Exosome composite material for treating periodontitis and preparation method thereof

Abstract

The application relates to the field of high polymer materials, in particular to an exosome composite material for treating periodontitis and a preparation method thereof. The exosome composite material comprises the following raw materials in parts by weight: 10-20 parts of a chitosan derivative, 10-20 parts of sodium hyaluronate, 1-5 parts of zein, 0.5-1 part of exosomes, 0.1-0.3 parts of phosphonodipeptide sodium and 60-70 parts of deionized water. The exosome composite material can inhibit periodontal pathogenic bacteria, reduce the expression of proinflammatory factors by inhibiting the signal pathways such as NF-kappa B, and thus relieve periodontal tissue inflammation.

Description

Technical Field

[0001] This invention relates to the field of polymer materials, and more particularly to an exosome composite material for treating periodontitis and its preparation method. Background Technology

[0002] Periodontitis, a chronic infectious disease caused by plaque biofilm, is the leading cause of tooth loss in adults worldwide. Its pathological process involves not only soft tissue destruction such as gingival redness, bleeding, and periodontal pocket formation, but also alveolar bone, causing irreversible bone resorption. While current basic treatments (such as supragingival scaling and subgingival scaling) effectively remove plaque, their effectiveness in clearing pathogenic microorganisms from deep periodontal pockets, regenerating damaged soft and hard tissues, and regulating excessive host inflammatory responses remains unsatisfactory. Therefore, developing a novel drug delivery system capable of local delivery and possessing both antibacterial and anti-inflammatory properties as well as promoting regeneration has become an important research direction in the field of periodontitis treatment.

[0003] Against this backdrop, local delivery systems based on biopolymers have shown great potential, as they can be designed to adapt to the specific anatomical environment of periodontal pockets, achieving controlled drug release and long-term retention. Among these, natural polymers are highly favored due to their good biocompatibility, biodegradability, and ease of functionalization. Chitosan, a cationic polysaccharide obtained from the deacetylation of chitin, is an outstanding representative of this type of material. It possesses antibacterial, hemostatic, and bioadhesive properties, and can bind to negatively charged bacterial cell membranes through electrostatic interactions, disrupting their integrity. However, the antibacterial spectrum and anti-inflammatory efficacy of chitosan alone are limited, and its solubility depends on an acidic environment, restricting its application under physiological conditions. To endow it with more powerful targeted therapeutic functions, chemical grafting modification of the polymer chain has become a key strategy. Spirulina acid, a pentacyclic triterpenoid acid compound extracted from natural plants, has attracted much attention due to its excellent anti-inflammatory and osteomodulatory activities. Studies have shown that scintiline can effectively inhibit the expression of pro-inflammatory factors such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) by regulating key inflammatory signaling pathways such as NF-κB and MAPK, thereby alleviating excessive immune responses in periodontal tissues. Simultaneously, it can promote osteoblast differentiation and mineralization, and inhibit osteoclast formation and activity, bidirectionally regulating bone metabolism balance at the microscopic level and creating a favorable microenvironment for alveolar bone regeneration. However, scintiline itself has poor water solubility, short retention time at the lesion site, and is easily cleared systemically, limiting its full potential for local therapeutic effects.

[0004] Suppressing inflammation and eliminating pathogens are the first steps in treatment, but achieving functional regeneration of periodontal soft and hard tissues is the ultimate goal. Exosomes, as nanoscale vesicles secreted by cells, carry abundant bioactive substances such as proteins and nucleic acids, and can efficiently regulate the proliferation, migration, and differentiation of recipient cells, playing the role of "cell messengers" in tissue repair. Integrating exosomes (especially those derived from mesenchymal stem cells) into periodontal local treatment systems provides a novel cell-free therapeutic strategy for promoting periodontal ligament stem cell activation, angiogenesis, and bone remodeling. However, exosomes themselves have application bottlenecks such as poor storage stability, easy inactivation in complex inflammatory environments, and short local retention time.

