A PP-BST 3-x Implant and its preparation method and application

Abstract

The present invention discloses a PP-BST 3‑x The invention relates to an implant and its preparation method and application, which belongs to the field of medical material technology. The invention constructs oxygen vacancies (BST) on the surface of strontium ion-doped barium titanate. 3‑x ), under ultrasonic conditions, the internal electron movement of the material is enhanced, making the electron-hole separation easier, thereby accelerating the reaction of electrons with surrounding substances and improving the efficiency of ROS generation. And the PP-BST prepared by the present invention 3‑x On the one hand, the implant can polarize macrophages through electrical potential and inhibit inflammation; on the other hand, it can regulate Sr release through ultrasound, thereby inhibiting osteoclast differentiation in an infected environment, achieving dual-pathway osteogenesis, and improving the high osteoclast environment brought about by the bacterial microenvironment while inhibiting bacteria, thereby promoting bone regeneration, accelerating bone healing ability, and reducing patient recovery time.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and specifically relates to a PP-BST 3-x Implant, preparation method and application thereof. Background Art

[0002] The primary cause of infection in implants is bacterial adhesion and colonization on their surfaces, leading to the formation of a biofilm. Bacterial infection makes it difficult for bone tissue around the implant to regenerate. Once a biofilm forms on the implant surface, persistent inflammation and necrosis of the surrounding tissue can ultimately lead to implant failure. Because microorganisms within biofilms can resist host immune attacks and are less sensitive to antibiotics, their removal is particularly challenging clinically.

[0003] Currently, surgical removal of infected implants is still considered the standard of care, but it significantly prolongs the patient's hospital stay and greatly increases medical costs. In order to circumvent the inevitable obstacles of surgical resection, various implant surface modification strategies have been developed to combat bacterial infections. Implant surface modification is the antibacterial modification of the implant surface to reduce bacterial adhesion and inhibit plaque biofilm formation, thereby preventing early postoperative infection. Although this strategy has achieved remarkable success in antibacterial treatment, the antibacterial coatings currently applied to implant surfaces are mostly traditional antibacterial agents. The relatively long sterilization time and potential bacterial resistance of traditional antibacterial agents have largely hindered their widespread application in clinical practice. In addition, the current antibacterial coatings on implant surfaces still have defects such as structural stability, controllability, and poor timeliness.

[0004] Recently, sonodynamic therapy (SDT) has received widespread attention in the treatment of implant infections due to its advantages such as bacterial resistance, minimal invasiveness, deep tissue penetration and immediate therapeutic effects. However, the sonosensitizers currently used in SDT are insufficient to kill bacteria in clinical settings. Existing materials have low efficiency in generating ROS and difficulty in electron-hole separation. In addition, existing defect engineering only focuses on the generation of ROS, but does not pay attention to the fact that the bacterial infection environment induces osteoclast activity, thereby inhibiting local bone regeneration and inducing implant loosening. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a PP-BST 3-x The invention relates to an implant and a preparation method and application thereof, so as to solve the technical problems of low ROS generation efficiency and low bone regeneration efficiency of existing materials.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is to provide a PP-BST 3-x The method for preparing an implant comprises the following steps:

[0007] S1. Dissolve the titanium source in an organic solvent, then add alkali solution until no sediment is formed to obtain a titanium-containing suspension. The addition of alkali solution will promote the hydrolysis reaction of the titanium source to generate Ti(OH)4 precipitation; dissolve the barium source and strontium source in water, then add the mixed solution to the titanium-containing suspension, react at 170-190℃ and 1-5MPa for 46-50h, then wash, and place the washed product at 70-90℃ to dry for 22-26h to obtain BST nanoparticles. Finally, place the BST nanoparticles in a reducing atmosphere and react at 450-550℃ for 10-14h to obtain BST 3-x Nanoparticles; the molar ratio of Ti, Ba and Sr elements in the titanium source, barium source and strontium source is 0.5-1.5:2-4:0.1-0.5;

[0008] S2. Dopamine (DA) is dissolved in a buffer solution, and the PEKK scaffold is then completely immersed in the dopamine solution for 22-26 hours to form a uniform polydopamine (pDA) coating on the surface of the PEKK scaffold through oxidative polymerization of dopamine. The scaffold is then washed with water to remove unreacted dopamine monomers and other residues, thereby obtaining a PEKK@pDA scaffold (PP).

