Implant piezoelectric nano coating as well as preparation method and application thereof

Abstract

The invention discloses an implant piezoelectric nano coating as well as a preparation method and application thereof. The nanometer coating is composed of nanometer flower-shaped barium titanate and gold nanoparticles loaded on the surface of the nanometer flower-shaped barium titanate. According to the invention, gold nanoparticles are deposited on nano barium titanate at a high temperature by using a method of reducing tetrachloroauric acid by using sodium citrate, and nBTOatAu prepared by using the method has obvious osteogenesis and antibacterial properties. The invention provides a high-reactivity, catalytic and non-invasive immediate treatment mode for the peri-implantitis, and has wide application potential in the aspects of post-implant disinfection, peri-implantitis treatment and implant survival rate improvement.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a dental implant piezoelectric nano-coating and a preparation method and application thereof. BACKGROUND

[0002] The plaque accumulation around the infected dental implant can continuously replicate, leading to the occurrence of soft tissue inflammation and alveolar bone destruction around the dental implant. This condition is called peri-implantitis, which is a worldwide problem that has not been solved. Current implant infection therapy focuses on the use of antibiotics, scraping and surgery to remove bacteria. However, complications such as bacterial drug resistance, plaque accumulation, postoperative implant exposure, and even implant shedding have prompted dentists to explore the best treatment method, and many antibacterial strategies based on implant surface modification have been developed, such as loading metal ions, or modifying with quaternary ammonium salt-based polymers. However, their clinical application is usually limited by dose dependence, short-term antibacterial efficacy, or potential side effects on adjacent tissues. Therefore, there is an urgent need for a biocompatible implant surface piezoelectric type electroactive material with on-demand, non-invasive, and efficient antibacterial properties and osteogenesis-promoting properties. Piezoelectric type electroactive materials convert mechanical stimulation into electrical signals, exhibiting a piezoelectric effect similar to that of natural bone.

[0003] Patent CN202011158393.0 discloses a surface metal-loaded piezoelectric material and a preparation method and application thereof. The piezoelectric material is composed of nano-piezoelectric particles with a particle size of 100-500 nm and metal nanoparticles with a diameter of 1-30 nm loaded on the surface thereof; the piezoelectric particles are barium titanate, lithium niobate or lead titanate; and the metal is Au, Pt, Pd, Al or Ni. The preparation method of the surface metal-loaded piezoelectric material comprises the following steps: (1) dispersing the piezoelectric particles in anhydrous ethanol, adding a mercapto reagent, stirring uniformly, and then ultrasonic treatment; and then centrifuging, washing, and drying to obtain mercapto piezoelectric particles; (2) dispersing the mercapto piezoelectric particles in a dispersing agent, adding a metal salt solution and a methanol aqueous solution, stirring uniformly, adjusting the pH of the system to 9-10, and then ultrasonic treatment; and then centrifuging, washing, and drying to obtain the surface metal-loaded piezoelectric material. The metal-loaded piezoelectric material prepared by the method has certain antibacterial properties, but the performance is not strong, and it does not have osteogenesis-promoting properties. SUMMARY

[0004] The present application aims to provide a dental implant piezoelectric nano-coating and a preparation method and application thereof.

[0005] A dental implant piezoelectric nano-coating, which is composed of nano-flower-shaped barium titanate and gold nanoparticles loaded on the surface thereof.

[0006] The preparation method of the dental implant piezoelectric nano-coating is carried out according to the following steps:

[0007] (1) Titanium dioxide preparation: polish the titanium sheet with sandpaper, then ultrasonically clean it with acetone, anhydrous ethanol and deionized water for 10-20 min, respectively, put it into a drying oven, seal it in a bag and store it for later use; place the titanium sheet in an aqueous solution containing 0.3-0.7wt% hydrofluoric acid for anodic oxidation, the anode is a pure titanium sheet test piece, and the cathode is a carbon electrode; after oxidation, ultrasonically clean it with ultrapure water for 3-7 min, dry it, and mark it as TiO2 nanotube;

[0008] (2) Preparation of barium titanate nanoflower: place the TiO2 nanotube in a polytetrafluoroethylene reaction kettle, add 0.05-0.15M barium hydroxide solution, place it in a stainless steel reaction kettle and tighten the screw locking device; heat to 200-240℃ at a rate of 4-6℃ / min and keep for 70-90 min, take it out after cooling in the furnace, dry it for later use, and mark it as nBTO;

[0009] (3) Preparation of gold-loaded barium titanate nanoflower: place the sodium citrate solution, tetrachloroauric acid solution and nBTO in a conical flask containing ultrapure water, ultrasonically treat it for 0.5-1.5 min, then heat and stir it in a constant-temperature magnetic stirring oil bath to boiling for 8-12 min, the solution color gradually changes to wine red, cool it to room temperature to obtain a seedling piezoelectric nanocoating, mark it as nBTO@Au.

