A Ti-BTx implant body, a preparation method and application thereof

Abstract

The application discloses a Ti-BTx implant, a preparation method and application thereof, and belongs to the technical field of material preparation. The application constructs oxygen vacancies (BTx) on the surface of barium titanate (BT) through defect engineering, increases the effect of the material on the influence of bacteria electrons, inhibits the electron transfer of the bacterial electron transfer chain, and further inhibits the proliferation of bacteria and hinders the formation of bacterial biofilm, thereby reducing the implant infection. As long as the bacteria contact the Ti-BTx implant, the proliferation of the bacteria can be inhibited for a long time, and the time for preventing infection is prolonged. Moreover, the Ti-BTx implant prepared by the application can also promote the proliferation of bone cells. Although the BTx influences the electron transfer of bacteria, the electron transfer of cells is located in mitochondria rather than cell membranes, so the BTx can promote the proliferation of bone cells through potential change, improves the bone healing capacity, and reduces the recovery time of patients.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical materials, and particularly relates to a Ti-BTx implant and a preparation method and application thereof. BACKGROUND

[0002] Bone implant infection is a disastrous surgical complication, and the main cause of implant-induced infection is the adhesion and colonization of bacteria on the surface of the implant, and then the formation of a biofilm. Once a biofilm is formed on the surface of the implant, the implant will eventually fail due to continuous inflammation and surrounding tissue necrosis. Due to bacterial drug resistance and limited drug penetration at the infection site, the efficacy of systemic antibiotic administration is compromised, and bacterial infection severely affects bone regeneration, resulting in delayed healing and unbearable pain at the patient's fracture site. Therefore, endowing the implant with antibacterial properties, especially hindering biofilm formation, has become the key to solving implant infection.

[0003] Currently, antibiotics are used before and after surgery to prevent implant infection, but the effect is not good. Therefore, some studies use gelatin and other materials to load antibiotics on the surface of the implant to achieve slow release and antibacterial effect, thereby preventing implant infection. This solves the problem of low efficiency of systemic antibiotic use, but it still cannot avoid antibiotic resistance and the emergence of superbugs. In addition, some studies use reactive oxygen species (ROS) for antibacterial purposes, but they not only kill bacteria, but also accelerate cell aging, which is not conducive to tissue regeneration. Moreover, the production of ROS often requires external intervention, such as ultrasonic or light stimulation. However, there is currently no method for early diagnosis of infection in clinical practice, so the use of ROS for antibacterial purposes is still far from clinical application. Neither antibiotics nor ROS can kill bacteria to a certain extent, but they will inhibit bone tissue regeneration. Therefore, it is necessary to develop an implant coating that targets bacteria without affecting cell proliferation, can long-term inhibit bacterial proliferation, and thus prevent implant infection and promote bone tissue regeneration. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a Ti-BTx implant and a preparation method and application thereof to solve the technical problem that the existing implant cannot achieve both osteogenesis and antibacterial effect.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is to provide a preparation method of a Ti-BTx implant, comprising the following steps:

[0006] S1, acid pickling treatment is performed on a titanium sample to remove the natural oxide layer on the surface, and then the titanium sample is placed in an electrolyte for electrolytic oxidation for 1.5-2.5 h to obtain a porous titanium implant;

[0007] S2, dissolving the titanium salt in an organic solvent, then adjusting the pH value to 5-7 to avoid fast hydrolysis of the solution, then adding the barium salt solution to react for 0.5-2h, and finally adding ammonia water to react for 5-60min to obtain the barium titanate sol; the ratio of the titanium salt, the barium salt and the ammonia water is 0.1-0.3mol:0.1-0.3mol:10-50mL;

[0008] S3, immersing the porous titanium implant into the barium titanate sol to react for 12-48h to ensure that the sol is fully infiltrated into the porous structure, then taking out the sample and drying at room temperature for 10-14h to make the sol gradually gelatinize on the surface of the porous titanium, ensuring that the surface is uniformly dried and a gel layer is formed, then placing the dried sample in a heat treatment at 200-600℃ for 3.5-6.5h, and finally slowly cooling to room temperature to avoid damage to the structure of the barium titanate coating caused by sharp temperature change, to obtain the Ti-BTx implant.

