repeated 3-6 times.

10. Use of the bone-targeted in situ bioactive coating on the surface of implanted titanium according to any one of claims 1 to 4 as an implant material in the field of orthopedics or dentistry.

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