Injectable hydrogel for treating periodontitis and preparation method thereof

Abstract

The invention discloses injectable hydrogel for treating periodontitis and a preparation method of the injectable hydrogel. The injectable hydrogel is prepared from protocatechuic acid grafted carboxymethyl chitosan hydrogel and a zinc-metformin complex loaded in the protocatechuic acid grafted carboxymethyl chitosan hydrogel. The hydrogel aims to become a safe and effective treatment method so as to reduce oxidative stress and tissue damage caused by periodontitis. And the hydrogel is designed into an injectable form, so that the clinical operation is facilitated, and the comfort of a patient is improved. Therefore, the treatment effect and life quality of patients with chronic periodontitis are improved, and inconvenience and discomfort in the treatment process are reduced.

Description

Technical Field

[0001] The invention relates to an injectable hydrogel for treating periodontitis and a preparation method thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Periodontitis is a persistent inflammatory disease induced by pathogenic microorganisms, including the host's immune response. Chronic periodontitis seriously affects people's quality of life, leading to alveolar bone loss, tooth loss, masticatory dysfunction and poor aesthetics. The imbalance between reactive oxygen and antioxidant stress systems leads to an imbalance in the oxidative stress system, which induces periodontitis and further aggravates the loss of periodontal tissue attachment.

[0004] The long-term effects of supragingival scaling and subgingival curettage are limited by the regeneration of microorganisms and their removal in deep subgingival pockets. Adjunctive treatments, such as the use of antimicrobial, antioxidant, and bone-promoting biomaterials, can promote periodontal tissue regeneration.

[0005] Although traditional supragingival scaling and subgingival curettage can remove some periodontal pathogens, their long-term effects are limited by the regeneration of microorganisms and the difficulty of removal in deep subgingival pockets. Therefore, a new treatment method is needed to make up for these shortcomings. Summary of the invention

[0006] In view of the shortcomings of the existing technology, the purpose of the invention is to solve some key problems in the treatment of chronic periodontitis, including: making up for the deficiencies of basic treatment and promoting the regeneration of periodontal tissue. By using biomaterials with antibacterial, antioxidant and bone-promoting properties, the invention aims to promote the regeneration of periodontal tissue to reduce alveolar bone loss, tooth loss and other problems.

[0007] The technical solution adopted by the present invention is as follows: In a first aspect of the present invention, an injectable hydrogel for treating periodontitis is provided, wherein the injectable hydrogel comprises: a protocatechuic acid grafted carboxymethyl chitosan hydrogel, and a zinc-metformin complex loaded in the protocatechuic acid grafted carboxymethyl chitosan hydrogel.

[0008] In one or some embodiments of the present invention, the ratio of protocatechuic acid grafted carboxymethyl chitosan hydrogel to zinc-metformin complex is 1 mL: 4-16 mmol. The concentration of protocatechuic acid grafted carboxymethyl chitosan hydrogel is 10-20%, i.e., 10-20 mg / 0.1 mL.

[0009] In one or some embodiments of the present invention, the protocatechuic acid grafted carboxymethyl chitosan is prepared by the following method: The protocatechuic acid is dissolved in isopropanol, and then EDC and NHS are added, and nitrogen is introduced into the reaction system to react; after the reaction, it is concentrated, and then DMF is added to dissolve it to obtain activated protocatechuic acid; Dissolve carboxymethyl chitosan in water; The activated protocatechuic acid is added to the prepared carboxymethyl chitosan solution for reaction; after the reaction, the obtained solution is added to ethanol, the precipitated product is separated by centrifugation, washed, and dried to obtain protocatechuic acid grafted carboxymethyl chitosan, which is dissolved in water to obtain protocatechuic acid grafted carboxymethyl chitosan hydrogel.

[0010] In a second aspect of the present invention, a method for preparing the injectable hydrogel according to the first aspect is provided, the method comprising the following steps: The hydrogel can be obtained by dispersing protocatechuic acid grafted carboxymethyl chitosan in an aqueous solution of a zinc-metformin complex.

[0011] In one or some embodiments of the present invention, the feed ratio of the protocatechuic acid grafted carboxymethyl chitosan to the zinc-metformin complex aqueous solution is (50-200) mg: (0.5-2) mL; the concentration of the zinc-metformin complex aqueous solution is 4-16 mmol / L. It has been verified by experiments that the 4-16 mmol / L zinc-metformin complex aqueous solution significantly increases the alkaline phosphatase ALP expressed by the body and has a high antibacterial ability.