[0005] Based on the above background, this invention focuses on the complex pathological microenvironment of periodontitis and the unmet clinical needs. Through the design of polymer materials and the construction of composite systems, it provides a novel treatment strategy for periodontitis, which has important theoretical value and application prospects in the interdisciplinary field of biomaterials and oral regenerative medicine. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the primary objective of this invention is to provide an exosome composite material for treating periodontitis, which can inhibit periodontal pathogens and reduce the expression of pro-inflammatory factors by inhibiting signaling pathways such as NF-κB, thereby alleviating periodontal tissue inflammation.

[0007] Another objective of this invention is to provide a simple method for preparing an exosome composite material for treating periodontitis.

[0008] The objective of this invention is achieved through the following technical solution: An exosome composite material for treating periodontitis comprises the following raw materials in parts by weight: 10-20 parts chitosan derivative, 10-20 parts sodium hyaluronate, 1-5 parts zein, 0.5-1 part exosomes, 0.1-0.3 parts sodium phosphonodeptide, and 60-70 parts deionized water.

[0009] The present invention adds sodium phosphonodipeptide as a protective agent, which helps to maintain the structural integrity and bioactivity of exosomes during preparation and storage, thereby ensuring their sustained therapeutic effect in the periodontal region.

[0010] In addition, this invention introduces zein, which has good film-forming and bioadhesive properties, enabling the composite material to adhere tightly to the inner wall of the periodontal pocket, forming a local protective film, prolonging the action time of the drug and exosomes, and achieving continuous and controllable release.

[0011] Preferably, the preparation process of the chitosan derivative is as follows: (1) Take chitosan and add it to deionized water, adjust the pH, and prepare a chitosan solution; (2) Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the spiculic acid solution, and stir to obtain an activated spiculic acid mixed solution; (3) The chitosan acid mixture is added to the chitosan solution, and the chitosan derivative is obtained after the reaction.

[0012] This invention grafts spiculosic acid onto the water-soluble chitosan molecular chain, utilizing the hydrophilic skeleton of chitosan to achieve stable dispersion of spiculosic acid in the aqueous phase, significantly improving its local bioavailability and overcoming the problems of poor absorption and short action time caused by the high lipid solubility of spiculosic acid.

[0013] Preferably, in step (1), the pH is adjusted to 5-6; and the concentration of the chitosan solution is 1-2 wt%.

[0014] Preferably, in step (2), the mass ratio of the spiculinic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(0.8-1.2):(1-1.5); the solvent of the spiculinic acid solution is composed of ethanol and water in a volume ratio of 1:(0.5-1); the concentration of the spiculinic acid solution is 40-60 mg / mL; and the stirring time is 30-40 min.

[0015] Preferably, the volume ratio of the chitosan solution and the styraciferic acid mixture in step (3) is 1:(0.3-0.5); the reaction time is 8-12h.

[0016] Preferably, the exosomes are bone marrow mesenchymal stem cell exosomes.

[0017] Preferably, the method for preparing the bone marrow mesenchymal stem cell exosomes is as follows: Bone marrow mesenchymal stem cells were seeded into a culture medium, and the supernatant was collected, centrifuged at differential speed, and freeze-dried to obtain bone marrow mesenchymal stem cell exosomes.

[0018] Preferably, the culture medium is DMEM medium containing 10% FBS; the seeding density of the bone marrow mesenchymal stem cells is (3-7) × 10⁻⁶. 5 per mL.

[0019] Preferably, the bone marrow mesenchymal stem cells are P3-P5 generation bone marrow mesenchymal stem cells.

[0020] The preparation method of the above-mentioned exosome composite material for treating periodontitis includes the following steps: S1. Add sodium hyaluronate to 2 / 4 of deionized water to obtain an aqueous solution of sodium hyaluronate; add chitosan derivative to 1 / 4 of deionized water to obtain an aqueous solution of chitosan derivative; add the aqueous solution of chitosan derivative to the aqueous solution of sodium hyaluronate to obtain a mixed solution; S2. Add zein to the remaining deionized water, then mix it thoroughly with the mixture, exosomes, and sodium phosphonodipeptide.

