Methods for making and using probiotic-loaded electrospun film compositions for treating periodontitis

Abstract

The application discloses a preparation and application method of a probiotic-loaded electrospun film composition for treating periodontitis, and comprises the following steps: S1, first, an electrospun film (PLGA-nHA) doped with hydroxyapatite nanoparticles (nHA) and polylactic acid (PLGA) is prepared through electrospinning; S2, Lactobacillus plantarum DP189 is cultured; and S3, a preparation of the probiotic-loaded electrospun film. The probiotic-loaded electrospun film composition can improve the symptoms of periodontal tissues of rats, such as reducing inflammatory response, reducing the content of inflammatory factor TNF-alpha, increasing the content of bone saliva protein BSP, and improving the absorption degree of alveolar bone of rats. The probiotic-loaded electrospun film composition can be used for preparing a biological preparation for treating periodontitis, and can also be applied to treating other various diseases in the oral cavity, and has a very wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the biomedical technology field, in particular to a preparation and application method of a probiotic-loaded electrospun film composition for treating periodontitis. BACKGROUND

[0002] Periodontitis is a chronic infectious oral disease caused by plaque microorganisms, which can cause inflammation of gingival epithelial tissue, collagen fiber dissolution and destruction of gingival and periodontal membrane, and alveolar bone absorption, resulting in tooth loosening and shedding, and is the primary cause of tooth loss in adults in China. The occurrence and development of periodontitis are closely related to periodontal microecological imbalance. The basic treatment method of periodontitis mainly relies on mechanical method to remove plaque biofilm, and is often supplemented by local or systemic medication, but the frequent colonization of periodontal pathogenic bacteria poses a challenge to conventional periodontitis treatment methods. Probiotics are generally derived from normal microbial flora and can regulate the local ecological balance of the oral cavity and improve the immune condition of the whole body. In the treatment of periodontitis, probiotics can be used as an alternative and supplementary biological agent to regulate periodontal plaque metabolism and control the progression of periodontitis. At present, the main method is to take probiotic lozenges to assist in the treatment of periodontitis, but the flow of gingival crevicular fluid will flush the initially colonized probiotics out of the periodontal pocket, so the colonization of probiotics in the periodontal pocket is not very obvious. Therefore, it is necessary to develop an ideal probiotic-loaded electrospun film, load the target probiotics on the degradable electrospun film, and place it in the periodontal pocket to increase the colonization of probiotics in the periodontal pocket, so as to adjust the periodontal microecology and achieve the purpose of treating periodontitis. SUMMARY

[0003] The present application aims to provide a preparation and application method of a probiotic-loaded electrospun film composition for treating periodontitis, so as to solve the problems in the background.

[0004] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a preparation method of a probiotic-loaded electrospun film composition for treating periodontitis, comprising the following steps:

[0005] S1, first, prepare an electrospun film (PLGA-nHA) doped with hydroxyapatite nanoparticles (nHA) and polylactic acid (PLGA) by electrospinning;

[0006] S2, culture Lactobacillus plantarum DP189;

[0007] S3, preparation of the probiotic-loaded electrospun film.

[0008] Preferably, step S1 comprises the following specific steps:

[0009] S11: first, 0.1-0.2 g of hydroxyapatite nanoparticles (nHA) is weighed, then 1-2 g of polylactic acid (PLGA) is weighed, and both are added to 8-12 mL of hexafluoroisopropanol solvent, stirred at room temperature for 2-3 days until the polylactic acid (PLGA) is completely dissolved and the hydroxyapatite nanoparticles (nHA) are fully dispersed in the solution;

[0010] S12: a 20 mL syringe with a 16G needle is used to suck the solution, and after the solution is sucked, the syringe is placed on a step syringe pump;

[0011] The positive electrode of the high-voltage power supply clamps the syringe needle, and the negative electrode of the high-voltage power supply clamps the receiving end aluminum foil;

[0012] Turn on the high-voltage power supply, the voltage is 15-20 kv, and the syringe pump advances at a speed of 1-2 mL / h, and the spinning starts;

[0013] S13: The obtained electrospun film (PLGA-nHA) electrospun film is placed in a 30-50 ℃ oven to remove the solvent;

[0014] After the solvent is completely removed, the film is placed in a dopamine release solution for polydopamine coating treatment, the dopamine release solution has a concentration of 2-5 mg / ml, the solvent is Tris-HCl solvent, and the concentration is 10-20 mM, and then the film is placed in a shaker at room temperature for 12-24 h.

