GTR film as well as preparation method and application thereof

Abstract

The invention discloses a GTR film as well as a preparation method and application thereof, belongs to the technical field of biomedical materials, and solves the problem that the antibacterial effect of a GTR film in the prior art is not ideal enough. The method comprises the following steps: uniformly dispersing BaTiO3 into a Tris buffer solution, dissolving dopamine hydrochloride powder into a reaction system, uniformly stirring, centrifuging to obtain a precipitate, cleaning, and drying to obtain PDA-coated BaTiO3 nanoparticles; adding the PDA-coated BaTiO3 nanoparticles into a silver-ammonia solution, stirring at room temperature, centrifuging, taking precipitate, cleaning and drying to obtain Ag-coated PDA-coated BaTiO3 particles; and uniformly dispersing the Ag-coated PDA-coated BaTiO3 particles in a hexafluoroisopropanol solution of poly-L-lactic acid, carrying out electrostatic spinning on the reaction system to obtain a fiber membrane, and sequentially carrying out ventilation, high-temperature annealing and natural cooling to room temperature to obtain the GTR membrane. The GTR film provided by the invention has double physical clues of piezoelectric and photothermal effects, has the effects of rapid sterilization and long-acting bacteriostasis, and is simple in preparation process.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and more specifically to a GTR membrane and its preparation method and application. Background Art

[0002] Periodontitis causes the loss of hard and soft tissues, and even leads to tooth loss, affecting the quality of life of patients. Guided tissue regeneration (GTR) technology is a key surgical strategy for periodontal treatment. The GTR membrane is a barrier membrane placed between the gum and the tooth after flap surgery to prevent the faster-growing epithelial cells and soft tissue-derived cells from growing towards the root, and to gain time for the undifferentiated cells derived from the periodontal ligament. If the undifferentiated cells can occupy the blood clot and the exposed root surface first, they can differentiate into different tissue-forming cells such as osteoblasts and fibroblasts, thus forming a complete set of periodontal tissues including bone tissue, periodontal ligament and cementum.

[0003] During GTR surgery, physical scraping during the operation can only remove most of the irritants, and there are still many bacteria colonized in places that the surgical instruments cannot reach. Drugs are often needed to cooperate to improve the sterilization effect. During the long healing process, due to the open environment of the oral cavity, various opportunistic pathogens colonize and reproduce, and the barrier membrane is prone to bacterial infection as an implant material. This situation not only affects the surgical effect, but may even lead to surgical failure. Currently, commercially available barrier membranes are mainly used as physical barriers and lack appropriate antibacterial ability.

[0004] Therefore, a barrier membrane with long-term antibacterial effect is urgently needed to solve the limitations of traditional options. Summary of the Invention

[0005] The present invention provides a GTR membrane and its preparation method and application to solve the problem that the antibacterial effect of the GTR membrane in the prior art is not ideal enough.

[0006] In a first aspect, the present invention provides a preparation method of a GTR membrane, including the following steps: uniformly dispersing BaTiO3 into Tris buffer solution, dissolving dopamine hydrochloride powder into the reaction system, stirring evenly, centrifuging to obtain a precipitate, cleaning and drying to obtain PDA@BaTiO3 nanoparticles; adding the PDA@BaTiO3 nanoparticles into silver ammonia solution, stirring at room temperature, centrifuging to obtain a precipitate, cleaning and drying to obtain Ag@PDA@BaTiO3 particles; uniformly dispersing the Ag@PDA@BaTiO3 particles in a hexafluoroisopropanol solution of poly-L-lactic acid, electrospinning the reaction system to obtain a fibrous membrane, and successively performing ventilation, high-temperature annealing, and natural cooling to room temperature to obtain the GTR membrane.

[0007] As a possible implementation method, the preparation method of the silver ammonia solution includes the steps of: dissolving AgNO3 in deionized water, adding ammonia water drop by drop while oscillating until the initially formed precipitate just dissolves.

[0008] As a possible implementation method, the concentration of the Tris buffer solution is 0.01 - 0.05 mol / L, the pH is 8.5, and the particle size of the dispersed phase is 10 mm; and / or, the concentration of silver ions in the silver ammonia solution is 0.04 - 0.08 mol / mL; and / or, the mass-volume fraction of the solution of L-lactic acid in hexafluoroisopropanol is 8% - 12%.