[0021] The present invention has the following advantages over the prior art: 1. The exosome composite material for treating periodontitis provided by the present invention comprises chitosan derivatives, sodium hyaluronate, sodium phosphonodipeptide, zein, exosomes and other raw materials. The exosome composite material can inhibit periodontal pathogens and reduce the expression of pro-inflammatory factors by inhibiting signaling pathways such as NF-κB, thereby alleviating periodontal tissue inflammation.

[0022] 2. The chitosan derivative of the present invention is prepared by grafting spiculosic acid onto the molecular chain of water-soluble chitosan. This modification method utilizes the hydrophilic skeleton of chitosan to achieve stable dispersion of spiculosic acid in the aqueous phase, significantly improving its local bioavailability and overcoming the problems of poor absorption and short action time caused by the high lipid solubility of spiculosic acid.

[0023] 3. This invention also adds sodium phosphonyl dipeptide as a protective agent, which helps maintain the structural integrity and bioactivity of exosomes during preparation and storage, thereby ensuring their sustained therapeutic effect in the periodontal region.

[0024] 4. This invention also introduces zein, which has good film-forming and bioadhesive properties, enabling the composite material to adhere tightly to the inner wall of the periodontal pocket, forming a local protective film, prolonging the action time of the drug and exosomes, and achieving continuous and controllable release. Attached Figure Description

[0025] Figure 1 The images show the morphological characteristics of bone marrow mesenchymal stem cells obtained in this invention, where a is a morphological image of P3 generation bone marrow mesenchymal stem cells, b is a morphological image of P4 generation bone marrow mesenchymal stem cells, and c is a morphological image of P5 generation bone marrow mesenchymal stem cells. Figure 2 The figures show the results of the cytotoxicity experiment, where a represents the cytotoxicity of the chitosan derivative, b represents the cytotoxicity of the blank control, and c represents the cytotoxicity of the positive control. Figure 3 The image shows the infrared spectrum of the chitosan derivative obtained in Example 1, where curve a is the infrared spectrum of chitosan and curve b is the infrared spectrum of the chitosan derivative. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0027] The P3-P5 generation bone marrow mesenchymal stem cells in Examples 1-3 of this invention were prepared by the following method: Human bone marrow fluid and PBS buffer were mixed thoroughly at a volume ratio of 1:1 and centrifuged at 1500 rpm for 10 min. An equal volume of Ficoll separation buffer was added to the diluted bone marrow fluid, and the mixture was centrifuged at 2500 rpm for 30 min. Mononuclear cells with a white membrane at the interface were aspirated, and the cells were gently washed with PBS. The cells were then resuspended in LG-DMEM medium containing 10% FBS and the cell density was adjusted to 1 × 10⁻⁶ cells / mL. 5 1 / mL, and inoculated on a substrate with a bottom area of ​​25 cm² 2 The cells were cultured in culture flasks at 37°C in a cell culture incubator with 5% CO2, with the medium changed every 2 days. When the cells reached 85% confluence, they were digested with trypsin-EDTA solution and passaged at a ratio of 1:3 to obtain passaged P3-P5 generation bone marrow mesenchymal stem cells for later use.

[0028] Example 1 This embodiment provides a chitosan derivative, which is prepared by the following method: (1) Take chitosan and add it to deionized water, adjust the pH to 5.5, and prepare a chitosan solution with a concentration of 1.5wt%; (2) Add spiculic acid to ethanol / water solution (v / v=1:0.7) to prepare a spiculic acid solution with a concentration of 50 mg / mL; add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the spiculic acid solution with a mass ratio of 1:1:1.2 and stir at room temperature for 35 min to obtain an activated spiculic acid mixed solution; (3) The chitosan solution and the spiculosic acid mixture were added to the chitosan solution at a volume ratio of 1:0.4, and reacted at room temperature for 10 h. The chitosan derivative was obtained by dialysis and freeze drying.