[0009] Based on the technical scheme, the application can be further improved as follows:

[0010] Further, before the acid washing treatment, the titanium sample is further subjected to ultrasonic cleaning with ethanol and water (ultrasonic power: 1-3w, ultrasonic time: 5-15min) to remove impurities on the surface of the sample.

[0011] Further, the acid washing treatment is: washing the titanium sample with 5-10wt% hydrofluoric acid solution for 3-5 times, and each washing time is 5-60s.

[0012] Further, the electrolytic oxidation process is: immersing the titanium sample as an anode and an inert metal as a cathode into an electrolyte, and the distance between the anode and the cathode is 5-20cm to ensure uniform current distribution, then electrolytic oxidation under a direct current voltage of 40-60v for 1.5-2.5h.

[0013] Further, the electrolyte is a mixture of ammonium fluoride, ethylene glycol and water in a ratio of 0.3-0.7g:90-100mL:3-7mL.

[0014] Further, the titanium salt is butyl titanate (Ti(OBu)4), isopropyl titanate or titanium tetrachloride; and the barium salt is barium nitrate (Ba(NO3)2), barium carbonate or barium acetate.

[0015] Further, the reagent used for adjusting the pH value is glacial acetic acid.

[0016] Further, the heat treatment process is: first, preheating the sample at 180-220℃ for 1.5-2.5h to remove residual solvents and further solidify the gel layer; then, setting a protective atmosphere for the reaction system and sintering at 550-650℃ for 2-4h.

[0017] Further, the gas used in the protective atmosphere is argon or hydrogen.

[0018] Further, the heating rate in the preheating stage is 2-5℃ / min, and the heating rate in the sintering stage is 3-10℃ / min.

[0019] Further, the cooling rate is 3-5℃ / min.

[0020] The application further discloses the Ti-BTx implant prepared by the preparation method.

[0021] The application further discloses application of the Ti-BTx implant in preparation of osteogenic materials.

[0022] The application has the following beneficial effects:

[0023] 1. The application adopts a non-antibiotic strategy to prevent implant infection, utilizes the fast proliferation characteristics of bacteria in the process of biofilm formation, constructs oxygen vacancies (BTx) on the surface of barium titanate (BT) through defect engineering, increases the effect of the material on the electron of bacteria, completely interferes with the electron transfer chain of bacteria, inhibits the electron transfer of the electron transfer chain of bacteria, and then inhibits the proliferation of bacteria and hinders the formation of bacterial biofilm, thereby reducing implant infection.

[0024] 2. The Ti-BTx implant prepared in the application can not only inhibit bacterial proliferation, but also promote the proliferation of bone cells. Although the BTx affects the electron transfer of bacteria, the electron transfer of cells is located in mitochondria rather than cell membranes, so the BTx can promote the proliferation of bone cells through potential change. The different mechanisms of the oxygen vacancy piezoelectric material on bacteria and cells achieve two functions of one material. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 SEM image of the Ti-BTx implant;

[0026] Figure 2 SEM surface morphology of the Ti-BTx implant after magnification;

[0027] Figure 3 Influence of sintering time on the membrane potential of bacteria;

[0028] Figure 4 Comparison of the antibacterial ability of Ti and Ti-BTx;

[0029] Figure 5 Influence of Ti and Ti-BTx on the outer membrane potential of bacteria;

[0030] Figure 6 Cell live and dead staining diagram;

[0031] Figure 7Results for Ti-BTx osteogenic ability. DETAILED DESCRIPTION

[0032] The following describes the specific embodiments of the present application, so that those skilled in the art can understand the present application. The specific conditions are not specified in the examples, and are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market. However, it should be clear that the present application is not limited to the scope of the specific embodiments, and that for those skilled in the art, any changes that are obvious within the spirit and scope of the present application as defined and determined by the appended claims are all included in the protection of the present application.