[0009] S3, BST 3-x (Ba 0.9 Sr 0.1 TiO 3-x ) nanoparticles were uniformly dispersed in water, and then the PEKK@pDA scaffold was placed on BST 3-x Soak in the suspension for 10-14 hours to allow BST to 3-x The nanoparticles were fixed to the surface of the PP scaffold by physical adsorption or chemical bonding, and finally dried at 70-90 ° C for 10-14 h to obtain PP-BST. 3-x Implant.

[0010] On the basis of the above technical solution, the present invention can also be improved as follows:

[0011] Furthermore, the titanium source is Ti(C4H9O)4, Ti[OCH(CH3)2]4 or TiCl4, the barium source is Ba(OH)2˖8H2O, Ba(NO3)2 or BaCO3, and the strontium source is Sr(NO3)2, SrCl2 or SrCO3.

[0012] Furthermore, the organic solvent is ethanol.

[0013] Furthermore, the alkali solution is aqueous ammonia (NH3·H2O), sodium bicarbonate (NaHCO3) or ammonium hydroxide (NH4OH).

[0014] Furthermore, the washing liquid used for washing is at least one of acetic acid, ethanol and deionized water.

[0015] Furthermore, the reducing atmosphere is a mixture of H2 and Ar in a volume ratio of 90-97:3-10.

[0016] Furthermore, the buffer is Tris-HCl buffer, PBS buffer or sodium bicarbonate buffer.

[0017] Furthermore, the concentration of the dopamine solution is 2-4 mg / mL, and the pH value is 8-9.

[0018] Further, BST 3-x The concentration of the suspension is 2-4 mg / mL.

[0019] The present invention also discloses PP-BST prepared by the above preparation method. 3-x Implant.

[0020] The present invention also discloses PP-BST 3-x Application of implants in the preparation of osteogenic materials.

[0021] The beneficial effects of the present invention are:

[0022] 1. Aiming at the problems of low ROS generation efficiency and difficulty in electron-hole separation in existing materials, the present invention constructs oxygen vacancies (BST) on the surface of strontium ion-doped barium titanate. 3-x ), the presence of oxygen vacancies makes it easier for electrons and holes to separate, thereby accelerating the reaction between electrons and surrounding substances, improving the efficiency of ROS production and making the bacteria killing effect stronger.

[0023] 2. To address the problem of difficulty in regenerating bone tissue around implants after bacterial infection, the PP-BST prepared by the present invention 3-x On the one hand, the implant can polarize macrophages through electric potential to inhibit inflammation; on the other hand, it can regulate Sr release through ultrasound, thereby inhibiting osteoclast differentiation in an infected environment, achieving dual-pathway osteogenesis. 3-x The implant can inhibit bacteria while improving the high osteoclast environment caused by the bacterial microenvironment, promoting bone regeneration, accelerating bone healing ability, and reducing patient recovery time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 BST 3-x TEM images of

[0025] Figure 2 PEKK@pDA scaffold (PP) and PP-BST 3-x SEM comparison images of implants;

[0026] Figure 3 PEKK@pDA scaffold (PP) and PP-BST3-x Comparison of the compressive Young's modulus of the implants;

[0027] Figure 4 PEKK@pDA scaffold (PP) and PP-BST 3-x Comparison of contact angle data between implants and water;

[0028] Figure 5 The ability of different Sr components to generate ROS;

[0029] Figure 6 PP-BST 3-x Ability to produce ROS (·OH);

[0030] Figure 7 PP-BST 3-x Produces singlet oxygen (1O 2 ) ability;