[0029] Example 2 This embodiment provides a chitosan derivative, which is prepared by the following method: (1) Take chitosan and add it to deionized water, adjust the pH to 5, and prepare a chitosan solution with a concentration of 1wt%; (2) Add spiculic acid to ethanol / water solution (v / v=1:0.5) to prepare a spiculic acid solution with a concentration of 40mg / mL; add EDC and NHS to the spiculic acid solution with a mass ratio of spiculic acid solution, EDC and NHS of 1:0.8:1, stir at room temperature for 30min to obtain an activated spiculic acid mixed solution. (3) The chitosan solution and the spiculosic acid mixed solution were added to the chitosan solution at a volume ratio of 1:0.3, and reacted at room temperature for 8-h. The chitosan derivative was obtained by dialysis and freeze drying.

[0030] Example 3 This embodiment provides a chitosan derivative, which is prepared by the following method: (1) Take chitosan and add it to deionized water, adjust the pH to 6, and prepare a chitosan solution with a concentration of 2wt%; (2) Add spiculic acid to ethanol / water solution (v / v=1:1) to prepare a spiculic acid solution with a concentration of 60mg / mL; add EDC and NHS to the spiculic acid solution with a mass ratio of 1:1.2:1.5 and stir at room temperature for 40min to obtain an activated spiculic acid mixed solution. (3) The chitosan solution and the spiculosic acid mixture were added to the chitosan solution at a volume ratio of 1:0.5, and reacted at room temperature for 12 hours. The chitosan derivative was obtained by dialysis and freeze-drying.

[0031] Example 4 This embodiment provides a bone marrow mesenchymal stem cell exosome, which is prepared by the following method: With 4×10 5 At a seeding density of 10 cells / mL, P3 generation bone marrow mesenchymal stem cells were seeded into DMEM medium containing 10% FBS and cultured for 3 days in a cell culture incubator at 37°C and 5% CO2. The supernatant was collected, centrifuged at 300×g for 10 min, and the supernatant was retained. It was then centrifuged again at 2000×g for 10 min and the supernatant was retained. It was then centrifuged at 10000×g for 30 min and the supernatant was retained. After filtering through a 0.22 μm filter membrane, it was centrifuged at 100000×g for 2 h and the precipitate was retained. After adding PBS and mixing well, it was centrifuged again at 100000×g for 2 h and the precipitate was retained. This was the relatively pure exosome. 100 μL of PBS was added and the cells were frozen at -80°C.

[0032] Example 5 This embodiment provides a bone marrow mesenchymal stem cell exosome, which is prepared by the following method: With 3×10 5 At a seeding density of 10 cells / mL, P4 generation bone marrow mesenchymal stem cells were seeded into DMEM medium containing 10% FBS and cultured for 3 days in a cell culture incubator at 37°C and 5% CO2. The supernatant was collected, centrifuged at 300×g for 10 min, and the supernatant was retained. It was then centrifuged again at 2000×g for 10 min and the supernatant was retained. It was then centrifuged at 10000×g for 30 min and the supernatant was retained. After filtering through a 0.22 μm filter membrane, it was centrifuged at 100000×g for 2 h and the precipitate was retained. After adding PBS and mixing well, it was centrifuged again at 100000×g for 2 h and the precipitate was retained. This was the relatively pure exosome. 100 μL of PBS was added and the cells were frozen at -80°C.