[0010] The sandpaper polishing is polishing with 600#, 1000# and 1500# sandpaper in sequence.

[0011] The diameter of the titanium sheet is 1-2 cm, and the thickness is 0.05-0.15 cm.

[0012] The oxidation uses a direct current stabilized power supply, the working voltage is 15-25V, and the oxidation time is 20-40 min.

[0013] The concentration of the sodium citrate solution is 30-50mM, and the concentration of the tetrachloroauric acid solution is 5-15mM; the volume ratio of the sodium citrate solution, tetrachloroauric acid solution and ultrapure water is (2-4):(1-3):80.

[0014] The application of the seedling piezoelectric nanocoating in antibiosis.

[0015] The application of the seedling piezoelectric nanocoating in promoting osteogenesis.

[0016] Beneficial effects of the present invention: The present invention utilizes a method of reducing tetrachloroauric acid with sodium citrate to deposit gold nanoparticles on nano-barium titanate at high temperature. The nBTO@Au prepared by this method has significant osteogenic properties, and the bactericidal activity of nBTO@Au is enhanced under ultrasonic response. The present invention provides a highly reactive, catalytic, and non-invasive instant treatment mode for peri-implantitis, which has broad application potential in post-implant disinfection, peri-implantitis treatment, and improving implant survival rates. The coating prepared by the present invention has a significant antibacterial effect, reduces the probability of infection, alleviates the patient's pain clinically, and has a significant osteogenic effect, which is expected to break through the bottleneck that the current titanium implant has no osteogenic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Surface electron microscope images of Ti, BTO, and nBTO@Au.

[0018] Figure 2 The results of alkaline phosphatase staining.

[0019] Figure 3 This is the result of extracellular matrix mineralization staining.

[0020] Figure 4 Results of plate colony plating. DETAILED DESCRIPTION

[0021] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0022] Example 1

[0023] A method for preparing a piezoelectric nano-coating for an implant is carried out according to the following steps:

[0024] (1) Preparation of titanium dioxide: A titanium sheet (diameter 1.5 cm, thickness 0.1 cm) was polished with 600#, 1000#, and 1500# sandpaper in sequence to obtain a smooth and uniform surface; then, the titanium sheet was ultrasonically cleaned with acetone, anhydrous ethanol, and deionized water for 15 min, respectively, and placed in a drying oven for thorough drying, and then sealed in a bag for later use; the titanium sheet was placed in an aqueous solution containing 0.5 wt% hydrofluoric acid for anodization, with the anode being a pure titanium sheet specimen and the cathode being a carbon electrode. A DC stabilized power supply was used, the operating voltage was 20 V, and the oxidation time was 30 min; the obtained sample was ultrasonically cleaned with ultrapure water for 5 min, dried, and recorded as TiO2 nanotubes;

[0025] (2) Preparation of nanoflower-shaped barium titanate: TiO2 nanotubes prepared in step (1) were placed in a polytetrafluoroethylene reaction kettle, 30 mL of 0.1 M barium hydroxide solution was added, and the stainless steel reaction kettle was placed in a screw lock device; heated to 220°C at a rate of 5°C / min and kept for 80 min, and the sample was taken out after furnace cooling, dried and reserved, and recorded as nBTO;

[0026] (3) Preparation of gold-loaded nanoflower-shaped barium titanate: gold nanoparticles were prepared by sodium citrate reduction of tetrachloroauric acid; 7.5 ml of sodium citrate (40 mM), 5 ml of tetrachloroauric acid (10 mM) and 4 titanium pieces with nBTO coating were placed in a conical flask containing 190 ml of ultrapure water, ultrasonic instrument was used for ultrasonic for 1 min, and then heated and stirred in a constant temperature magnetic stirring oil bath to boiling for 10 min, the solution color gradually changed to wine red, and the implant piezoelectric nano coating was obtained after cooling to room temperature, and recorded as nBTO@Au.

