Application of bacterium-derived vesicles derived from lactobacillus bulgaricus in preparation of medicament for accelerating movement of orthodontic teeth

Abstract

The invention relates to the technical field of biological medicines, in particular to application of lactobacillus bulgaricus-derived bacterial vesicles in preparation of a medicament for accelerating orthodontic tooth movement. In the orthodontic pressure applying process, BEVs derived from lactobacillus bulgaricus is injected under the movable tooth periosteum, cell factor generation and osteoclast formation in alveolar bones can be induced, orthodontic tooth movement is further remarkably accelerated, negative effects on the survival condition of mice in an experiment are avoided, safety and effectiveness are achieved, and the method is suitable for popularization and application. And postoperative bleeding and other postoperative risks cannot be generated.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of bacteria-derived vesicles derived from Lactobacillus bulgaricus in the preparation of a medicament for accelerating orthodontic tooth movement. Background Art

[0002] Dental correction, commonly known as orthodontics, is a treatment process that improves the position of teeth and jaws. For many patients, seeing the correction effect quickly is one of their main concerns. However, the traditional tooth movement speed is subject to physiological limitations and takes a long time. Therefore, during orthodontic treatment, achieving rapid tooth movement and shortening the treatment time can reduce the time required for the entire correction process, which is an obvious benefit for patients who want to have straight teeth as soon as possible; it can also improve the patient's quality of life, reduce discomfort such as tooth pain, gingival swelling, etc., and make it easier for patients to adapt to the correction process; in addition, by accelerating orthodontic tooth movement, the risk of tooth recurrence can be reduced. This is because accelerated treatment can better control the position and movement trajectory of the teeth, thereby reducing the possibility of the teeth returning to their original position.

[0003] Orthodontic tooth movement is a complex process involving multiple aspects such as teeth, periodontal tissues and jaws. The immune microenvironment plays an important role in orthodontic tooth movement and affects the effect and speed of tooth movement. During orthodontic treatment, teeth are subjected to external forces and the tissues around the teeth undergo inflammatory reactions. Cells and molecules in the immune microenvironment play an important role in orthodontic tooth movement. Among them, immune cells such as macrophages, dendritic cells and lymphocytes regulate the intensity and duration of the inflammatory response by releasing inflammatory mediators and cytokines. In addition, immune cells are also involved in the process of bone remodeling, playing a key regulatory role in tooth movement.

[0004] Bacterial extracellular vesicles (BEVs) are nanoscale lipid bilayer vesicles produced by Gram-positive or Gram-negative bacteria, with a diameter of generally 20-400nm, carrying a variety of bacterial substances, such as lipids, proteins, nucleic acids and metabolites. With the continuous deepening of research, the clinical application potential of BEVs has been continuously explored and attracted the interest of many researchers. As a small molecule active substance derived from bacteria, BEVs have the advantages of stable structure and long effective circulation time. The bacterial substances carried by BEVs can effectively regulate the vitality of immune cells, suggesting its application potential in disease prevention. Studies have found that BEVs can effectively mediate the secretion of various cytokines by immune cells, such as interferon, interleukin, etc. These cytokines can regulate the activity, proliferation and differentiation of immune cells. At present, there is no report on whether BEVs can accelerate the application of orthodontic tooth movement. Summary of the invention

[0005] To investigate whether BEVs can accelerate orthodontic tooth movement.

[0006] The technical solution of the present invention is: application of bacterial-derived vesicles derived from Lactobacillus bulgaricus in the preparation of an agent for accelerating orthodontic tooth movement.

[0007] The steps for preparing bacterial-derived vesicles from Lactobacillus bulgaricus are as follows:

[0008] Inoculate Lactobacillus bulgaricus into Lactobacillus delbrueckii culture medium, i.e., MRS culture medium, and culture at 37° C. for 20 h, collect the supernatant in the culture, and centrifuge the collected supernatant at 3000 g for 15 min to remove cells and cell debris;

[0009] The supernatant was then centrifuged at 10,000 g for 30 min, and then filtered through a 0.45 μm syringe filter to obtain a filtrate. The filtrate was added to an ultracentrifuge tube and centrifuged at 120,000 g for 90 min using an ultracentrifuge to precipitate bacterial-derived vesicles BEVs.

[0010] The precipitated bacterial-derived vesicles BEVs were resuspended in phosphate buffered saline (PBS), and centrifuged again at 120,000 g for 90 min using an ultracentrifuge to remove impurities; then, the washed bacterial-derived vesicles BEVs were resuspended in a PBS solution.