[0033] Example 6 This embodiment provides a bone marrow mesenchymal stem cell exosome, which is prepared by the following method: With 7×10 5 At a seeding density of 10 cells / mL, P5 generation bone marrow mesenchymal stem cells were seeded into DMEM medium containing 10% FBS and cultured for 3 days in a cell culture incubator at 37°C and 5% CO2. The supernatant was collected, centrifuged at 300×g for 10 min, and the supernatant was retained. It was then centrifuged again at 2000×g for 10 min and the supernatant was retained. It was then centrifuged at 10000×g for 30 min and the supernatant was retained. After filtering through a 0.22 μm filter membrane, it was centrifuged at 100000×g for 2 h and the precipitate was retained. After adding PBS and mixing well, it was centrifuged again at 100000×g for 2 h and the precipitate was retained. This was the relatively pure exosome. 100 μL of PBS was added and the cells were frozen at -80°C.

[0034] Example 7 This embodiment provides an exosome composite material for treating periodontitis, comprising the following raw materials in parts by weight: 14 parts of chitosan derivative obtained in Example 1, 16 parts of sodium hyaluronate, 3 parts of zein, 0.7 parts of exosomes obtained in Example 4, 0.2 parts of sodium phosphonodeptide, and 68 parts of deionized water.

[0035] This embodiment also provides a method for preparing the above-mentioned exosome composite material for treating periodontitis, including the following steps: S1. Add sodium hyaluronate to 2 / 4 of the deionized water and stir until completely dissolved to obtain an aqueous solution of sodium hyaluronate; add chitosan derivative to 1 / 4 of the deionized water and stir until completely dissolved to obtain an aqueous solution of chitosan derivative; slowly add the aqueous solution of chitosan derivative to the aqueous solution of sodium hyaluronate and stir until homogeneous to obtain a mixed solution; S2. Dissolve zein in a small amount of 75wt% ethanol aqueous solution, and slowly add it dropwise to the remaining deionized water under stirring. Remove the ethanol by rotary evaporation to obtain a zein aqueous dispersion. Add the zein aqueous dispersion to the mixture under stirring, then add sodium phosphonodipeptide, stir until completely dissolved, adjust the pH to 6, cool in an ice bath at 4°C for 15 min, slowly add exosomes, and let stand overnight at 4°C.

[0036] Example 8 This embodiment provides an exosome composite material for treating periodontitis, comprising the following raw materials in parts by weight: 10 parts of chitosan derivative obtained in Example 2, 10 parts of sodium hyaluronate, 1 part of zein, 0.5 parts of exosomes obtained in Example 5, 0.1 parts of sodium phosphonodeptide, and 60 parts of deionized water.

[0037] This embodiment also provides a method for preparing the above-mentioned exosome composite material for treating periodontitis, including the following steps: S1. Add sodium hyaluronate to 2 / 4 of the deionized water and stir until completely dissolved to obtain an aqueous solution of sodium hyaluronate; add chitosan derivative to 1 / 4 of the deionized water and stir until completely dissolved to obtain an aqueous solution of chitosan derivative; slowly add the aqueous solution of chitosan derivative to the aqueous solution of sodium hyaluronate and stir until homogeneous to obtain a mixed solution; S2. Dissolve zein in a small amount of 75wt% ethanol aqueous solution, and slowly add it dropwise to the remaining deionized water under stirring. Remove the ethanol by rotary evaporation to obtain a zein aqueous dispersion. Add the zein aqueous dispersion to the mixture under stirring, then add sodium phosphonodipeptide, stir until completely dissolved, adjust the pH to 6, cool in an ice bath at 4°C for 15 min, slowly add exosomes, and let stand overnight at 4°C.

[0038] Example 9 This embodiment provides an exosome composite material for treating periodontitis, comprising the following raw materials in parts by weight: 20 parts of chitosan derivative obtained in Example 3, 20 parts of sodium hyaluronate, 5 parts of zein, 1 part of exosome obtained in Example 6, 0.3 parts of sodium phosphonodeptide, and 70 parts of deionized water.