[0012] In one or some embodiments of the present invention, the protocatechuic acid grafted carboxymethyl chitosan is prepared by the following method: The protocatechuic acid is dissolved in isopropanol, and then EDC and NHS are added, and nitrogen is introduced into the reaction system to react; after the reaction, it is concentrated, and then DMF is added to dissolve it to obtain activated protocatechuic acid; Dissolve carboxymethyl chitosan in water; The activated protocatechuic acid is added to the prepared carboxymethyl chitosan solution to react; after the reaction, the obtained solution is added to ethanol, and the precipitated product is separated by centrifugation, washed, and dried to obtain the protocatechuic acid grafted carboxymethyl chitosan.

[0013] Preferably, the molar ratio of protocatechuic acid, EDC, NHS and carboxymethyl chitosan is (1-5): (1-5): (1-5) (1-5).

[0014] Preferably, the activation reaction time is 18 to 24 hours.

[0015] Preferably, the activated protocatechuic acid reacts with carboxymethyl chitosan for 10 to 14 hours.

[0016] In one or some embodiments of the present invention, the zinc-metformin complex is obtained by the following method: Metformin hydrochloride and zinc chloride are dissolved in anhydrous ethanol respectively; then the metformin hydrochloride solution is added into the zinc chloride solution to react until microcrystals are precipitated, and the crystal product is collected after precipitation, and the zinc-metformin complex is obtained after drying.

[0017] The saliva of patients with periodontitis shows a trend of excessive copper content and decreased zinc content. The saliva of patients with periodontitis shows a trend of excessive copper content and decreased zinc content. This is because the increase in copper content will change the permeability of the gingival epithelium and impair the absorption of zinc on the mucosa.

[0018] Preferably, the molar ratio of metformin hydrochloride to zinc chloride is 2:1.

[0019] Preferably, the reaction temperature is 40-60° C. and the reaction time is 5-7 h.

[0020] In the third aspect of the present invention, there is provided use of the injectable hydrogel according to the first aspect and / or the injectable hydrogel prepared according to the second aspect in preparing a product for treating periodontitis.

[0021] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects: The present invention starts from clinical problems and uses the advantages of cross-disciplinary studies from the perspective of biomaterials to construct a safe, effective, easy-to-operate, and comfortable injectable hydrogel, aiming to make up for the shortcomings of basic periodontal treatment in clinical practice.

[0022] The hydrogel of the present invention is intended to be a safe and effective treatment method to reduce oxidative stress and tissue damage caused by periodontitis. The hydrogel of the present invention is designed to be injectable to facilitate clinical operation and improve patient comfort. The treatment effect and quality of life of patients with chronic periodontitis are thereby improved, while reducing the inconvenience and discomfort during treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 Schematic diagram of the preparation principle of the injectable hydrogel of the present invention.

[0025] Figure 2 These are macroscopic photographs and scanning electron microscope photographs of the injectable hydrogel of the present invention.

[0026] Figure 3 This is the Fourier infrared spectroscopy analysis of the injectable hydrogel of the present invention.

[0027] Figure 4 This is an analysis of the results of the CCK-8 experiment, hemolysis experiment, and staining experiment of the injectable hydrogel of the present invention.

[0028] Figure 5 The present invention is an analysis of the alkaline phosphatase and alizarin red staining and quantitative detection results of the injectable hydrogel of the present invention.

[0029] Figure 6 The results of the antioxidant capacity analysis of the injectable hydrogel of the present invention are shown in FIG.

[0030] Figure 7 The results of the antibacterial effect analysis of the injectable hydrogel of the present invention are shown in FIG.

[0031] Figure 8 The present invention is an analysis of the therapeutic effect of the injectable hydrogel on periodontitis.

[0032] Fig. 9 The invention relates to the detection and analysis of active substances in periodontal tissues of the injectable hydrogel of the present invention.

[0033] Fig.10 Experimental effect of caffeic acid grafted carboxymethyl chitosan-zinc metformin in the treatment of periodontitis in rats. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0037] Example 1 1) Preparation of zinc-metformin complex using metformin hydrochloride and zinc chloride as raw materials: dissolve metformin hydrochloride and zinc chloride in anhydrous ethanol at a concentration ratio of 2:1. Then slowly add the metformin hydrochloride solution to the zinc chloride solution. Keep the entire reaction system at 50 °C for full reaction, and continue magnetic stirring for 6 hours until microcrystals precipitate. Collect the crystalline product after precipitation, and freeze-dry to obtain the zinc-metformin complex.