[0011] The beneficial effects of the present invention are as follows: during the application of orthodontic pressure, the subperiosteal injection of BEVs derived from Lactobacillus bulgaricus can induce cytokine production and osteoclast formation in the alveolar bone, thereby significantly accelerating orthodontic tooth movement, and has no negative impact on the survival of mice in the experiment. It is safe and effective, and does not cause postoperative bleeding or other postoperative risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shown are schematic diagrams comparing CT scan results of maxillary bone samples of the BEVs treatment group and the PBS control group on day 0, day 7 and day 14, respectively, in the embodiment, wherein (a) is a schematic diagram of CT sections of maxillary bone samples of the BEVs treatment group and the PBS control group on day 0, day 7 and day 14, respectively, and (b) is a schematic diagram of three-dimensional reconstructed CT images of maxillary bone samples of the BEVs treatment group and the PBS control group on day 0, day 7 and day 14, respectively;

[0013] Figure 2 Shown is a schematic diagram of the comparison of the orthodontic tooth movement distance of mice in the PBS control group and the BEVs injection group on the 7th day and the 14th day respectively in the embodiment;

[0014] Figure 3 The following are comparative schematic diagrams of the detection results of IL-6, IL-1β and TNF-α cytokine genes in the PBS control group and the BEVs injection group in the embodiment on the 7th day, respectively, wherein (a) is a comparative schematic diagram of the detection results of the IL-6 cytokine gene, (b) is a comparative schematic diagram of the detection results of the IL-1β cytokine gene, and (c) is a comparative schematic diagram of the detection results of the TNF-α cytokine gene;

[0015] Figure 4 The images shown are HE staining and TRAP staining images of the alveolar bone around the mobile teeth of mice in the blank control group, PBS control group and BEVs injection group at the 7th day, respectively, wherein (a) is the HE staining image of the alveolar bone around the mobile teeth of mice at the 7th day, and (b) is the TRAP staining image of the alveolar bone around the mobile teeth of mice at the 7th day; P: dental pulp, D: dentin, AB: alveolar bone, PDL: periodontal ligament;

[0016] Figure 5 Shown are statistical graphs of the number of osteoclasts in the alveolar bone unit area around the mobile teeth of mice in the blank control group, PBS control group and BEVs injection group on the 7th day respectively. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0018] Example

[0019] (1) Establishment of mouse orthodontic model:

[0020] First, an orthodontic tooth movement model was established in mice. After the mice were anesthetized, a 0.20 mm ligature was used to ligate the left first molar, and a nickel-titanium tension spring was used to apply a force of about 30 g to ligate the two maxillary central incisors of the mice as support. The ligature was bonded to the central incisor with resin to pull the first molar to move mesially. In order to relieve the discomfort of the mice, soft food was used for feeding, and the tooth movement appliance was checked every day. The mice without the orthodontic tooth movement model were used as the blank control group (Blank).

[0021] (2) Extraction of bacterial vesicles (BEVs) from Lactobacillus bulgaricus:

[0022] Lactobacillus bulgaricus (purchased from Beina Biotech, catalog number: #BNCC187941) was inoculated into Lactobacillus delbrueckii culture medium, i.e., MRS culture medium, and cultured at 37°C for 20 h. The supernatant in the culture was collected and centrifuged at 3000g for 15 min to remove cells and large cell debris. The supernatant was then centrifuged at 10000g for 30 min and filtered through a 0.45 μm syringe filter. The filtrate was added to an ultracentrifuge tube and centrifuged at 120000g for 90 min using an ultracentrifuge (Beckman, Germany) to precipitate BEVs. The precipitated BEVs were resuspended with an appropriate amount of PBS and centrifuged again at 120000g for 90 min using an ultracentrifuge to remove impurities. Then, the washed BEVs were resuspended in a phosphate buffer solution (PBS) and identified by electron microscopy and nanotracking particle analysis.