[0039] This embodiment also provides a method for preparing the above-mentioned exosome composite material for treating periodontitis, including the following steps: S1. Add sodium hyaluronate to 2 / 4 of the deionized water and stir until completely dissolved to obtain an aqueous solution of sodium hyaluronate; add chitosan derivative to 1 / 4 of the deionized water and stir until completely dissolved to obtain an aqueous solution of chitosan derivative; slowly add the aqueous solution of chitosan derivative to the aqueous solution of sodium hyaluronate and stir until homogeneous to obtain a mixed solution; S2. Dissolve zein in a small amount of 75wt% ethanol aqueous solution, and slowly add it dropwise to the remaining deionized water under stirring. Remove the ethanol by rotary evaporation to obtain a zein aqueous dispersion. Add the zein aqueous dispersion to the mixture under stirring, then add sodium phosphonodipeptide, stir until completely dissolved, adjust the pH to 6, cool in an ice bath at 4°C for 15 min, slowly add exosomes, and let stand overnight at 4°C.

[0040] Comparative Example 1 Based on Example 7, chitosan was used instead of the chitosan derivative prepared in Example 1 to form Comparative Example 1.

[0041] Comparative Example 2 Based on Example 7, sodium phosphonodipeptide was omitted to form Comparative Example 2.

[0042] Experimental Example 1 The morphology of the P3-P5 generation bone marrow mesenchymal stem cells obtained in this invention was observed using an inverted microscope. The results are shown in [reference needed]. Figure 1 .

[0043] Figure 1 The images show the morphological characteristics of bone marrow mesenchymal stem cells obtained in this invention (magnification 20×), where a is a morphological image of P3 generation bone marrow mesenchymal stem cells, b is a morphological image of P4 generation bone marrow mesenchymal stem cells, and c is a morphological image of P5 generation bone marrow mesenchymal stem cells. Figure 1 It can be seen that the cells have a uniform morphology and exhibit a long spindle-shaped structure, which is consistent with the typical characteristics of bone marrow mesenchymal stem cells.

[0044] Experiment Example 2 The cytotoxicity of the chitosan derivatives prepared in Example 1 was analyzed, and the experimental steps are as follows: 100 μL of a density of 3 × 10 5 L929 fibroblasts in logarithmic growth phase were added to a cell culture plate and cultured in DMEM complete medium at 37°C and 5% CO2 for 12 h. The medium was then discarded, and 200 μL of DMEM complete medium containing 50 μg / mL chitosan derivative prepared in Example 1 was added. The cells were cultured for another 24 h, and cell morphology was observed under a microscope. The blank control was DMEM serum-free medium, and the positive control was DMEM complete medium supplemented with 5% phenol. Results are shown in [link to results]. Figure 2 .

[0045] Figure 2 The figures show the results of cytotoxicity experiments, where a represents the cytotoxicity of the chitosan derivative, b represents the cytotoxicity of the blank control, and c represents the cytotoxicity of the positive control. Figure 2It can be seen that the chitosan derivative prepared in Example 1 has no obvious cytotoxicity.

[0046] Experimental Example 3 The chitosan derivatives prepared in Example 1 were analyzed by Fourier transform infrared spectroscopy (FT-IR), and the results are shown in [reference needed]. Figure 3 .

[0047] Figure 3 The image shows the infrared spectrum of the chitosan derivative obtained in Example 1. Curve a is the infrared spectrum of chitosan, and curve b is the infrared spectrum of the chitosan derivative. (Observation) Figure 3 It can be seen that: compared to chitosan, chitosan derivatives have a lower content of 1630 cm⁻¹. -1 (Amide I band) and 1550 cm -1 (Amide II band) shows characteristic peaks of amide bonds, 2870-2920 cm⁻¹ -1 The significantly enhanced CH stretching vibration peak indicates that the chitosan derivative was successfully prepared.

[0048] Experiment Example 4 The mixture obtained by adding sodium phosphonyl dipeptide in step S2 of the examples or comparative examples at a volume ratio of 1:10 was mixed with PBS buffer and stored at 4°C for 7 days. The protein concentration was detected using a BCA kit. The number of exosome particles was detected by nanoflow cytometry after the sample was diluted 10 times. The results are shown in Table 1.