[0009] As a possible implementation method, calculated in g / mL, the ratio of BaTiO3 to the Tris buffer solution is 1:100 - 120; and / or, the mass ratio of dopamine hydrochloride to BaTiO3 is 1:4 - 5; and / or, calculated in g / mL, the ratio of PDA@BaTiO3 nanoparticles to the silver ammonia solution is 1:40 - 50.

[0010] As a possible implementation method, the cleaning includes the steps of: washing 3 times with deionized water; and / or, the drying includes the steps of: freeze-drying for 24 - 36 h.

[0011] As a possible implementation method, the electrospinning includes the steps of: placing a syringe filled with the reaction system 15 cm away from the receiver, operating at a flow rate of 1 mL / h, and performing electrospinning at a voltage of 15 kV; and / or, the conditions for the electrospinning are: the speed of the receiver is 1500 r / min, and the total working time is 210 min; and / or, the conditions for the high-temperature annealing are: high-temperature annealing at 105 °C for 10 h.

[0012] In a second aspect, the present invention provides a GTR membrane prepared by the preparation method according to any one of the possible implementation methods in the first aspect.

[0013] In a third aspect, the present invention provides an application of the GTR membrane according to any one of the possible implementation methods in the second aspect in the preparation of an implant material after periodontal flap surgery.

[0014] The piezoelectric effect refers to the phenomenon that when certain crystals or polymer materials (such as barium titanate, zinc oxide, polyvinylidene fluoride, etc.) are subjected to pressure, vibration or deformation, the internal charge distribution changes, resulting in the formation of a potential difference on the surface of the material. Piezoelectric antibacterial is an emerging antibacterial strategy that utilizes the property of piezoelectric materials to generate an electric field under mechanical stress to inhibit or kill bacteria. This electromechanical coupling property provides a unique physical approach for antibacterial applications: when a piezoelectric material comes into contact with bacteria and is subjected to external mechanical stimuli (such as body fluid flow, human movement or ultrasonic waves), the piezoelectric field generated on its surface can directly damage the integrity of the bacterial cell membrane, or interfere with bacterial metabolism and cause its death by catalyzing the production of free radicals such as reactive oxygen species (ROS).

[0015] In recent years, the research on near-infrared (NIR) responsive photothermal materials in the field of antibacterial has also attracted much attention. Near-infrared photothermal materials can absorb light energy of specific wavelengths and efficiently convert it into heat energy, generating instantaneous high temperatures (above 50 - 60 °C) locally, directly damaging the bacterial cell membrane structure, denaturing proteins and causing DNA damage. In addition, these materials (such as noble metal nanostructures, carbon-based materials, semiconductor polymers, etc.) can also be combined with photothermal effects and chemical / photodynamic synergistic antibacterial mechanisms through surface functionalization or composite design to further enhance the antibacterial efficiency and reduce the required light power.

[0016] Compared with traditional antibiotics and chemical disinfectants, piezoelectric materials and near-infrared responsive photothermal antibacterial materials have significant advantages. First, their action mechanisms rely on physical electric fields, free radical reactions or thermal effects, which can effectively avoid the problem of pathogen drug resistance; second, piezoelectric materials do not require an external power source and can be activated only by mechanical energy in the environment. Compared with traditional photosensitizers excited by ultraviolet or visible light, near-infrared light has a deeper tissue penetration depth (up to centimeter level), low absorption and scattering characteristics in biological tissues, providing the possibility for the treatment of deep infections. Therefore, combining the piezoelectric effect and the photothermal effect and applying them to the GTR membrane is a promising strategy, which is beneficial to achieving efficient and long-term antibacterial effects of the membrane.

[0017] The present invention provides a GTR membrane with piezoelectric and photothermal effect dual physical cues, which has rapid sterilization and long-term antibacterial effects. Its preparation process is simple. By coating Ag@PDA with photothermal effect on barium titanate and then doping it into PLLA for electrospinning to form a membrane, the piezoelectric performance of the membrane material is improved and excellent photothermal conversion performance is additionally obtained. During GTR surgery, near-infrared light is used to quickly kill bacteria, avoiding the use of additional antibiotics. During the subsequent healing process, the piezoelectric effect continuously plays a role to maintain the antibacterial effect. Description of the Drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 Optical photograph of the electrospun membrane provided by the embodiment of the present invention.