[0023] (3) BEVs processing and orthodontic tooth movement measurement:

[0024] The mice with orthodontic tooth movement model were randomly divided into two groups, with 6 mice in each group: one group was a PBS control group, which was locally injected under the mobile periosteum once every 3 days, with 10ul for each mouse each time; the other group was a BEVs treatment group, which was locally injected under the mobile periosteum once every 3 days, with 20ug / 10ul for each mouse each time; the experiment was sampled on the 0th day, the 7th day and the 14th day, that is, the mice were euthanized, the maxilla of the mice was separated, the soft tissue was removed, and the orthodontic appliance was removed; all fresh maxillary samples were scanned using the same Micro-CT (vivaCT80, Switzerland) under the same conditions, with the parameters of voltage 55kV, current 72μA, voxel 15.6μm, and the CT scanning results of the maxillary samples on the 0th day, the 7th day and the 14th day are shown in Figure 2. Figure 1 As shown, Figure 1 (a) is a schematic diagram of the CT sections of the maxillary bone samples of the BEVs treatment group and the PBS control group on day 0, day 7, and day 14, respectively;

[0025] The scanned CT data were randomly renamed, and the high points of the distal surface of the first molar and the mesial surface of the second molar were marked. The distance between the two points was measured as the orthodontic tooth movement distance of the mouse, and then imported into InVivoDental (Anatomage, USA) for three-dimensional reconstruction. Figure 1 (b);

[0026] Figure 2 FIG. 1 is a schematic diagram showing the comparison of the orthodontic tooth movement distances of mice in the PBS control group and the BEVs injection group on the 7th day and the 14th day, respectively. Figure 1 and Figure 2It can be seen that the BEVs injection group can significantly promote orthodontic tooth movement on the 7th and 14th days, and the difference is statistically significant (*p<0.05; **p<0.01);

[0027] (4) Tissue cytokine detection:

[0028] Maxillary bone tissues of mice in the PBS control group and the BEVs injection group were collected, and 1 ml of TRIzol was added to homogenize the tissue samples. The samples were shaken and aspirated evenly, and total RNA was extracted by the guanidine thiocyanate-phenol single-phase solution method, i.e., the TRIzol method. Total RNA was reverse transcribed according to the reverse transcription kit steps, and real-time fluorescence quantitative polymerase chain reaction (PCR) was performed according to the kit steps. cDNA was diluted, 2 μl of cDNA was added to each well of a 96-well plate, and a reverse transcription-polymerase chain reaction (RT-PCR) reaction system was configured. 8 μl of the reaction system was added to each well, the film was sealed, and the plates were centrifuged at 800 rpm for 5 minutes. The expression of various cytokine genes was detected using an ABI fluorescence quantitative polymerase chain reaction instrument, i.e., a fluorescence quantitative PCR instrument, produced by Applied Biosystems, Inc. in the United States. The expression of IL-6, IL-1β, and TNF-α cytokine genes in tissues was detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR), with GAPDH as the internal reference gene and 2 -△△CT The relative expression of target genes was calculated by this method.

[0029] Figure 3 : is a schematic diagram comparing the detection results of IL-6, IL-1β and TNF-α cytokine genes in the PBS control group and the BEVs injection group on the 7th day in the embodiment, Figure 3 As can be seen from (a), (b) and (c), on the 7th day, the expression levels of IL-6, IL-1β and TNF-α inflammatory factors in the EBVs treatment group were significantly higher than those in the PBS injection group, and the difference was statistically significant (**p<0.01; ***p<0.001);

[0030] (5) Histological staining:

[0031] Maxillary bone tissues of mice in the blank control group, PBS control group, and BEVs injection group were collected and fixed in 4% paraformaldehyde for 48 hours and then decalcified in 14% EDTA (pH=7.2-7.4) decalcification solution. The decalcification solution was changed every 3-4 days for 2-3 weeks. The decalcified tissue samples were dehydrated with 75%, 85%, 95%, and 100% gradient alcohol, treated with xylene I, II, and III, and then embedded in paraffin and prepared paraffin sections. The sections were 4 μm thick, placed on adhesive slides, and stored at room temperature.

[0032] HE staining: paraffin sections were dewaxed with xylene and gradient alcohol to water, stained with hematoxylin for 2 minutes, washed with water 3-5 times, and the staining was observed under a microscope; eosin staining was performed for 40 seconds, placed in 80% ethanol for 7 seconds, and then placed in anhydrous ethanol, and the staining was observed under a microscope; the sections were sequentially treated with xylene I and II for 5 minutes each, sealed with neutral gum, stored at room temperature, and photographed using an upright microscope. The HE staining comparison of the alveolar bone around the mobile teeth of mice on the 7th day was obtained as shown in the figure below. Figure 4 (a);