[0049] Table 1 As shown in Table 1, compared to Comparative Example 2, the solution obtained in Example 7 had a higher protein content, and the purity, particle number, and median particle size of the exosomes did not change significantly, indicating that the solution obtained in Example 7 had better exosome activity. These results demonstrate that the addition of sodium phosphonodeptide helps maintain good exosome activity and improves exosome stability.

[0050] Experimental Example 5 *Porphyromonas gingivalis*, *Aggregobacter actinomycetes*, and *Fusobacterium nucleatum* were cultured in an anaerobic environment on TBS liquid medium for 48 h, and then diluted with TBS medium to obtain 1×10⁻⁶ samples. 6 Prepare a seed culture of CFU / mL for later use.

[0051] Take 100 μL of the above seed culture and spread it evenly on TSB agar medium. After the bacterial culture is fully absorbed, use a 3 mm punch to make holes in the culture dish and add 20 μL of the exosome composite material prepared in the example or comparative example into the holes. Incubate anaerobically at 37 °C. After 24 h, count the number of viable pathogenic bacteria. The control group is distilled water. Calculate the inhibition rate of pathogenic bacteria. Inhibition rate (%) = (number of bacteria in control group - number of bacteria in experimental group) / number of bacteria in control group × 100%. The results are shown in Table 2.

[0052] Table 2 As shown in Table 2, compared to Example 7, Comparative Example 1 used chitosan, while Comparative Example 2, by omitting sodium phosphonyl dipeptide, uniformly resulted in a lower inhibition rate. These results indicate that the combined use of chitosan derivatives and sodium phosphonyl dipeptide can enhance the inhibitory effect on pathogenic bacteria.

[0053] Experimental Example 6 The recipe for high-sugar periodontitis consists of: 30g skim milk powder, 5g flour, 55g sucrose, 3g yeast powder, 1.5g liver powder, a small amount of salt, and fresh vegetables.

[0054] Forty 6-week-old male SD rats were acclimatized for 7 days, and five were randomly selected as the normal control group.

[0055] Except for the normal group, the remaining rats were anesthetized with sodium pentobarbital solution. A 0.2 mm orthodontic ligature wire was used to ligate the interproximal spaces between the first and second molars in the right maxilla of the rats, with the wire being pressed as far as possible into the gingiva. The rats were fed a high-sugar diet, and the ligature wires were observed every 7 days for any dislodgement. If dislodgement was observed, the ligatures were re-ligated. After 3 weeks, the periodontal condition of each rat was observed: gingival redness and swelling, bleeding on probing, and increased exposure of the root neck indicated successful establishment of the periodontitis model.

[0056] Rats that successfully modeled the disease were randomly divided into the following groups and administered the drug, with 5 rats in each group: Model group: 20 μL of sterile water was injected into the periodontal pocket once a week for a total of 4 weeks; Example 7 group: 20 μL of the exosome composite material of Example 7 was injected into the periodontal pocket once a week for 4 weeks; Example 8 group: 20 μL of the exosome composite material of Example 8 was injected into the periodontal pocket once a week for 4 weeks; Example 9 group: 20 μL of the exosome composite material of Example 9 was injected into the periodontal pocket once a week for 4 weeks; Comparative Example 1: 20 μL of the exosome composite material of Comparative Example 1 was injected into the periodontal pocket once a week for 4 weeks; Comparative Example 2: 20 μL of the exosome composite material of Comparative Example 2 was injected into the periodontal pocket once a week for 4 weeks; After the final treatment, rats in each group were anesthetized and euthanized. Specimens of the right maxillary molars, alveolar bone, and jawbone were collected. The alveolar bone damage in each group was assessed by scanning and reconstructing three-dimensional images of the rat maxilla. The distances from the cementoenamel junction (CEJ) to the alveolar ridge crest (ABC) of the first molars (mesial, distal, and central) were measured, and the average values ​​were calculated. The expression of TNF-α and IL-6 in the periodontal tissues of each rat was detected using a kit. The experimental results are shown in Table 3.