[0031] Figure 8 Comparison of bacterial communities on different materials in plate experiments;

[0032] Figure 9 Live and dead cell staining diagrams for different materials;

[0033] Figure 10 The staining diagrams are for M1 and M2 macrophages;

[0034] Figure 11 This is the trap staining result;

[0035] Figure 12 The staining results of the osteogenic marker RUNX2 are shown. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0037] Example 1

[0038] A PP-BST 3-x The method for preparing an implant comprises the following steps:

[0039] S1. Dissolve 4 mL of Ti[OCH(CH3)2]4 in 10 mL of ethanol and stir for 30 minutes. Then, add sodium bicarbonate until no sediment is formed (Ti(OH)4 precipitates) to obtain a titanium-containing suspension. Then transfer the titanium-containing suspension to a Teflon-lined autoclave, dissolve 10 g of Ba(NO3)2 and 8 g of SrCl2 in 20 mL of water at 50°C, stir for 10 minutes, add the mixture to the titanium-containing suspension, and react at 170°C and 5 MPa for 50 hours. Then, wash with acetic acid, ethanol, and deionized water in sequence, and dry the washed product at 90°C for 22 hours to obtain BST nanoparticles. Finally, place the BST nanoparticles in a reducing atmosphere (H2:Ar=90:10, v:v) and react at 450°C for 14 hours to obtain BST. 3-x Nanoparticles;

[0040] S2. Dopamine (DA) was dissolved in sodium bicarbonate buffer. The PEKK scaffold was then completely immersed in the dopamine solution (concentration: 2 mg / mL, pH: 8) and stirred for 22 h. The scaffold was then washed with water to obtain the PEKK@pDA scaffold.

[0041] S3, BST 3-x (Ba 0.9 Sr 0.1 TiO 3-x ) nanoparticles were ultrasonically dispersed in water, and then the PEKK@pDA scaffold was placed on the BST 3-x The suspension (concentration of 4 mg / mL) was allowed to stand for 10 h, and finally dried at 90 ° C for 10 h to obtain PP-BST. 3-x Implant.

[0042] Example 2

[0043] A PP-BST 3-x The method for preparing an implant comprises the following steps:

[0044] S1. Dissolve 3 mL of TiCl4 in 10 mL of ethanol and stir for 30 minutes, then add ammonium hydroxide until no sediment is formed (Ti(OH)4 precipitation) to obtain a titanium-containing suspension; then transfer the titanium-containing suspension to a Teflon-lined autoclave, dissolve 9 g of BaCO3 and 0.6 g of SrCO3 in 20 mL of water at 50°C, stir for 10 minutes, add the mixture to the titanium-containing suspension, react at 190°C and 1 MPa for 46 hours, then wash with acetic acid, ethanol and deionized water in sequence, and dry the washed product at 70°C for 26 hours to obtain BST nanoparticles. Finally, place the BST nanoparticles in a reducing atmosphere (H2:Ar=97:3, v:v) and react at 550°C for 10 hours to obtain BST 3-x Nanoparticles;

[0045] S2. Dopamine (DA) was dissolved in PBS buffer. The PEKK scaffold was then completely immersed in the dopamine solution (4 mg / mL, pH 9) and stirred for 26 h. The scaffold was then washed with water to obtain the PEKK@pDA scaffold.

[0046] S3, BST 3-x (Ba 0.9 Sr 0.1 TiO 3-x ) nanoparticles were ultrasonically dispersed in water, and then the PEKK@pDA scaffold was placed on the BST 3-x The suspension (concentration of 2 mg / mL) was allowed to stand for 14 h, and finally dried at 70 ° C for 14 h to obtain PP-BST. 3-x Implant.