[0020] Figure 2 SEM image of the electrospun membrane provided by the embodiment of the present invention.

[0021] Figure 3 Open-circuit voltage measurement result of the GTR membrane provided by the embodiment of the present invention.

[0022] Figure 4 Photothermal performance test result of the GTR membrane provided by the embodiment of the present invention.

[0023] Figure 5 Antibacterial performance test result of the GTR membrane provided by the embodiment of the present invention.

[0024] Figure 6 Biocompatibility test result of the GTR membrane provided by the embodiment of the present invention, where Control is the control group. Detailed implementation manners

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] To solve the problem that the antibacterial effect of the GTR membrane in the prior art is not ideal enough, the embodiment of the present invention provides an experiment on the preparation and performance test of a GTR membrane.

[0027] The following will further elaborate on the technical solutions of the present invention with reference to specific embodiments.

[0028] Embodiment 1

[0029] This embodiment provides an experiment on the preparation of a GTR membrane.

[0030] Dissolve 302.5 mg of Tris (>99.9% (T), Aladdin, China) in 250 mL of deionized water to form a Tris buffer solution (pH 8.5, 10 mM); uniformly disperse 2.5 g of BaTiO3 (200 nm) powder into the 250 mL of the prepared Tris buffer solution, and ultrasonicate for 30 min to make it uniformly dispersed. Subsequently, dissolve 0.5 g of dopamine hydrochloride powder into the reaction system, stir at 60 °C for 12 h, then centrifuge, and wash the precipitate with deionized water three times. Freeze-dry for 24 h to obtain PDA@BaTiO3 nanoparticles.

[0031] Add 1 g of the prepared PDA@BaTiO3 nanoparticles to 50 mL of 0.4 M silver ammonia solution (dissolve 340 mg of AgNO3 in 50 mL of deionized water, then add ammonia water drop by drop while shaking until the initially formed precipitate just dissolves), stir at room temperature for 2 h, so that the Ag + in the solution is in-situ reduced to Ag nanoparticles by polydopamine and loaded onto the surface of PDA@BaTiO3. Centrifuge, and wash the precipitate with deionized water three times to remove the unreacted AgNO3. Finally, freeze-dry for 24 h to obtain Ag@PDA@BaTiO3 particles, and its structure is a core-shell structure.

[0032] Dissolve poly-L-lactic acid (PLLA, molecular weight 260,000, Jinan Daigang Biotechnology Co., Ltd.) in hexafluoroisopropanol (Aladdin) to prepare a solution with a mass-volume fraction of 10%, stir for 2 h to ensure that PLLA is completely dissolved in hexafluoroisopropanol. Add the prepared Ag@PDA@BaTiO3 particles, ultrasonically disperse for 2 h to make the nanoparticles uniformly dispersed. Place a 10 mL syringe with a 19-gauge needle 15 cm away from the receiver, work at a flow rate of 1 mL / h, perform electrospinning at a voltage of 15 kV, the speed of the receiver is 1500 r / min, and the total working time is 210 min. Place the electrospun fiber membrane in the fume hood overnight to allow the residual organic solvents to fully evaporate. Then anneal the membrane at 105 °C for 10 h and then naturally cool to room temperature. The obtained material is PLLA+AgPBT, that is, the GTR membrane.

[0033] Dope barium titanate (BTO) nanoparticles and the prepared PDA@BaTiO3 nanoparticles into a 10% (mass-volume fraction) PLLA solution respectively, and prepare PLLA+BTO membranes and PLLA+PBT membranes respectively with the same electrospinning parameters, and prepare pure PLLA membranes with the same electrospinning parameters.

[0034] The optical pictures of PLLA, PLLA+BTO, PLLA+PBT, and PLLA+AgPBT are as Figure 1As shown. Scanning electron microscopy was performed on PLLA, PLLA + BTO (PLLA / BT), PLLA + PBT (PLLA / PBT), and PLLA + AgPBT in this example respectively, and the SEM results are as shown in Figure 2 . As can be seen from Figure 1 and Figure 2 , with the loading of different nanoparticles, the color of the membrane changes from light to dark; the nanoparticles are uniformly dispersed in the PLLA fibers, proving the successful preparation of the GTR membrane.