[0015] After coating, the film is washed with deionized water to obtain a polydopamine-modified PLGA-nHA film (pDA-PLGA-nHA).

[0016] Preferably, step S2 includes the following specific steps:

[0017] The Lactobacillus plantarum DP189 is cultured in a MRS medium at 37℃ for at least 16 h until the concentration of the Lactobacillus plantarum DP189 is 5×108 CFU / mL.

[0018] Preferably, step S3 includes the following specific steps:

[0019] S31: The electrospun film is prepared into an electrospun film sample with a diameter of 14 mm by using a punch, and the electrospun film sample needs to be ultraviolet irradiated in a clean bench for 2 h before use to prepare a sterile electrospun film sample;

[0020] S32: The sterile electrospun film sample is laid flat in a 24-well plate, and the inoculation density is 1×10 51ml of L. plantarum DP189 culture solution is placed on the surface of the electrospun film, and the L. plantarum DP189 electrospun film is prepared.

[0021] The application method of the probiotic-loaded electrospun film composition for treating periodontitis comprises the following steps:

[0022] In the first step, the receptor is anesthetized by using 10% chloral hydrate (3.5 mL / kg), and the oral cavity is disinfected by using alcohol;

[0023] In the second step, after disinfecting the oral cavity with alcohol, the teeth and gums of the receptor are dried with a sterile cotton swab, and a 1.5 mm x 4.0 mm L. plantarum DP189-loaded electrospun film is attached to the periodontal tissue.

[0024] In the third step, the receptor is fasted and watered for 6 hours after the attachment operation.

[0025] Compared with the prior art, the beneficial effects of the present application are that the L. plantarum DP189 strain-loaded electrospun film composition can improve the symptoms of periodontal tissue, such as reducing inflammatory response, reducing the content of inflammatory factor TNF-α, increasing the content of bone saliva protein BSP, and improving the absorption degree of alveolar bone. Therefore, the L. plantarum DP189-loaded electrospun film composition provided by the present application can be used to prepare a biological preparation for treating periodontitis, and the L. plantarum DP189-loaded electrospun film composition provided by the present application can also be applied to treat other various diseases in the oral cavity, and has a very broad application prospect.

[0026] The purpose of the present application is to use a polylactic acid-nano hydroxyapatite (PLGA-nHA) electrospun film as a carrier, to place the film in a dopamine Tris buffer solution for polydopamine coating treatment, and then obtain a polydopamine modified PLGA-nHA film (pDA-PLGA-nHA), so as to construct a pDA-PLGA-nHA electrospun film with good biocompatibility, anti-inflammatory and bone regeneration induction. The whole process is simple to operate, the raw materials are easy to obtain and low in price, and the biocompatibility is good, and the cell is non-toxic and harmless. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is an electron micrograph of the electrospun film in the present application.

[0028] Figure 2 It is an electron micrograph of the probiotic-loaded electrospun film at different culture time points in the present application.

[0029] Figure 3The H&E staining result image of the rat teeth and periodontal tissue after different treatments in the application.

[0030] Figure 4 The periodontal tissue immunohistochemical staining result image in the application.

[0031] Figure 5 The Micro-CT scanning result showing the alveolar bone regeneration in the application. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0033] Please refer to Figures 1-5 The application provides a technical solution: a preparation method of a probiotic-loaded electrospun film composition for treating periodontitis, comprising the following steps:

[0034] S1, first, an electrospun film (PLGA-nHA) doped with hydroxyapatite nanoparticles (nHA) and polylactic acid (PLGA) is prepared by electrospinning;

[0035] S2, Lactobacillus plantarum DP189 is cultured;

[0036] S3, preparation of the electrospun film loaded with L. plantarum DP189.

