Composition for periodontal treatment and preparation method thereof

Abstract

A composition for periodontal treatment and a method of making the same. The composition comprises a polymer carrier, microspheres with an antibiotic core encapsulated by a polymer shell containing an oxygen generator and nanoparticles with catalase-like activity, and the oxygen generator (including peroxide compounds, oxygen storage materials and microalgae) in the shell of the microspheres is an oxygen source. A hydrogen peroxide generator configured to generate hydrogen peroxide or release oxygen through the oxygen storage material and the microalgae when the peroxide compound is hydrolyzed; wherein along with the hydrolysis of the oxygen generating agent and the degradation of the polymer shell, the antibiotics positioned in the core of the microsphere are gradually released; nanoparticles with catalase-like activity encapsulated in the carrier are used for decomposing the hydrogen peroxide to generate oxygen and water. The periodontal treatment composition is characterized in that the sequence release of oxygen and antibiotics is achieved through the special structural design of the microspheres, the oxygen generated when the peroxide compound is hydrolyzed and the hydrogen peroxide is decomposed contributes to the maintenance of an oxygen-enriched microenvironment in the periodontal pocket in which the polymer is placed, and at the same time, the oxygen-enriched microenvironment of the periodontal pocket is released. After the peroxide compound is hydrolyzed, pores are formed in the shell of the microsphere. Subsequently, the antibiotics in the microsphere core will be released and exhibit enhanced antibacterial and anti-biofilm effects due to the mitigation of the hypoxic microenvironment.

Description

Technical Field

[0001] The present invention relates to a composition for treating periodontitis and a method for preparing the composition, and particularly, although not exclusively, to a composition for treating periodontitis based on the sequential release of oxygen and antibiotics. Background Art

[0002] Periodontal disease has a high prevalence in both developing and developed countries, making it a public health problem. Periodontitis, an inflammatory disease associated with dysbiosis and affecting the periodontal supporting tissues, is the main cause of tooth loss in adults and seriously affects the quality of life of patients. Currently, non-surgical periodontal treatment includes scaling and root planing as well as the use of antibiotics. Sometimes, due to residual bacteria and the unchanged hypoxic microenvironment in the deep periodontal pocket, the inflammation of the periodontal tissue is not significantly improved by mechanical treatment alone and is prone to recurrence. The adjunctive use of antibiotics has been proven to be effective in clinical practice, but there is the problem of antibiotic resistance, and studies have shown that the hypoxic microenvironment of biofilms may lead to their strong resistance to many antibiotics. Therefore, the development of new strategies with translational potential is crucial to eliminate residual biofilms, improve the dysbiosis of the periodontal microenvironment, and prevent the rapid recolonization of pathogens.

[0003] Plaque microorganisms are the initiating factors for the development and progression of periodontitis. As periodontitis progresses, the main components of subgingival plaque shift from Gram-positive aerobic bacteria to Gram-negative anaerobic bacteria. It has been reported that the oxygen content at the bottom of the periodontal pocket gradually decreases with increasing depth. The average partial pressure of oxygen (pO2) in moderate periodontal pockets with a depth of 5-6 mm is 15.7 mmHg, while the average pO2 in deeper periodontal pockets with a depth of 7-10 mm is 12 mmHg. These results suggest that the hypoxic microenvironment within deep periodontal pockets may facilitate the further proliferation of anaerobic periodontal pathogens. On the other hand, oxygen deficiency may enhance the inflammatory response because insufficient oxygen affects the production of reactive oxygen species (ROS) in immune cells, further hindering the clearance of pathogens. Therefore, the hypoxic microenvironment within deep periodontal pockets is associated with the microbial and immunological aspects of the inflammatory process of periodontitis and may be a potential target for periodontal therapy.

[0004] Porphyromonas gingivalis (P.gingivalis) is considered to be a key pathogen of periodontitis. It can grow in a stable state under low levels of oxygen (6-10%), but cannot survive prolonged exposure to 20% oxygen. Studies have shown that hyperbaric oxygen therapy significantly reduces the gingival index, probing depth, and attachment loss in patients with aggressive periodontitis. However, hyperbaric oxygen therapy has problems such as cumbersome treatment process and expensive equipment. Therefore, the local application of oxygen-producing materials to increase the oxygen in the periodontal pocket to a certain level will effectively improve the periodontal microenvironment and reduce the content of anaerobic pathogens associated with periodontitis. It is expected to become a new strategy for periodontal treatment. Summary of the Invention

[0005] According to a first aspect of the present invention, there is provided a composition for periodontal treatment, comprising:

[0006] - a polymer carrier encapsulating microspheres comprising an oxygen generator shell and an antibiotic core, wherein the oxygen generator is configured to generate hydrogen peroxide when the peroxide compound is hydrolyzed or release oxygen through the oxygen storage material and the microalgae, and the antibiotic is subsequently released under the oxygen-rich microenvironment; and

[0007] - nanoparticles also encapsulated in the polymer carrier, wherein the nanoparticles are configured to have catalase-like activity and decompose the hydrogen peroxide to produce oxygen and water;

[0008] The composition can treat periodontitis through a sequential release of oxygen and antibiotics. After periodontal treatment, the oxygen generated when the peroxide compound is hydrolyzed and the hydrogen peroxide is decomposed or released by the oxygen storage material and microalgae helps maintain an oxygen-rich microenvironment within the periodontal pocket where the composition is placed. Subsequently, the antibiotics located in the microsphere core are released through the pores formed by the hydrolysis of the peroxide. The released antibiotics further exhibit enhanced antibacterial and anti-biofilm effects in this oxygen-rich microenvironment.