[0035] Example 2

[0036] This example provides a performance test experiment for the GTR membrane.

[0037] The piezoelectric properties of the membrane were verified by testing the electrical output performance of the membrane under ultrasonic stimulation, and the open-circuit voltage of the membrane was measured by an electrochemical workstation. The piezoelectric properties of PLLA, PLLA + BTO (PLLA + BT), PLLA + PBT, and PLLA + AgPBT in Example 1 were verified in this example, and the results are as shown in Figure 3 . Figure 3 It can be seen that compared with the pure PLLA membrane, the piezoelectric output of the composite membrane with added nanoparticles is enhanced. The piezoelectric performance of PLLA is weak. After combining with the piezoelectric ceramic BTO, the material has both excellent piezoelectric output and the softness of the organic piezoelectric material. The reason for the further improvement of the piezoelectric output of PLLA + PBT may be that the polydopamine layer on the surface of barium titanate reduces the interfacial energy gap between BTO and PLLA. The piezoelectric output of the PLLA + AgPBT group is the largest, probably because the addition of Ag as a conductive phase is beneficial to the transfer of electrons.

[0038] To study the in vitro photothermal conversion effect of the electrospun membrane, 808 nm near-infrared laser irradiation was used, and an infrared thermal imager was used to measure the temperature change of the GTR membrane in a PBS wet environment under irradiation with different power densities, and the results are as shown in Figure 4 . As can be seen from Figure 4 , the temperature changes after irradiation for 1 / 3 / 5 / 7 / 10 min at power densities of 1.5, 1.8, and 2.0 W / cm 2 were observed. The temperature change of the membrane is positively correlated with the power density of the near-infrared light. In contrast, the temperature of the PLLA + AgPBT membrane at 2.0 W / cm 2 rose from 23 °C to 38.2 / 52.1 / 54.8 / 58.0 / 60.1 °C respectively after near-infrared irradiation for 1 / 3 / 5 / 7 / 10 min. Therefore, the GTR membrane prepared in Example 1 has good photothermal conversion efficiency, and because of 2.0 W / cm 2Under such conditions, the PLLA+AgPBT film can be heated to above 50 °C within 5 min, which is a temperature suitable for antibacterial purposes. Therefore, for subsequent bacterial experiments, near-infrared light with a power of 2.0 W / cm 2 was selected as the experimental parameter.

[0039] Staphylococcus aureus was taken out from a -80 °C ultra-low temperature refrigerator and placed in a laminar flow hood for operation. After the bacterial strain melted, a sterilized inoculation loop was used to pick up the frozen bacterial mass in the test tube and place it into 20 mL of Luria-Bertani (LB) medium, and cultured overnight at 37 °C and 150 rpm. The overnight bacterial liquid was taken, and its OD 630 was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the bacterial liquid concentration was diluted to 1×10 6 for standby. The GTR film prepared in Example 1 was cut into 1.5 cm × 1.5 cm, soaked in 75% alcohol for 30 min, and then placed in the laminar flow hood and irradiated with ultraviolet light for 12 h for standby. Sterilized films were added into a 24-well plate, and then 500 μL of the diluted bacterial liquid was added. In the control group (Control), only 500 μL of the diluted bacterial liquid was added. The above samples were respectively subjected to ① ultrasonic treatment at 10 6 CFU / mL for 5 / 10 / 20 min (US+), ② near-infrared light irradiation for 5 / 10 / 20 min (NIR), and ③ ultrasonic treatment for the same time after near-infrared treatment for 5 / 10 / 20 min (NIR / US+). After the treatment was completed, plating was carried out. 100 μL of the incubated bacterial liquid (diluted to 10 3 CFU / mL) was dropped onto the agar plate, and then it was evenly spread in this area with a disposable spreading rod. The coated agar plate was placed in an incubator at 37 °C and incubated overnight. After the incubation was completed, the agar plate was placed under a colony counter for photographing. The colony statistical chart is as shown in Figure 5 As shown, it can be seen from Figure 5 that the film with piezoelectric effect and photothermal effect has excellent antibacterial effects after ultrasonic treatment and near-infrared light treatment. According to calculations, after 5 min of ultrasonic treatment, the material provided by the present invention achieved an antibacterial rate of approximately 18.5% through the piezoelectric effect, while a 98% antibacterial rate can be achieved by single near-infrared irradiation for 5 min. When most bacteria were killed by near-infrared irradiation and then ultrasonic treatment was continued, the antibacterial efficiency of the film was greatly improved, almost reaching 100%.