[0037] Preferably, step S1 comprises the following specific steps:

[0038] S11: first, 0.1-0.2 g of hydroxyapatite nanoparticles (nHA) is weighed, then 1-2 g of polylactic acid (PLGA) is weighed, and the two are added to 8-12 mL of hexafluoroisopropanol solvent, stirred at room temperature for 2-3 days, until the polylactic acid (PLGA) is completely dissolved and the hydroxyapatite nanoparticles (nHA) are fully dispersed in the solution;

[0039] S12: a 20 mL syringe with a 16G needle is used to suck the solution, and after the solution is sucked, the syringe is placed on a step syringe pump;

[0040] The positive electrode of a high-voltage power supply is clamped at the needle of the syringe, and the negative electrode of the high-voltage power supply is clamped at the receiving end aluminum foil;

[0041] The high-voltage power supply is turned on, the voltage is 15-20 kv, the syringe pump is pushed at a speed of 1-2 mL / h, and the spinning starts;

[0042] S13: The obtained electrospun film (PLGA-nHA) electrospun film is placed in an oven at 30-50 °C to remove the solvent;

[0043] After the solvent is completely removed, the film is placed in a dopamine release solution for polydopamine coating treatment, the dopamine release solution has a concentration of 2-5 mg / ml, the solvent is Tris-HCl solvent, and the concentration is 10-20 mM, and then the film is placed in a shaking bed at room temperature for 12-24 h shaking;

[0044] After the coating is completed, the polydopamine modified PLGA-nHA film (pDA-PLGA-nHA) is obtained by washing with deionized water.

[0045] Preferably, step S2 comprises the following specific steps:

[0046] The Lactobacillus plantarum DP189 is cultured in the MRS medium at 37 °C for at least 16 h, until the concentration of the Lactobacillus plantarum DP189 is 5x108 CFU / mL.

[0047] Preferably, step S3 comprises the following specific steps:

[0048] S31: The electrospun sample is prepared into a diameter of 14 mm by using a punch, and the electrospun sample needs to be ultraviolet irradiated for 2 h in an ultraclean bench before use, to prepare a sterile electrospun film sample;

[0049] S32: The sterile electrospun film sample is laid flat in a 24-well plate, and 1x10 5 CFU of the Lactobacillus plantarum DP189 culture solution 1 ml is inoculated on the surface of the electrospun sample, and the preparation of the Lactobacillus plantarum DP189 electrospun film is completed.

[0050] The application method of the probiotic electrospun film composition for treating periodontitis comprises the following steps:

[0051] Firstly, the recipient is anesthetized with 10% chloral hydrate (3.5 mL / kg), and the oral cavity is disinfected with alcohol;

[0052] Secondly, after disinfecting the oral cavity with alcohol, the teeth and gums of the recipient are dried with a sterile cotton swab, and a 1.5 mm x 4.0 mm Lactobacillus plantarum DP189 electrospun film is attached to the periodontal tissue;

[0053] Thirdly, the recipient is fasted and watered for 6 h after the operation.

[0054] The Lactobacillus plantarum (L. plantarum) DP189 strain of the present application has been preserved in China Center for Type Culture Collection on March 25, 2019, and the preservation number is CCTCC NO: M2019199.

[0055] The purpose of the present application is to use a polylactic acid-nano hydroxyapatite (PLGA-nHA) electrospun film as a carrier, to place the film in a dopamine Tris buffer solution for polydopamine coating treatment, and then to obtain a polydopamine modified PLGA-nHA film (pDA-PLGA-nHA), so as to construct a pDA-PLGA-nHA electrospun film with good biocompatibility, anti-inflammatory and bone regeneration inducing properties. The whole process is simple to operate, the raw materials are easy to obtain and low in price, and the biocompatibility is good, which is non-toxic and harmless to cells.

[0056] The present application adopts the method of silk thread ligation to establish a rat periodontitis animal model. After the modeling is successful, 10% chloral hydrate (3.5 mL / kg) is used for anesthesia, and the whole body is fully disinfected by alcohol and then placed in a sterile operation table. The rat is fixed on the dissection board, the oral cavity is disinfected by alcohol, the steel wire is removed with a needle holder, the rat model teeth and gums are dried with a sterile cotton swab, and a 1.5mm*4.0mm electrospun film loaded with L. plantarum DP189 is attached to the periodontal pocket. After implantation, the rat is fasted and watered for 6 hours, and then sacrificed after 14 days. The effect of the electrospun film combination loaded with L. plantarum DP189 strain on the periodontal tissue of the rat periodontitis model is investigated. The research results show that the electrospun film combination loaded with L. plantarum DP189 strain can improve the symptoms of the rat periodontal tissue, such as reducing the inflammatory response, reducing the content of inflammatory factor TNF-α, increasing the content of bone saliva protein BSP, and improving the absorption degree of rat alveolar bone. Therefore, the electrospun film combination loaded with L. plantarum DP189 provided by the present application can be used to prepare a biological preparation for treating periodontitis. At the same time, the electrospun film combination loaded with L. plantarum DP189 provided by the present application can also be applied to treat other various diseases in the oral cavity, and has a very broad application prospect.