[0047] Example 3

[0048] A PP-BST 3-x The method for preparing an implant comprises the following steps:

[0049] S1. 3.41 mL of Ti(C4H9O)4 was dissolved in 10 mL of ethanol and stirred for 30 min. Ammonia water was then added until no more sediment was formed (Ti(OH)4 precipitated) to obtain a titanium-containing suspension. The titanium-containing suspension was then transferred to a Teflon-lined autoclave. 9.45 g of Ba(OH)2˖8H2O and 0.7 g of Sr(NO3)2 were dissolved in 20 mL of water at 50 °C. After stirring for 10 min, the mixture was added to the titanium-containing suspension and reacted at 180 °C and 3 MPa for 48 h. The mixture was then washed with acetic acid, ethanol, and deionized water in sequence. The washed product was dried at 80 °C for 24 h to obtain BST nanoparticles. Finally, the BST nanoparticles were placed in a reducing atmosphere (H2:Ar=95:5, v:v) and reacted at 500 °C for 12 h to obtain BST. 3-x Nanoparticles, the TEM image of which is as follows Figure 1 As shown, the formation of oxygen vacancies is also shown;

[0050] S2. Dopamine (DA) was dissolved in Tris-HCl buffer. The PEKK scaffold was then completely immersed in the dopamine solution (concentration 3 mg / mL, pH 8.5) and stirred for 24 h. The scaffold was then washed with water to obtain the PEKK@pDA scaffold (PP).

[0051] S3, BST 3-x (Ba 0.9 Sr 0.1 TiO 3-x) nanoparticles were ultrasonically dispersed in water, and then the PEKK@pDA scaffold was placed on the BST 3-x The suspension (concentration of 3 mg / mL) was allowed to stand for 12 h, and finally dried at 80 ° C for 12 h to obtain PP-BST. 3-x Implant.

[0052] PEKK@pDA scaffold (PP) and PP-BST 3-x SEM images of implants Figure 2 As shown in the figure, it can be seen that the PP porous implant was successfully prepared, and BST 3-x It is ellipsoidal and evenly distributed on the PP surface.

[0053] Example 4

[0054] The difference between this example and example 3 is that the amount of Sr(NO3)2 added is adjusted to 0.32g (5%), and the remaining components and preparation steps are the same as those in example 3 to obtain PP-BST. 3-x Implant.

[0055] Example 5

[0056] The difference between this example and example 3 is that the amount of Sr(NO3)2 added is adjusted to 0.64g (10%), and the remaining components and preparation steps are the same as those in example 3 to obtain PP-BST. 3-x Implant.

[0057] Example 6

[0058] The difference between this embodiment and embodiment 3 is that the amount of Sr(NO3)2 added is adjusted to 0.96g (15%), and the remaining components and preparation steps are the same as those in embodiment 3 to obtain PP-BST. 3-x Implant.

[0059] Comparative Example

[0060] The difference between this comparative example and Example 3 is that Sr(NO3)2 is omitted, and the remaining components and preparation steps are the same as those in Example 3 to obtain a PP-BT implant.

[0061] The PP-BST used in the following experiments 3-x The implant is PP-BST prepared in Example 3 3-x implants

[0062] Experimental Example 1 Mechanical Properties

[0063] Verification of PP-BST by universal mechanical testing 3-x Mechanical properties of implants, test methods refer to ISO 10993 standard. Figure 3As shown, PP-BST 3-x The Young's modulus of the implant is 248 MPa, which is lower than that of bone, causing less wear on the bone and more conducive to long-term use.

[0064] PP-BST verified by testing 3-x The contact resistance of the implant with water is tested by cleaning the surface of the material and then drying it to ensure that there is no contaminant or grease on the surface. Pure water is dropped on the surface of the material, 3-5 drops of water are dropped on each sample to ensure the repeatability of the test, and the angle formed by the water drop on the surface is measured by a contact angle meter. The results are as follows: Figure 4 As shown in the figure, it can be seen that compared with the PP bracket, the BST 3-x The scaffold of the material has good contact with water, which is conducive to bone growth into the prosthesis.