[0033] Example 1

[0034] A method for preparing a Ti-BTx implant, comprising the following steps:

[0035] S1, sequentially ultrasonic cleaning the titanium sample with ethanol and deionized water (ultrasonic power is 1w, ultrasonic time is 15min), then rinsing the titanium sample with 5wt% hydrofluoric acid solution for 5 times, each time for 5s, then immersing the titanium sample as an anode and platinum as a cathode (the distance between the cathode and the anode is 20cm) into an electrolyte (ammonium fluoride: ethylene glycol: water = 0.7g: 90mL: 7mL), electrolytic oxidation under a direct current voltage of 40v for 2.5h, finally rinsing the sample surface with deionized water and placing it in nitrogen, drying at 120℃ for 2h, to obtain a porous titanium implant;

[0036] S2, dissolving 0.3mol of butyl titanate in 50mL of ethanol, then adding glacial acetic acid to adjust the pH value of the solution to 7; then dissolving 0.1mol of barium nitrate in 50mL of deionized water, slowly dropping the barium nitrate solution into the butyl titanate solution and reacting for 2h, finally adding 50mL of ammonia water and reacting for 60min, to obtain a barium titanate sol;

[0037] S3, immersing the porous titanium implant into the barium titanate sol and reacting for 12h, then taking out the sample and drying at room temperature for 10h; then first increasing the temperature from room temperature to 220℃ at a heating rate of 5℃ / min, placing the sample at this temperature for 1.5h of preheating, then setting the reaction system to a hydrogen atmosphere, and continuing to increase the temperature to 550℃ at a heating rate of 3℃ / min, and sintering at this temperature for 3h, after the heat treatment, slowly cooling to room temperature at a cooling rate of 3℃ / min, finally washing the reactant with water, to obtain a Ti-BTx implant.

[0038] Example 2

[0039] A preparation method of a Ti-BTx implant, comprising the following steps:

[0040] S1, sequentially ultrasonic cleaning titanium samples with ethanol and deionized water (ultrasonic power is 3w, ultrasonic time is 5min), then flushing the titanium sample with 10wt% hydrofluoric acid solution for 3 times, each time for 60s, then immersing the titanium sample as anode and platinum as cathode (the distance between anode and cathode is 5cm) into electrolyte (ammonium fluoride: ethylene glycol: water = 0.3g: 100mL: 3mL), electrolytic oxidation under 60v direct current for 1.5h, finally flushing the sample surface with deionized water, placing it in nitrogen, drying at 80℃ for 12h, obtaining a porous titanium implant;

[0041] S2, dissolving 0.1mol butyl titanate in 50mL ethanol, then adding glacial acetic acid to adjust the pH value of the solution to 5; then dissolving 0.3mol barium nitrate in 50mL deionized water, slowly dropping the barium nitrate solution into the butyl titanate solution and reacting for 0.5h, finally adding 10mL ammonia water and reacting for 5min, obtaining barium titanate sol;

[0042] S3, immersing the porous titanium implant into the barium titanate sol and reacting for 48h, then taking out the sample and drying at room temperature for 14h; then first increasing the temperature from room temperature to 180℃ at a heating rate of 2℃ / min, placing the sample at the temperature for preheating for 2.5h, then setting the reaction system as argon atmosphere, and continuing to increase the temperature to 650℃ at a heating rate of 10℃ / min, and sintering at the temperature for 2h, after the heat treatment, slowly cooling to room temperature at a cooling rate of 5℃ / min, finally washing the reactant with water, obtaining a Ti-BTx implant.

[0043] Example 3

[0044] A preparation method of a Ti-BTx implant, comprising the following steps:

[0045] S1, sequentially ultrasonic cleaning titanium samples with ethanol and deionized water (ultrasonic power is 2w, ultrasonic time is 10min), then flushing the titanium sample with 8wt% hydrofluoric acid solution for 4 times, each time for 30s, then immersing the titanium sample as anode and platinum as cathode (the distance between anode and cathode is 10cm) into electrolyte (ammonium fluoride: ethylene glycol: water = 0.5g: 95mL: 5mL), electrolytic oxidation under 50v direct current for 2h, finally flushing the sample surface with deionized water, placing it in nitrogen, drying at 100℃ for 7h, obtaining a porous titanium implant;

[0046] S2, 0.2 mol of butyl titanate was dissolved in 50 mL of ethanol, then glacial acetic acid was added to adjust the pH value of the solution to 6; then 0.2 mol of barium nitrate was dissolved in 50 mL of deionized water, and the barium nitrate solution was slowly added dropwise into the butyl titanate solution and reacted for 1 h, and finally 30 mL of ammonia water was added and reacted for 30 min to obtain a barium titanate sol;