[0057] The experiments of the present application are as follows. In the following examples, the test materials used are purchased from conventional biochemical reagent companies unless otherwise specified. In the quantitative test of the following examples, three repeated experiments are set, and the average value is taken as the result.

[0058] Experimental materials:

[0059] Liquid MRS medium: solvent is water, containing peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, sodium acetate 5 g / L, sodium citrate 5 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L, Tween-80 1 mL / L, glucose 20 g / L; pH 6.6.

[0060] SD male rats (body weight 180-220 g): Changchun Yis Experimental Animal Technology Co., Ltd.

[0061] Basic feed: Changchun Yis Experimental Animal Technology Co., Ltd.

[0062] Spun film: PLGA-nHA nanofiber membrane was prepared by blending electrospinning. The obtained PLGA-nHA electrospun film was placed in a 30-50 ℃ oven to remove the solvent, and after the solvent was completely removed, the film was placed in a dopamine Tris buffer solution for polydopamine coating treatment, the concentration of dopamine Tris buffer solution was 2-5 mg / ml, the concentration was 10-20 mM, and then placed in a shaking bed at room temperature for 12-24 h shaking. After coating, the polydopamine (pDA) modified PLGA-nHA film (pDA-PLGA-nHA) was obtained by washing with deionized water.

[0063] Example The influence of L. plantarum DP189 strain, electrospun film, combination of L. plantarum DP189 strain and electrospun film on rat periodontitis model

[0064] I. Establishment of animal model and grouping

[0065] Experimental selection 60 weight about 180±220g SD rats, Changchun City Yis Experimental Animal Technology Co. Ltd. After adaptive feeding for 1 week, 12 rats were randomly selected as blank control group. The rest of the rats were established experimental periodontitis model. The method of ligation wire ligation was used to establish the experimental periodontitis animal model of rats, and the rats were regularly checked whether the ligation steel wire in the oral cavity was dropped. If it was dropped, it was re-ligated, and the induction time was 2 weeks. After 2 weeks of modeling, the rats were examined according to the clinical diagnostic criteria of periodontitis: (1) the color, shape and texture of the gums; (2) whether the periodontal probing bleeds; (3) whether there is the formation of deep periodontal pocket; (4) whether there is attachment loss, and the formation of attachment loss is the index to distinguish periodontitis and gingivitis. The above four standards indicate that the modeling is successful, and the rats that are not successful in modeling are excluded, and the model successful rats are screened out, and randomly divided into 5 groups (10 rats each): (1) blank control group: 10% chloral hydrate (3.5 mL / kg) was used for anesthesia, and no subsequent operation was performed, so as to exclude the interference of anesthetic effect on experimental results; (2) periodontitis group: 10% chloral hydrate (3.5 mL / kg) was used for anesthesia, and no subsequent operation was performed, so as to exclude the interference of anesthetic effect on experimental results; (3) electrospinning silk group: 10% chloral hydrate (3.5 mL / kg) was used for anesthesia, and the whole body was fully disinfected with alcohol before being placed in a sterile operating table. The rats were fixed on the dissection plate, and after the oral cavity was disinfected with alcohol, the steel wire was removed with a needle holder. The tooth and modeling tooth sulcus were dried with a sterile cotton swab, and the 1.5 mm×4 mm electrospinning film was attached to the periodontal pocket; (4) probiotic group: 10% chloral hydrate (3.5 mL / kg) was used for anesthesia, and the whole body was fully disinfected with alcohol before being placed in a sterile operating table. The rats were fixed on the dissection plate, and after the oral cavity was disinfected with alcohol, the steel wire was removed with a needle holder. The tooth and modeling tooth sulcus were dried with a sterile cotton swab, and the probiotic was applied to the periodontal pocket; (5) probiotic + electrospinning film group: 10% chloral hydrate (3.5 mL / kg) was used for anesthesia, and the whole body was fully disinfected with alcohol before being placed in a sterile operating table. The rats were fixed on the dissection plate, and after the oral cavity was disinfected with alcohol, the steel wire was removed with a needle holder. The tooth and modeling tooth sulcus were dried with a sterile cotton swab, and the 1.5 mm×4 mm electrospinning film of L. plantarum DP189 was attached to the periodontal pocket. After 14 days, the rats were sacrificed, the complete maxilla of the rats was washed repeatedly with physiological saline, the blood stains were washed, and the 10% neutral formalin buffer solution was used for fixation, and the HE staining, immunohistochemistry and Micro-CT detection were carried out.