[0038] 2) Grafting of protocatechuic acid onto carboxymethyl chitosan via EDC / NHS: Protocatechuic acid (3.3 mmol) was dissolved in 40 mL of isopropanol, and then EDC (3.3 mmol) and NHS (3.3 mmol) were added. Nitrogen was introduced into the reaction system and the reaction was allowed to proceed for 24 h. Rotary evaporation was performed, and then DMF was added to dissolve the activated PCA. Carboxymethyl chitosan (3.3 mmol) was dissolved in 40 mL of deionized water. The activated protocatechuic acid was added dropwise to the prepared carboxymethyl chitosan solution and stirred at room temperature for 12 h. The resulting solution was added dropwise to ethanol, the precipitated product was separated by centrifugation, and then rinsed three times with 75% ethanol. Protocatechuic acid-grafted carboxymethyl chitosan was obtained after freeze-drying.

[0039] 3) Disperse protocatechuic acid grafted carboxymethyl chitosan (100 mg) in an aqueous solution of zinc-metformin complex (1 mL, 0, 4, 8, 16 mmol / L, respectively) to obtain a hydrogel.

[0040] In this example, zinc-metformin was loaded into protocatechuic acid-grafted carboxymethyl chitosan hydrogel, and the injectable hydrogel was successfully prepared by the above method. Figure 2 Macroscopic and scanning electron microscope images of the hydrogel.

[0041] The existence of each component of the hydrogel was verified by Fourier transform infrared spectroscopy analysis, such as Figure 3 .

[0042] The CCK-8 test showed that after 2 days of co-culture with the hydrogel extract, human periodontal ligament stem cells maintained good vitality. The hemolysis test showed that the hydrogels had good blood compatibility, and the hemolysis rate of each group was less than 2% ( Figure 4 B). After co-culturing human periodontal ligament stem cells with hydrogel extract for 2 days, live / dead cell staining was performed and it was found that the hydrogel extract had no significant effect on cell growth. This proves that the hydrogel has good cell compatibility. Figure 4 .

[0043] Alkaline phosphatase and alizarin red staining and quantitative detection showed that the hydrogel significantly increased the staining area and expression of alkaline phosphatase and calcium nodules of periodontal ligament stem cells. Figure 5. It was demonstrated that the hydrogel had good ability to induce osteogenic differentiation in vitro.

[0044] In order to evaluate the antioxidant capacity of the hydrogel, the DPPH and ABTS methods were used for detection. The hydrogel showed good DPPH and ABTS free radical scavenging ability, such as Figure 6 .

[0045] The antibacterial effect of the hydrogel on Staphylococcus aureus, Escherichia coli and Fusobacterium nucleatum was tested by colony counting experiments. The experiment found that the hydrogel can significantly inhibit the growth of these three bacteria. Figure 7 .

[0046] Regarding the efficacy of hydrogel on periodontitis, the present invention constructed a rat periodontitis model for verification. Then, the rats were randomly divided into four groups: (1) sham operation group; (2) periodontitis group; (3) minocycline group; (4) hydrogel group. Except for the healthy group, the drug was injected once every other day at the mesial, middle and distal ends of the second molar of the rats. The periodontitis group was injected with 50 μL of hydrogel and an equal amount of saline, and the minocycline group was injected with 50 μL of minocycline hydrochloride paste, and the injection was continued for 4 weeks. The treatment effect was analyzed after 28 days of treatment, and the periodontal tissues were collected.

[0047] To examine the regeneration of alveolar bone, micro-CT scans of the hard tissues including maxillary bones and teeth were performed 4 weeks later. Then, the distance between the alveolar enamel junction and the apex of the alveolar ridge was measured at 6 anatomical sites of the maxillary second molar, including the buccal and lingual surfaces (3 points on each side, mid, mid, and distal regions). After hydrogel treatment, alveolar bone resorption was reduced compared with the sham group. By measuring the distance from the cementoenamel junction to the apex of the alveolar ridge in each experimental group, it was found that the distance from the cementoenamel junction to the apex of the alveolar ridge was significantly lower in the hydrogel treatment group and the positive control treatment group (minocycline hydrochloride paste treatment) than in the periodontitis group. The periodontal tissues were fixed, decalcified, dehydrated, transparentized, embedded, and finally sectioned. The tissues were then stained using the H&E staining method. HE staining showed that rats in the periodontitis group had typical pathological manifestations, including inflammatory cell infiltration of the periodontal ligament, hyperplasia of the junctional epithelium to the root, and macrophage infiltration around the alveolar ridge. Masson staining and osteoclast staining were used to evaluate collagen deposition and osteoclast activity. It was found that after treatment with the hydrogel, the fibers in the gingival and periodontal connective tissues were arranged more regularly, and the number of osteoclasts around the alveolar ridge decreased, such as Figure 8 The levels of TNF-α and IL-1β in periodontal tissues were detected by immunohistochemical staining, and then the expression in the tissues was quantitatively analyzed. After treatment with hydrogel, the gingival inflammation of rats was alleviated and the expression of inflammatory factors was significantly reduced, such as Fig. 9 .