[0047] S3, the porous titanium implant was immersed in the barium titanate sol and reacted for 30 h, then the sample was taken out and dried at room temperature for 12 h; then the temperature was first increased from room temperature to 200℃ at a heating rate of 3℃ / min, the sample was preheated at this temperature for 2 h, then the reaction system was set to an argon atmosphere, and the temperature was continued to increase to 600℃ at a heating rate of 6℃ / min, and sintered at this temperature for 4 h, after the heat treatment was completed, it was slowly cooled to room temperature at a cooling rate of 4℃ / min, and finally the reaction was washed with water to obtain a Ti-BTx implant.

[0048] Example 4

[0049] The difference between this example and Example 3 is that the sintering time of step S3 is adjusted to 2 h, and the rest of the implementation conditions are the same as those of Example 3, to obtain a Ti-BTx implant.

[0050] Comparative Example

[0051] The difference between this comparative example and Example 3 is that the sintering time of step S3 is adjusted to 1 h, and the rest of the implementation conditions are the same as those of Example 3, to obtain a Ti-BTx implant.

[0052] Experimental Example 1 Structural Characterization

[0053] Example 3 was taken as an example to characterize the Ti-BTx implant, and the SEM image thereof is shown in Figure 1 As can be seen from the figure, Ti has a micro-porous structure, which is beneficial to the fixation of the implant and the adhesion of cells. The Figure 1 was enlarged to further observe the details of the Ti-BTx implant, as shown in Figure 2 As can be seen from the figure, the Ti-BTx implant is successfully synthesized, and its morphology is close to spherical and uniformly distributed on the surface of the titanium implant, which is also beneficial to the uniform effect of BTx on the bone.

[0054] The sintering time of step S3 is different, and the proportion of oxygen vacancies in the final product is also different. The Ti-BTx implants obtained in Example 3, Example 4 and the comparative example were compared in terms of the effect on the potential of bacteria, and the results are shown in Figure 3As shown, the Ti-BTx formed by treatment at different times has different membrane potential changing abilities on bacteria. The more green the color, the greater the effect on membrane potential. It can be seen that the Ti-BTx implant formed when the sintering time is 4h (Example 3) has a higher membrane potential changing ability, which increases the effect of the Ti-BTx material on bacterial electrons, completely interferes with the bacterial electron transport chain, and inhibits the electron transfer of the bacterial electron transport chain.

[0055] The Ti-BTx implant used in the following experiments is the Ti-BTx implant prepared in Example 3.

[0056] Experimental Example 2 Test of the performance of inhibiting biofilm formation

[0057] The effect of Ti-BTx on Staphylococcus aureus (ATCC 25923) was evaluated by plate spreading method. The specific process was as follows: Ti-BTx implants were placed in 48-well plates and exposed to bacterial suspension (bacterial concentration was 2×10 7 CFU / mL) for 8 h, and then the colony-forming units (CFU) were quantified by plating on agar plates at 37°C for 12 h. For further verification, bacteria were added to Ti and Ti-BTx plates and cultured at 37°C for 1 day. Live / dead staining was then performed using the live / dead BacLight activity kit. Figure 4 As shown, compared with the control group (Ti group) in which no oxygen vacancies were formed, the Ti-BTx group (Ti-BTx implant prepared in Example 3) had a more obvious effect of inhibiting bacterial proliferation, indicating that the Ti-BTx implant prepared in the present invention has a good ability to inhibit biofilm formation in a simulated in vivo environment.

[0058] The bacterial membrane potential of Staphylococcus aureus was evaluated using DiBAC4 (3). A bacterial suspension of Staphylococcus aureus (5 × 10 7 CFU mL -1 ) were applied to Ti and Ti-BTx surfaces respectively and incubated for 6 h. Then the culture medium was replaced with LB medium containing DiBAC4 (3) (5 μM) and incubated at 37 °C for another 2 h. The outer membrane potential of bacteria was detected and evaluated by fluorescence imaging, as shown in Figure 2. Figure 5 As shown, the bacterial outer membrane potential was found to change after contact with Ti-BTx implants, which was due to the inhibition of electron transport by BTx.