[0066] The electron microscope photograph of the electrospinning film is shown in Figure 1 ),

[0067] (a), (b), (c) SEM images of PLGA-nHA fiber membrane. (d), (e), (f) SEM images of pDA coated PLGA-nHA fiber membrane.

[0068] The surface morphology of the obtained fiber membrane was observed by scanning electron microscope. After pDA coating, the PLGA-nHA fiber membrane did not change significantly, and the fiber without beads was relatively smooth, with a diameter of about 200 nm.

[0069] The results of co-culturing the electrospun film with L. plantarum DP189 (see Figure 2 ) showed that,

[0070] (a) electrospun film co-cultured with L. plantarum DP189 for 1 day; (b) electrospun film co-cultured with L. plantarum DP189 for 3 days; (c) electrospun film co-cultured with L. plantarum DP189 for 5 days; (d) L. plantarum DP189.

[0071] The morphological changes of the fiber membrane were observed by scanning electron microscope, and L. plantarum DP189 was fixed on the pDA coated electrospun film. In addition, it was found that the number of rod-shaped cells on the fiber membrane increased with the increase of co-culturing time, i.e. the number of L. plantarum DP189 gradually increased. It is shown that L. plantarum DP189 can be loaded on the electrospun film by co-culturing method, and the method can effectively load L. plantarum DP189 on the electrospun film is safe.

[0072] The results of periodontal tissue morphology analysis (see Figure 3 ) showed that,

[0073] M1 and M2 are teeth, and M1 is a model tooth; red asterisk is enamel cementum junction, yellow arrow is periodontal pocket, and green arrow is inflammatory cell.

[0074] Hematoxylin-eosin staining was used to observe the gingival tissue around the teeth of each group under an optical microscope. The blank control group (Group 1) Figure 3 a) The gingival epithelium was complete, with no loss of attachment, and the junctional epithelium was tightly attached to the surface of the enamel, with no erosion, ulcer, inflammatory cell infiltration, and no abnormal changes in connective tissue. The periodontitis group (Group 2) Figure 3b) The binding epithelium of the gingival proliferated apically, the attachment loss and deep periodontal pocket were formed, a large amount of inflammatory exudates were observed in the pocket, the epithelium in the sulcus was eroded, a large amount of inflammatory cell infiltration was observed, and active osteoclastic bone absorption pits were observed on the cementum surface. The probiotic group and the spinning group Figure 3 d-e) Compared with the periodontitis group, the degree of gingival tissue inflammation and bone resorption was not significantly improved. The probiotic + spinning group compared with the periodontitis group Figure 3 c) The periodontal pocket depth and attachment loss were significantly reduced, the inflammatory cells and osteoclastic bone absorption pits were significantly reduced, the gingival inflammation and periodontal tissue destruction were relieved to varying degrees, indicating that the L. plantarum DP189 electrospun film had the potential to reduce gingival tissue inflammation and alveolar bone resorption.

[0075] The level of TNF-α in the gingival crevicular fluid of periodontitis patients was significantly higher than that of periodontal healthy people, and BSP was decreased, and after systematic treatment, the level of TNF-α was significantly reduced and BSP was slightly increased. Therefore, TNF-α and BSP play a key role in the occurrence, development and repair of periodontitis. The results of immunohistochemical staining are shown in Figure 4

[0076] a-e, the results of immunohistochemical staining of TNF-α in periodontal tissue of each group; f-j, the results of immunohistochemical staining of BSP in periodontal tissue of each group.

[0077] Compared with the blank group, there were a large number of TNF-α positive cells in the periodontal tissue of the periodontitis group, the number of TNF-α positive cells in the electrospinning group compared with the periodontitis group was not significantly reduced, the number of TNF-α positive cells in the probiotic group compared with the periodontitis group was slightly decreased, and the number of TNF-α positive cells in the probiotic + electrospinning group was significantly decreased; compared with the blank group, no BSP positive cells were observed in the periodontal tissue of the periodontitis group and the probiotic group, a small amount of BSP positive cells was observed in the electrospinning group, and the number of BSP positive cells in the probiotic + electrospinning group compared with the periodontitis group was significantly increased. The above results show that the L. plantarum DP189 electrospun film effectively controls the inflammation of periodontal tissue and has certain bone induction ability.