[0027] Example 2

[0028] A preparation method of an implant piezoelectric nano coating, which is carried out according to the following steps:

[0029] (1) Preparation of titanium dioxide: titanium pieces (diameter 1.0 cm, thickness 0.08 cm) were polished with 600#, 1000# and 1500# sandpaper in sequence to obtain a smooth and uniform surface; then ultrasonic cleaning was performed with acetone, anhydrous ethanol and deionized water for 10 min, respectively, and then the titanium pieces were placed in a drying oven for full drying and sealed in a bag for storage; the titanium pieces were placed in a 0.4wt% hydrofluoric acid solution for anodic oxidation, the anode was a pure titanium piece, the cathode was a carbon electrode, a direct current stabilized power supply was used, the working voltage was 15V, and the oxidation time was 40 min; the obtained sample was ultrasonically cleaned with ultrapure water for 4 min, dried, and recorded as TiO2 nanotubes;

[0030] (2) Preparation of nanoflower-shaped barium titanate: TiO2 nanotubes prepared in step (1) were placed in a polytetrafluoroethylene reaction kettle, 30 mL of 0.1 M barium hydroxide solution was added, and the stainless steel reaction kettle was placed in a screw lock device; heated to 220°C at a rate of 5°C / min and kept for 80 min, and the sample was taken out after furnace cooling, dried and reserved, and recorded as nBTO;

[0031] (3) Preparation of gold-loaded nanoflower-shaped barium titanate: Gold nanoparticles were prepared by sodium citrate reduction of tetrachloroauric acid. 7.5 ml of sodium citrate (40 mM), 5 ml of tetrachloroauric acid (10 mM), and 4 titanium sheets containing nBTO coating were placed in a conical flask containing 190 ml of ultrapure water. After ultrasonic treatment for 1 min, the solution was heated and stirred in a constant-temperature magnetic stirring oil bath until boiling for 8 min. The solution color gradually changed to wine red. After cooling to room temperature, the implant piezoelectric nanocoating was obtained, denoted as nBTO@Au.

[0032] Example 3

[0033] A method for preparing an implant piezoelectric nanocoating was performed according to the following steps:

[0034] (1) Preparation of titanium dioxide: The titanium sheet (diameter 2 cm, thickness 0.1 cm) was polished with 600#, 1000#, and 1500# sandpaper in sequence to obtain a smooth and uniform surface. Then it was ultrasonically cleaned with acetone, anhydrous ethanol, and deionized water for 20 min, respectively, and then placed in a drying oven for full drying and sealed in a bag for storage. The titanium sheet was placed in a solution containing 0.6wt% hydrofluoric acid for anodic oxidation. The anode was a pure titanium sheet specimen, and the cathode was a carbon electrode. A direct current stabilized power supply was used, with a working voltage of 25V and an oxidation time of 25 min. The obtained sample was ultrasonically cleaned with ultrapure water for 6 min and dried, denoted as TiO2 nanotube;

[0035] (2) Preparation of nanoflower-shaped barium titanate: The TiO2 nanotube prepared in step (1) was placed in a polytetrafluoroethylene reaction kettle, 30 mL of 0.15M barium hydroxide solution was added, and it was placed in a stainless steel reaction kettle and screwed tightly with a screw locking device. It was heated to 240℃ at a rate of 6℃ / min and kept for 85 min. After cooling in the oven, the sample was taken out and dried for use, denoted as nBTO.

[0036] (3) Preparation of gold-loaded nanoflower-shaped barium titanate: Gold nanoparticles were prepared by sodium citrate reduction of tetrachloroauric acid. 7.5 ml of sodium citrate (40 mM), 5 ml of tetrachloroauric acid (10 mM), and 4 titanium sheets containing nBTO coating were placed in a conical flask containing 190 ml of ultrapure water. After ultrasonic treatment for 1.5 min, the solution was heated and stirred in a constant-temperature magnetic stirring oil bath until boiling for 12 min. The solution color gradually changed to wine red. After cooling to room temperature, the implant piezoelectric nanocoating was obtained, denoted as nBTO@Au.

[0037] Experimental Example 1: Material characterization

[0038] The nBTO@Au prepared in Example 1 was used as the detection object.

[0039] Characterization method: The sample was fixed on a tray using a conductive adhesive and sputtered with a thin layer of platinum (Pt) to improve its conductivity. It was then placed in a field emission scanning electron microscope to observe the surface features of the sample.