[0059] Experimental Example 3 Osteogenesis Performance Test

[0060] MC3T3-E1 osteoblast-related cells were seeded onto the Ti-BTx surface and cultured in a 37°C incubator for 24 h. Cell viability and death staining ( Figure 6) As can be seen, green indicates live cells. In the Ti-BTx group, there were no obvious dead cells, and BTx also promoted cell proliferation. The osteogenic capacity of Ti and Ti-BTx was then assessed using ALP and ARS. MC3T3-E1 cells were seeded into each well of a 12-well plate. When cell confluence reached 70%, the α-MEM medium was replaced with osteogenic induction medium containing 10 mM β-glycerophosphate, 50 µg / mL ascorbic acid (Sigma), 50 µg / mL ascorbic acid, 10 mM β-glycerophosphate (Sigma), and 10 nM dexamethasone (Sigma) to promote the osteogenic capacity of MC3T3-E1 osteoblasts and implants. The osteoinduction medium was refreshed along with the materials every 3 days. On days 7 and 14, ALP activity was assessed using a 5-bromo-4-chloro-3-indoleyl phosphate / nitro blue tetrazolium (BCIP / NBT) ALP colorimetric kit (Beyotime, China). To evaluate the calcified extracellular matrix, the cells were fixed and treated with ARS dye (Beyotime, China), and ARS was measured on the 14th day. After removing the excess dye, the image was captured using a scanner. The more red the image, the more ARS and the better the osteogenic ability. The staining results show that the Ti-BTx implant has a good osteogenic ability (e.g. Figure 7 ).

Claims

1. A method of producing a Ti-BTx implant, characterized by, The method comprises the following steps: S1, acid washing a titanium sample, and then electrolyzing and oxidizing the titanium sample in an electrolyte for 1.5-2.5 h to obtain a porous titanium implant; S2, dissolving a titanium salt in an organic solvent, then adjusting the pH value to 5-7, then adding a barium salt solution and reacting for 0.5-2 h, and finally adding ammonia water and reacting for 5-60 min to obtain a barium titanate sol; the ratio of the titanium salt, the barium salt and the ammonia water is 0.1-0.3 mol: 0.1-0.3 mol: 10-50 mL; S3, immersing the porous titanium implant in the barium titanate sol and reacting for 12-48 h, then taking out the sample and drying at room temperature for 10-14 h, then preheating the dried sample at 180-220 ℃ for 1.5-2.5 h, then setting the reaction system to a protective atmosphere and sintering at 550-650 ℃ for 2-4 h, and finally cooling to room temperature to obtain a Ti-BTx implant.

2. The method of claim 1, wherein the Ti-BTx implant is prepared by, The acid washing treatment is: washing the titanium sample with a 5-10 wt% hydrofluoric acid solution for 3-5 times, and each washing time is 5-60 s.

3. The method of claim 1, wherein the Ti-BTx implant is prepared by, The electrolytic oxidation process is: immersing the titanium sample as an anode and an inert metal as a cathode in an electrolyte, the distance between the anode and the cathode is 5-20 cm, and then electrolyzing and oxidizing under a direct current voltage of 40-60 V for 1.5-2.5 h.

4. The method of claim 3, wherein the Ti-BTx implant is prepared by, The electrolyte is a mixture of ammonium fluoride, ethylene glycol and water in a ratio of 0.3-0.7 g: 90-100 mL: 3-7 mL.

5. The method of claim 1, wherein the Ti-BTx implant is prepared by, The titanium salt is butyl titanate, isopropyl titanate or titanium tetrachloride; and the barium salt is barium nitrate, barium carbonate or barium acetate.

6. The method of claim 1, wherein the Ti-BTx implant is prepared by, The heating rate in the preheating stage is 2-5 ℃ / min, and the heating rate in the sintering stage is 3-10 ℃ / min.

7. The method of claim 1, wherein the Ti-BTx implant is prepared by, The cooling rate is 3-5 ℃ / min.

8. A Ti-BTx implant, characterized in that, The Ti-BTx implant is prepared by the method of any one of claims 1-7.

9. Use of the Ti-BTx implant of claim 8 in preparing osteogenic materials.

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