[0048] In this study, attempts were made to use other polyphenolic acids such as caffeic acid and carboxymethyl chitosan to prepare caffeic acid grafted carboxymethyl chitosan, using the same preparation method as protocatechuic acid grafted carboxymethyl chitosan in Example 1. Experiments have shown that caffeic acid grafted carboxymethyl chitosan cannot form a hydrogel like protocatechuic acid grafted carboxymethyl chitosan. In subsequent animal experiments on periodontitis, caffeic acid grafted carboxymethyl chitosan was mixed with zinc metformin and loaded into poloxamer (PF127) to treat rat periodontitis. The results showed that the caffeic acid grafted carboxymethyl chitosan group could not effectively treat rat periodontitis, such as Fig.10 .

[0049] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An injectable hydrogel for the treatment of periodontitis, characterized in that: The injectable hydrogel comprises a protocatechuic acid grafted carboxymethyl chitosan hydrogel and a zinc-metformin complex loaded in the protocatechuic acid grafted carboxymethyl chitosan hydrogel.

2. The injectable hydrogel for treating periodontitis according to claim 1, characterized in that: The ratio of protocatechuic acid grafted carboxymethyl chitosan hydrogel to zinc-metformin complex is 1mL:4-16 mmol, and the concentration of protocatechuic acid grafted carboxymethyl chitosan hydrogel is 10-20%.

3. The method for preparing the injectable hydrogel according to claim 1 or 2, characterized in that: The method comprises the following steps: The hydrogel can be obtained by dispersing protocatechuic acid grafted carboxymethyl chitosan in an aqueous solution of a zinc-metformin complex.

4. The method for preparing the injectable hydrogel according to claim 3, characterized in that: The feeding ratio of the protocatechuic acid grafted carboxymethyl chitosan to the zinc-metformin complex aqueous solution is (50-200) mg: (0.5-2) mL; the concentration of the zinc-metformin complex aqueous solution is 4-16 mmol / L.

5. The method for preparing the injectable hydrogel according to claim 3, characterized in that: The protocatechuic acid grafted carboxymethyl chitosan is prepared by the following method: The protocatechuic acid is dissolved in isopropanol, and then EDC and NHS are added, and nitrogen is introduced into the reaction system to react; after the reaction, it is concentrated, and then DMF is added to dissolve it to obtain activated protocatechuic acid; Dissolve carboxymethyl chitosan in water; The activated protocatechuic acid is added to the prepared carboxymethyl chitosan solution to react; after the reaction, the obtained solution is added to ethanol, and the precipitated product is separated by centrifugation, washed, and dried to obtain the protocatechuic acid grafted carboxymethyl chitosan.

6. The method for preparing the injectable hydrogel according to claim 5, characterized in that: The molar ratio of protocatechuic acid, EDC, NHS and carboxymethyl chitosan is (1-5): (1-5): (1-5) (1-5).

7. The method for preparing the injectable hydrogel according to claim 5, characterized in that: The activation reaction time is 18~24h.

8. The method for preparing the injectable hydrogel according to claim 5, characterized in that: The activated protocatechuic acid reacts with carboxymethyl chitosan for 10-14 hours.

9. The method for preparing the injectable hydrogel according to claim 3, characterized in that: The zinc-metformin complex is obtained by the following method: Metformin hydrochloride and zinc chloride are dissolved in anhydrous ethanol respectively; then the metformin hydrochloride solution is added into the zinc chloride solution to react until microcrystals are precipitated, and the crystal product is collected after precipitation, and the zinc-metformin complex is obtained after drying.

10. Use of the injectable hydrogel according to claim 1 or 2 in the preparation of a product for treating periodontitis.

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