[0078] In order to evaluate the effect of L. plantarum DP189 electrospun film on alveolar bone resorption in periodontitis rats, the method of Micro-CT scanning was used to reconstruct the three-dimensional model of the teeth of each group of rats, and the changes of alveolar bone loss and bone structure parameters of each group of rats were analyzed.

[0079] The results of Micro-CT scanning of rat alveolar bone are shown in Figure 5 ​​

[0080] a, white line is the distance from enamel cementum junction to alveolar crest, namely attachment loss; b-f, Micro-CT three-dimensional reconstruction pictures of the model teeth of rats in each group.

[0081] The severity of periodontitis is mainly measured by the amount of attachment loss, the greater the attachment loss value, the more severe the alveolar bone absorption and periodontitis. Because three-dimensional measurement can more accurately evaluate the condition of alveolar bone changes compared with two-dimensional measurement, through three-dimensional reconstruction of the upper jaw of the rat model, the alveolar bone absorption condition of the teeth of rats in each group can be directly observed, and the alveolar bone absorption condition of the L. plantarum DP189 electrospun film is significantly improved compared with the periodontitis group, which indicates that the L. plantarum DP189 electrospun film has a protective effect on the alveolar bone absorption of periodontitis rats.

[0082] The application of the L. plantarum DP189 electrospun film is disclosed, and those skilled in the art can refer to the content of the present application and appropriately improve the process parameters for implementation. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The product of the present application has been described through a preferred embodiment, and relevant personnel can obviously modify or appropriately change and combine the product described in the present application without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0083] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method of preparing a probiotic-loaded electrospun film for treating periodontitis, characterized in that, Comprising the following steps: S1, first prepare a hydroxyapatite nanoparticle (nHA) and polylactic acid (PLGA) electrospun film (PLGA-nHA) by electrospinning; S2, L. plantarum DP189 culture; S3, preparation of L. plantarum DP189 electrospun film; Step S1 includes the following specific steps: S11: First, weigh 0.1-0.2 g of hydroxyapatite nanoparticles (nHA), then weigh 1-2 g of polylactic acid (PLGA), and add both to 8-12 mL of hexafluoroisopropanol solvent. Stir at room temperature for 2-3 days until the polylactic acid (PLGA) is completely dissolved and the hydroxyapatite nanoparticles (nHA) are fully dispersed in the solution; S12: Use a 16G needle and a 20 mL syringe to draw the solution. After drawing the solution, place the syringe on the step injection pump; Use the positive electrode of the high-voltage power supply to hold the syringe needle, and the negative electrode of the high-voltage power supply to hold the receiving end aluminum foil; Turn on the high-voltage power supply, the voltage is 15-20 kv, the injection pump advances at a speed of 1-2 mL / h, and the spinning begins; S13: Place the obtained electrospun film (PLGA-nHA) electrospun film in a 30-50 ℃ oven to remove the solvent; After the solvent is completely removed, place the film in a dopamine release solution for polydopamine coating treatment. The dopamine release solution has a concentration of 2-5 mg / ml, and the solvent is Tris-HCl solvent with a concentration of 10-20 mM. Then place it in a shaker at room temperature for 12-24 h. After coating, wash thoroughly with deionized water to obtain a polydopamine-modified PLGA-nHA film (pDA-PLGA-nHA).

2. The method of claim 1, wherein the preparation of the probiotic- loaded electrospun thin film for treating periodontitis composition is characterized by: Step S2 includes the following specific steps: L. plantarum DP189 is cultured in MRS medium at 37℃ for at least 16h, until the concentration of L. plantarum DP189 is 5×108 CFU / mL.

3. The method of claim 1, wherein the preparation of the probiotic- loaded electrospun thin film therapeutic composition for periodontitis is characterized by: Step S3 includes the following specific steps: S31: Use a punch to prepare an electrospun film sample with a diameter of 14 mm. The electrospun film sample needs to be ultraviolet irradiated for 2 h in a clean bench before use to prepare a sterile electrospun film sample; S32: Sterile electrospun film samples were plated into 24-well plates at a seeding density of 1 x 10 5 L. plantarum DP189 culture 1 ml was plated onto the surface of the electrospun film, and the L. plantarum DP189 electrospun film preparation was completed.

4. Use of the probiotic electrospun film composition for treating periodontitis according to claim 1 in the preparation of a drug for treating periodontitis.

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