[0057] Table 3 As shown in Table 3, compared with Comparative Examples 1-2, the exosome composite material prepared in Example 7 exhibits superior therapeutic effects for periodontitis. These results demonstrate that the combined use of chitosan derivatives and sodium phosphonodeptide can enhance the therapeutic effect on periodontitis.

[0058] In summary, the combined use of chitosan derivatives and phosphonyl dipeptide sodium can synergistically inhibit periodontal pathogens and reduce the expression of pro-inflammatory factors by inhibiting signaling pathways such as NF-κB, thereby alleviating periodontal tissue inflammation. Furthermore, phosphonyl dipeptide sodium can effectively maintain exosome activity and synergistically enhance tissue repair capabilities, thus achieving a multi-pronged comprehensive treatment for periodontitis.

[0059] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. An exosome composite material for treating periodontitis, characterized by, The raw materials include the following parts by weight: 10-20 parts chitosan derivative, 10-20 parts sodium hyaluronate, 1-5 parts zein, 0.5-1 part exosomes, 0.1-0.3 parts sodium phosphonodeptide, and 60-70 parts deionized water.

2. The exosome composite material for treating periodontitis as described in claim 1, characterized in that, The preparation process of the chitosan derivative is as follows: (1) Take chitosan and add it to deionized water, adjust the pH, and prepare a chitosan solution; (2) Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the spiculic acid solution, and stir to obtain an activated spiculic acid mixed solution; (3) The chitosan acid mixture is added to the chitosan solution, and the chitosan derivative is obtained after the reaction.

3. The exosome composite material for treating periodontitis as described in claim 2, characterized in that, In step (1), the pH is adjusted to 5-6; the concentration of the chitosan solution is 1-2 wt%.

4. The exosome composite material for treating periodontitis as described in claim 2, characterized in that, In step (2), the mass ratio of the spiculin solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(0.8-1.2):(1-1.5); the solvent of the spiculin solution is composed of ethanol and water in a volume ratio of 1:(0.5-1); the concentration of the spiculin solution is 40-60 mg / mL; and the stirring time is 30-40 min.

5. The exosome composite material for treating periodontitis as described in claim 2, characterized in that, The volume ratio of the chitosan solution and the styraciferic acid mixture in step (3) is 1:(0.3-0.5); the reaction time is 8-12h.

6. The exosome composite material for treating periodontitis as described in claim 1, characterized in that, The exosomes are bone marrow mesenchymal stem cell exosomes.

7. The exosome composite material for treating periodontitis as described in claim 6, characterized in that, The method for preparing the bone marrow mesenchymal stem cell exosomes is as follows: Bone marrow mesenchymal stem cells were seeded into a culture medium, and the supernatant was collected, centrifuged at differential speed, and freeze-dried to obtain bone marrow mesenchymal stem cell exosomes.

8. The exosome composite material for treating periodontitis as described in claim 7, characterized in that, The culture medium is DMEM medium containing 10% FBS; the seeding density of the bone marrow mesenchymal stem cells is (3-7) x 10 5 / mL.

9. The exosome composite material for treating periodontitis as described in claim 8, characterized in that, The bone marrow mesenchymal stem cells mentioned are P3-P5 generation bone marrow mesenchymal stem cells.

10. A method for preparing an exosome composite material for treating periodontitis according to any one of claims 1-9, characterized in that, Includes the following steps: S1. Add sodium hyaluronate to 2 / 4 of deionized water to obtain an aqueous solution of sodium hyaluronate; add chitosan derivative to 1 / 4 of deionized water to obtain an aqueous solution of chitosan derivative; add the aqueous solution of chitosan derivative to the aqueous solution of sodium hyaluronate to obtain a mixed solution; S2. Add zein to the remaining deionized water, then mix it thoroughly with the mixture, exosomes, and sodium phosphonodipeptide.

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