[0040] The results are shown in Figure 1 Ti surface, except for a small amount of scratches left by polishing, is basically smooth; the surface micro-morphology of BTO formed under hydrothermal environment at 220°C for 80 min presents a coral-like arrangement, and the morphology is uniform; the nBTO@Au can successfully grow in situ on BTO, the diameter of gold particles is relatively uniform, and the distribution is relatively uniform.

[0041] Experimental example 2: alkaline phosphatase staining

[0042] The nBTO@Au prepared in Example 1 was used as the detection object.

[0043] The BMSCs were inoculated on each group of samples at a cell density of 2×10 4 / ml, and osteogenic induction was started when the cell confluence reached 75%. After 7 days of induction culture, ALP kit was used for staining, and the specific operation steps were as follows:

[0044] (1) Remove the osteogenic induction medium and wash with PBS twice;

[0045] (2) Add 1ml of 4% paraformaldehyde solution to each well, and fix at room temperature for 30min;

[0046] (3) Remove the fixing solution and wash with PBS twice to ensure that the fixing solution is completely washed off;

[0047] (4) Add 500ul of alkaline phosphatase staining working solution to each well, and fix at room temperature for 10min;

[0048] (5) Remove the alkaline phosphatase staining working solution and wash with PBS twice to thoroughly wash away the excess staining solution;

[0049] (6) Observe and take pictures under a stereomicroscope.

[0050] The proportion of the alkaline phosphatase detection working solution is as follows: 3ml of color developing buffer, 10ul of BCIP (300X), and 20ul of NBT (150X).

[0051] The results show that according to the kit instructions, under the action of ALP in the cells, BCIP hydrolysis product reacts strongly with NBT to form insoluble dark blue to blue-violet compounds. The experimental results show that after 7 days of osteogenic induction, insoluble compounds are formed in each group, and the color depth is proportional to the ALP activity, as shown in Figure 2

[0052] ALP activity is nBTO@Au > BTO > Ti.

[0053] Experimental Example 3: Alizarin red staining

[0054] The nBTO@Au prepared in Example 1 was taken as the detection object.

[0055] The BMSCs were inoculated in each group of samples at a cell density of 2x10 4 / ml, and osteogenic induction was started when the cell confluence reached 75%. The induction culture was performed for 14 days, and then ARS kit was used for staining. The specific operation steps were as follows:

[0056] (1) The osteogenic induction medium was aspirated, and PBS was washed twice;

[0057] (2) 1ml of 4% paraformaldehyde solution was added to each well, and fixed at room temperature for 30min;

[0058] (3) The fixing solution was aspirated, and PBS was washed twice to ensure that the fixing solution was completely washed off;

[0059] (4) 500ul of alizarin red working solution was added to each well, and fixed at room temperature for 10min;

[0060] (5) The alizarin red working solution was aspirated, and PBS was washed twice to completely wash off the excess staining solution;

[0061] (6) Observed and photographed under a stereomicroscope.

[0062] Figure 3 The results showed that the extracellular matrix mineralization of each group of coating surface was induced for 14 days. Alizarin red staining solution mainly chelates with calcium ions in the solution to form insoluble complexes that can be directly observed under a microscope. The staining depth is positively correlated with the degree of extracellular matrix mineralization. The alizarin red staining results showed that the nBTO@Au group was the deepest, the BTO group was the second, and the Ti group was the shallowest.

[0063] In summary, we believe that the nBTO@Au piezoelectric coating constructed on the titanium surface has the ability to promote osteogenesis.

[0064] Experimental Example 4: Plate coating experiment

[0065] The nBTO@Au prepared in Example 1 was taken as the detection object.

[0066] The front and back of the materials required for the experiment were sterilized by ultraviolet light for 30min. Then using sterile forceps, each group of samples was placed into a 24-well plate with the treated surface facing up, and a micropipette was used to add 1mL of 1x10 6S. aureus suspension of CFU / mL, incubated at 37℃ for 6h, the ultrasonic group was treated by ultrasound, the ultrasonic time was 5min, the power was 1.0MHz, 1.5W / cm 2 , the duty cycle was 50%, after ultrasonic treatment, the sample was incubated for 24h, then the culture medium was discarded, the sample was washed with PBS for 3 times, 1ml PBS was added to each well, the well plate was placed in the ultrasonic instrument for 5min (300w, 40KHz), so that the bacteria adhered to the surface of the sample were resuspended in the PBS solution. The bacterial suspension adhered to the surface of the sample was collected, diluted to 10 5 times with PBS, and 100uL of the diluted solution was uniformly coated on the culture medium, and then the sample was incubated in the aerobic incubator at 37℃ until the observation node, and then the colony counting was performed.