[0040] The CCK-8 kit was used to study the cytotoxicity on PLLA, PLLA+BTO (PLLA+BT), PLLA+PBT, and PLLA+AgPBT films. The GTR film prepared in Example 1 was cut into a size of 1.5 cm × 1.5 cm, soaked in 75% ethanol, irradiated with ultraviolet light for 30 minutes, and then washed with phosphate buffer solution (PBS). In each well of a 24-well plate, 500 μL of cell suspension (5×105 Inoculate (cell / mL) into the well plate, place it in a humidified incubator containing 5% CO2, and incubate overnight at 37°C. After the cells adhere to the wall, wash them with PBS, add the sterilized membrane material, and add 500 μL of complete DMEM medium to each well. For the control group, add cells growing in the medium without the material. Incubate the plate for 24 hours, then take out the supernatant, wash it with PBS, and add cell culture medium containing 10% CCK-8. After further incubation for 2 hours, measure the absorbance of the medium at 450 nm using an Infinite F50 microplate reader (Tecan, USA) to obtain the results as Figure 6 shown. It can be seen from the results that PLLA, PLLA+BT, PLLA+PBT, and PLLA+AgPBT all have good biocompatibility.

[0041] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0042] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A method for preparing a GTR membrane, characterized in that, It includes the following steps: Disperse BaTiO3 evenly in Tris buffer solution, dissolve dopamine hydrochloride powder in the reaction system, stir evenly, centrifuge to obtain the precipitate, clean and dry to obtain PDA@BaTiO3 nanoparticles; Add the PDA@BaTiO3 nanoparticles into silver ammonia solution, stir at room temperature, centrifuge to obtain the precipitate, clean and dry to obtain Ag@PDA@BaTiO3 particles; Disperse the Ag@PDA@BaTiO3 particles evenly in the hexafluoroisopropanol solution of poly-L-lactic acid, electrospin the reaction system to obtain a fiber membrane, and successively carry out ventilation, high-temperature annealing, and natural cooling to room temperature to obtain the GTR membrane.

2. The preparation method according to claim 1, wherein The preparation method of the silver ammonia solution includes the steps: Dissolve AgNO3 in deionized water, then dropwise add ammonia water while oscillating until the initially formed precipitate just dissolves.

3. The preparation method according to claim 1, characterized in that, The concentration of the Tris buffer solution is 0.01 - 0.05 mol / L, the pH is 8.5, and the particle size of the dispersed substance is 10 mm; And / or, the concentration of silver ions in the silver ammonia solution is 0.04 - 0.08 mol / mL; And / or, the mass-volume fraction of the hexafluoroisopropanol solution of poly-L-lactic acid is 8% - 12%; 4. The preparation method according to claim 1, characterized in that, Calculated by g / mL, the ratio of BaTiO3 to the Tris buffer solution is 1:100 - 120; And / or, the mass ratio of dopamine hydrochloride to BaTiO3 is 1:4 - 5; And / or, calculated by g / mL, the ratio of the PDA@BaTiO3 nanoparticles to the silver ammonia solution is 1:40 - 50.

5. The preparation method according to claim 1, wherein The cleaning includes the steps: wash 3 times with deionized water; And / or, the drying includes the steps: freeze-dry for 24 - 36 h.

6. The preparation method according to claim 1, characterized in that, The electrospinning includes the steps: place the syringe filled with the reaction system 15 cm away from the receiver, work at a flow rate of 1 mL / h, and carry out electrospinning at a voltage of 15 kV; And / or, the conditions of the electrospinning are: the speed of the receiver is 1500 r / min, and the total working time is 210 min; And / or, the conditions of the high-temperature annealing are: high-temperature annealing at 105 °C for 10 h.

7. A GTR membrane prepared by the preparation method according to any one of claims 1 - 6.

8. Use of the GTR membrane according to claim 7 in the preparation of an implant material after periodontal flap surgery.

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