[0065] Experimental Example 2 ROS production ability test

[0066] The DPBF degradation experiment was conducted to investigate the effect of different Sr doping amounts (Examples 4-6) on the amount of ROS generated by surface oxygen vacancies. The specific steps were as follows: DPBF was dissolved in ethanol to prepare a 100μM DPBF solution, and the material to be tested was dissolved or dispersed in the ethanol solution to ensure uniform distribution; the DPBF solution and the sample solution were mixed in a certain proportion to ensure that the final concentration of DPBF was 100μM, and the mixed solution was sonicated for 10 minutes using an ultrasonic device (power 1W / cm²), and finally the absorbance change of DPBF at 410nm was measured using a UV-Vis spectrophotometer, the absorbance change over time was recorded, and the ratio to the initial value was calculated. The results are shown in Figure 2. Figure 5 As shown, even when the doping amount of Sr(NO3)2 is 10%, PP-BST 3-x ROS production by the implants remained unaffected.

[0067] To evaluate the production of ·OH, the control group (H2O), PP-BT, and PP-BST 3-x The sample was mixed with a solution containing MB and stimulated by US (1 MHz, 1.0 W / cm 2 , 50% duty cycle), and recorded the changes in MB absorption at 664 nm before and after US stimulation. Figure 6 As shown in the figure, it can be seen that PP-BST 3-x The lowest MB fluorescence intensity indicates that MB is degraded. 3-x It has the strongest ability to produce ·OH.

[0068] The control group, PP-BT, and PP-BST 3-x The sample was mixed with a solution containing SOSG, and the results were as follows Figure 7As shown, singlet oxygen (1O) was confirmed by SOSG at 525 nm. 2 ) exists, PP-BST 3-x The fluorescence intensity of the group increased, indicating that PP-BST 3-x It produces the most ROS, providing an important basis for sterilization. Figure 6 and Figure 7 The black line in the figure represents the control group, the red line represents the PP-BT group, and the blue line represents the PP-BST group. 3-x Group.

[0069] Experimental Example 3 Antibacterial Performance Test

[0070] The coated plate method was used to evaluate the PP-BST under different US irradiation conditions. 3-x The bactericidal effect on Staphylococcus aureus (ATCC25923) is as follows: PP-BST 3-x Implants were exposed to bacterial suspension (2 × 10 7 CFU / mL) for 6 h, and the experimental group was cultured at 1W / cm 2 The cells were irradiated with US for 9 minutes. The control group was not irradiated with US. The cells were cultured on agar plates at 37°C for 18 hours and the colony forming units (cfu) were quantified. Figure 8 shown.

[0071] For further verification, the bacteria were cultured on different supports for 5 days and then subjected to live / dead staining assay using the live / dead BacLight activity kit. Figure 9 As shown, PP-BST 3-x There was less green fluorescence (live bacteria) than in the control group, indicating that PP-BST 3-x The implant has good antibacterial ability in the simulated in vivo environment.

[0072] Experimental Example 4 Osteogenesis Performance Test

[0073] Evaluation of PP-BST by macrophage (M1, M2) staining 3-x Promote the polarization of M2 macrophages, such as Figure 10 As shown, red is M1 and green is M2; compared with the control group, PP-BST 3-x Having more M2 macrophages has a better inhibitory effect on inflammation and is beneficial to improving the inflammatory state of the microenvironment.

[0074] Assessment of PP-BST using trap staining 3-xTo evaluate the ability of different implants and bacterial suspensions to inhibit osteoclast differentiation, RAW 264.7 cells were co-cultured with the bacterial suspensions to evaluate their inhibitory effects on osteoclast formation. Osteoclast formation was induced by bacterial suspensions. For F-actin ring staining, cells were stained with Actin-Tracker Green to visualize the F-actin rings, and cell nuclei were labeled with DAPI. The stained cells were observed under a fluorescence microscope. Figure 11 As shown in the figure, the smaller the ring is, the stronger the ability to inhibit osteoclasts is. 3-x It has a stronger ability to inhibit osteoclast differentiation.