[0067] In order to evaluate the survival rate of single-species bacteria under the action of Au-TNT, the plate coating experiment was carried out, and the plate coating results of S. aureus are shown in Figure 4 Fig. 6. The number of bacteria on the BTO group and the nBTO@Au group was slightly reduced compared with the Ti group, which may be because the structure of the BTO coating layer affects the stress response of the bacteria, leading to the rupture of the cell membrane and the reduction of the number of bacteria. However, this antibacterial effect is too poor to eliminate bacteria and prevent the spread of infection. In contrast, the survival rate of bacteria in the US(+) group decreased to different degrees, and the antibacterial effect of the nBTO@Au+US group was the best. The results of our study show that after adding Au NPs, the ultrasonic treatment time is greatly shortened, and the antibacterial rate is obviously increased within 5min, which is mainly because the deposition of Au NPs on TNT prevents the recombination of electron / hole pairs, the ROS yield is higher, and the sonodynamic effect is enhanced. In addition, compared with the BTO+US group, the antibacterial rate of the nBTO@Au+US group is higher within the same treatment time, which further proves that ROS plays a key role in the antibacterial effect mediated by SDT.

[0068] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A piezoelectric nano coating for an implant, characterized in that: The nano coating consists of nano flower-shaped barium titanate and gold nanoparticles loaded on the surface of the nano coating.

2. The method for preparing the implant piezoelectric nanocoating according to claim 1, characterized in that: Follow these steps: (1) Preparation of titanium dioxide: polish the titanium sheet with sandpaper, then ultrasonically clean it with acetone, anhydrous ethanol, and deionized water for 10-20 minutes respectively, place it in a drying oven for drying, and seal it in a sealed bag for later use; place the titanium sheet in an aqueous solution containing 0.3-0.7wt% hydrofluoric acid for anodization, with the anode being a pure titanium sheet specimen and the cathode being a carbon electrode; after oxidation, ultrasonically clean it with ultrapure water for 3-7 minutes, dry it, and record it as TiO2 nanotubes; (2) Preparation of nano-flower-like barium titanate: The TiO2 nanotubes prepared in step (1) were placed in a polytetrafluoroethylene reactor, 0.05-0.15M barium hydroxide solution was added, and the reactor was placed in a stainless steel reactor sleeve and the spiral locking device was tightened; the reactor was heated to 200-240°C at a heating rate of 4-6°C / min and maintained for 70-90 minutes, and then the reactor was cooled and removed from the reactor, dried and set aside, and recorded as titanium sheets containing nBTO coating; (3) Preparation of gold-loaded nanoflower-like barium titanate: Sodium citrate solution, tetrachloroauric acid solution and titanium sheet containing nBTO coating were placed in a conical flask containing ultrapure water. After ultrasonication for 0.5-1.5 minutes, the solution was heated and stirred in a constant temperature magnetic stirring oil bath until boiling for 8-12 minutes. The color of the solution gradually turned into wine red. After cooling to room temperature, the implant piezoelectric nanocoating was obtained, which was recorded as nBTO@Au.

3. The method for preparing the implant piezoelectric nanocoating according to claim 2, characterized in that: The sandpaper polishing is performed in sequence using 600#, 1000# and 1500# sandpaper.

4. The method for preparing the implant piezoelectric nanocoating according to claim 2, characterized in that: The titanium sheet has a diameter of 1-2 cm and a thickness of 0.05-0.15 cm.

5. The method for preparing the implant piezoelectric nanocoating according to claim 2, characterized in that: The oxidation uses a DC stable power supply with an operating voltage of 15-25V and an oxidation time of 20-40min.

6. The method for preparing the implant piezoelectric nanocoating according to claim 2, characterized in that: The concentration of the sodium citrate solution is 30-50 mM, and the concentration of the tetrachloroauric acid solution is 5-15 mM; the volume ratio of the sodium citrate solution, the tetrachloroauric acid solution and ultrapure water is (2-4): (1-3):

80.

7. Use of the implant piezoelectric nanocoating according to claim 1 in antibacterial applications.

8. Use of the implant piezoelectric nanocoating according to claim 1 in promoting osteogenesis.

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