[0033] TRAP staining: paraffin sections were dewaxed with xylene and gradient alcohol to water, Trap staining solution was added, and incubated at 37°C for 60-90 minutes; rinsed twice with ddH2O, counterstained with methyl green for 20 minutes; rinsed twice with ddH2O, sealed with glycerol gelatin, and photographed under an upright microscope. The TRAP staining comparison of the alveolar bone around the mobile teeth of mice on the 7th day was obtained as shown in the figure below. Figure 4 (b);

[0034] right Figure 4 (b) Statistics and analysis are performed to obtain Figure 5 The results, Figure 5 is a statistical diagram of the number of osteoclasts in the alveolar bone unit area around the mobile teeth of mice in the PBS control group and the BEVs injection group on the 7th day, Figure 5 It can be seen that on the 7th day, the number of osteoclasts in the alveolar bone unit area around the mobile teeth in the EBVs treatment group was significantly higher than that in the PBS injection group, and the difference was statistically significant (**p<0.01; ***p<0.001). The increase in the number of osteoclasts indicates increased bone absorption and decreased bone density, which reduces the bone resistance to tooth movement and accelerates the tooth movement rate, which helps verify that the BEVs injection group in the embodiment can achieve faster tooth movement.

[0035] In this embodiment, a mouse orthodontic tooth movement model was constructed by selecting and establishing different animal experimental groups. After sampling, the samples were scanned by Micro-CT, and then the tooth movement distance was measured. Comparison of the measurement results showed that local injection of BEVs under the periosteum can increase the orthodontic tooth movement distance of mice, and have a certain accelerating effect on orthodontic tooth movement, providing a basis for the application of bacterial-derived vesicles BEVs derived from Lactobacillus bulgaricus in accelerating orthodontic tooth movement.

[0036] The embodiment accelerates the orthodontic tooth movement process by local subperiosteal injection of bacterial vesicles (BEVs) derived from Lactobacillus bulgaricus during orthodontic treatment in mice without negatively affecting the survival of the mice. The application is safe and effective, and does not cause postoperative bleeding or other postoperative risks, thereby enhancing the practicality of the present invention.

[0037] In the embodiment, BEVs derived from Lactobacillus bulgaricus are locally injected under the periosteum during orthodontic treatment. Lactobacillus bulgaricus is a probiotic that is widely used in food processing and other processes. The BEVs secreted by it are also widely present in foods to which the probiotics are added, proving that it is safer than general drugs and has a higher degree of acceptance among the public. Animal experiments on mice have shown that BEVs derived from Lactobacillus bulgaricus are also safe and effective during orthodontic tooth movement in mice.

[0038] The use of the bacterial-derived vesicles derived from Lactobacillus bulgaricus in the preparation of an agent for accelerating orthodontic tooth movement can achieve rapid tooth movement during orthodontic treatment, thereby shortening the treatment time, reducing the time required for the entire correction process, and reducing discomfort such as tooth pain and gingival swelling, making it easier for patients to adapt to the correction process; in addition, accelerating orthodontic tooth movement can more accurately control the movement of teeth, thereby achieving better treatment effects and helping patients recover normal chewing function and oral health more quickly.

[0039] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of those skilled in the art without departing from the spirit of the present invention.

Claims

1. Application of bacterial-derived vesicles from Lactobacillus bulgaricus in the preparation of agents for accelerating orthodontic tooth movement.

2. The use of the bacterial-derived vesicles derived from Lactobacillus bulgaricus according to claim 1 in the preparation of a medicament for accelerating orthodontic tooth movement, characterized in that: The method for preparing bacterial-derived vesicles derived from Lactobacillus bulgaricus comprises: Inoculate Lactobacillus bulgaricus into Lactobacillus delbrueckii culture medium, i.e., MRS culture medium, and culture at 37° C. for 20 h, collect the supernatant in the culture, and centrifuge the collected supernatant at 3000 g for 15 min to remove cells and cell debris; The supernatant was then centrifuged at 10,000 g for 30 min, and then filtered through a 0.45 μm syringe filter to obtain a filtrate. The filtrate was added to an ultracentrifuge tube and centrifuged at 120,000 g for 90 min using an ultracentrifuge to precipitate bacterial-derived vesicles BEVs. The precipitated bacterial-derived vesicles BEVs were resuspended in phosphate buffered saline (PBS), and centrifuged again at 120,000 g for 90 min using an ultracentrifuge to remove impurities; then, the washed bacterial-derived vesicles BEVs were resuspended in a PBS solution.

Images