[0075] Detection of PP-BST using osteoblast-related markers 3-x To evaluate osteogenic capacity, MC3T3-E1 cells were seeded into each well of a 12-well plate. After reaching 70% confluence, the α-MEM medium was replaced with 10 mM β-glycerophosphate and 50 μg mL -1 Osteogenesis was induced by osteoinduction medium containing ascorbic acid and 10 nM dexamethasone (both from Sigma). The osteoinduction medium was refreshed every 3 days along with the materials. For immunofluorescence analysis of RUNX2, each group of cells was treated with a specific primary antibody: anti-RUNX2, followed by a secondary antibody Alexa Fluor 594 (1:500, Invitrogen). The cytoskeleton was stained with FITC-labeled phalloidin, and the nucleus was stained with DAPI to facilitate microscopic imaging and comparison of osteogenic properties. The results are shown in Figure 2. Figure 12 As shown in the figure, the more gold the stronger the bone formation ability. It can be seen that the BSTx group with Sr is better than the BT group without Sr. 3-x It can inhibit osteoclasts, indicating that PP-BST 3-x Has stronger osteogenic properties.

Claims

1. A PP-BST 3-x The method for preparing an implant is characterized in that: The following steps are involved: S1. A titanium source is dissolved in an organic solvent, and then an alkali solution is added until no sediment is formed to obtain a titanium-containing suspension; a barium source and a strontium source are dissolved in water, and then the mixture is added to the titanium-containing suspension, and the mixture is reacted at 170-190°C and 1-5MPa for 46-50 hours, and then washed, and the washed product is placed at 70-90°C and dried for 22-26 hours to obtain BST nanoparticles. Finally, the BST nanoparticles are placed in a reducing atmosphere and reacted at 450-550°C for 10-14 hours to obtain BST 3-x Nanoparticles; the molar ratio of Ti, Ba and Sr elements in the titanium source, barium source and strontium source is 0.5-1.5:2-4:0.1-0.5; the gas used in the reducing atmosphere is H2 and Ar mixed in a volume ratio of 90-97:3-10; S2. Dopamine was dissolved in a buffer solution, and the PEKK scaffold was then immersed in the dopamine solution for 22-26 hours, followed by washing to obtain a PEKK@pDA scaffold. S3, BST 3-x The nanoparticles were dispersed in water and then the PEKK@pDA scaffold was placed on BST 3-x Soak in the suspension for 10-14 hours, and finally dry at 70-90 ° C for 10-14 hours to obtain PP-BST 3-x Implant.

2. The PP-BST according to claim 1 3-x The method for preparing an implant is characterized in that: The titanium source is Ti(C4H9O)4, Ti[OCH(CH3)2]4 or TiCl4, the barium source is Ba(OH)2˖8H2O, Ba(NO3)2 or BaCO3, and the strontium source is Sr(NO3)2, SrCl2 or SrCO3.

3. The PP-BST according to claim 1 3-x The method for preparing an implant is characterized in that: The organic solvent is ethanol.

4. The PP-BST according to claim 1 3-x The method for preparing an implant is characterized in that: The alkali solution is ammonia water, sodium bicarbonate or ammonium hydroxide.

5. The PP-BST according to claim 1 3-x The method for preparing an implant is characterized in that: The washing liquid used for the washing is at least one of acetic acid, ethanol and deionized water.

6. The PP-BST according to claim 1 3-x The method for preparing an implant is characterized in that: The buffer is Tris-HCl buffer, PBS buffer or sodium bicarbonate buffer.

7. The PP-BST according to claim 1 3-x The method for preparing an implant is characterized in that: The BST 3-x The concentration of the suspension is 2-4 mg / mL.

8. A PP-BST3-x implant, characterized in that: Using the PP-BST described in any one of claims 1 to 7 3-x The implant is prepared by a method for preparing the implant.

9. The PP-BST according to claim 8 3-x Application of implants in the preparation of osteogenic materials.

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