[0009] According to a first aspect, the nanoparticles having catalase-like activity are much more stable than natural catalase.

[0010] According to the first aspect, the polymer carrier is also a drug delivery system encapsulating the microspheres and the nanoparticles.

[0011] According to the first aspect, the shell of the microsphere is a hydrophobic polymer containing the oxygen generator, and the core of the microsphere includes the antibiotic dissolved or dispersed in water or a hydrophilic polymer.

[0012] According to the first aspect, the oxygen generator includes at least one of calcium peroxide (CPO, chemical formula CaO2), magnesium peroxide and other peroxide compounds, oxygen storage materials (including sodium percarbonate, manganese dioxide, hemoglobin, etc.) and microalgae, or a combination thereof, and the main function of this component is to provide an oxygen source.

[0013] According to a first aspect, the hydrophobic polymer comprises polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyphosphate (PPE) and / or polyorthoester (POE).

[0014] According to the first aspect, the antibiotics include fluoroquinolone antibiotics (such as ciprofloxacin), tetracycline antibiotics (such as tetracycline, minocycline and doxycycline), nitroimidazole antibiotics (such as metronidazole), penicillin antibiotics (such as amoxicillin, amoxicillin clavulanate potassium), glycopeptide antibiotics and / or macrolides, and the antibiotics are dissolved or dispersed in the water or hydrophilic material, wherein the hydrophilic material includes at least one of gelatin, collagen, fibrin, silk protein, hyaluronic acid, cellulose, chitosan, alginate, dextran, polyvinyl alcohol (PVA) and / or polyethylene glycol (PEG).

[0015] According to a first aspect, the polymeric dosage form comprises a hydrogel, a fiber, a ribbon, a film and / or a microparticle.

[0016] According to a first aspect, the hydrogel comprises chitosan hydrogel, poloxamer, poly (N-isopropylacrylamide), PEG and / or cellulose.

[0017] According to a first aspect, the nanoparticles include CeO2, MnO2, Mn3O4, Au, Pd, Pt and / or Prussian blue nanoparticles.

[0018] According to the first aspect, the composition is further configured to increase the oxygen content of the microenvironment in the periodontal pocket after periodontal treatment.

[0019] According to a second aspect of the present invention, there is provided a method for preparing the core-shell structured microspheres according to the first aspect, comprising the following steps:

[0020] -Microspheres with a ciprofloxacin core and a PCL / CPO2 shell were prepared using a double emulsion method (w / o / w);

[0021] - 10mg / mL ciprofloxacin hydrochloride (Cipro), but less than 20mg / mL, to avoid uneven distribution of Cipro during microsphere preparation and dissolution

[0022] A Cipro aqueous solution (w1) was prepared by dissolving a certain amount of PCL in 1 mL of distilled water (according to the literature, the upper limit of PCL concentration is 50% or 60% w / v%), and CPO was added in a certain ratio (the optimal CPO:PCL mass ratio was 1:1) to form a polymer oily solution (o); the Cipro solution (w1) prepared above was added to the mixed solution (o), and a homogenizer was used to mix the mixture.

[0023] Emulsification at 15000 rpm formed a stable w1 / o emulsion.

[0024] - Then, this stable w1 / o emulsion was slowly added to 60 mL of 0.5% PVA (w2) using a homogenizer at less than 10000 rpm.

[0025] The mixture was treated for 1 minute to disperse and form microspheres with Cipro as the core and PCL / CPO as the shell (PCA-CIP). The final microsphere dispersion was then stirred for 1-2 hours to evaporate the solvent and harden the microspheres. The microspheres were collected and washed three times with distilled water and once with anhydrous ethanol, and dried in a vacuum oven for 24 hours.

[0026] According to a third aspect of the present invention, there is provided a method for preparing the composition according to the first aspect, comprising:

[0027] - Chitosan was dissolved in 0.1 M acetic acid solvent and sodium β-glycerophosphate (β-GP) was dissolved in pure water and then stored in an ice bath;

[0028] The microspheres and CeO2 nanoparticles contained in the composition are dispersed in the β-GP solution, the above components are placed in a double-tube applicator, mixed and injected into the periodontal pocket, thereby obtaining the composition according to the first aspect. According to a fourth aspect of the present invention, there is provided a method for preparing the composition according to the first aspect, comprising: a polymer carrier encapsulating an oxygen generator comprising a peroxide compound, wherein the oxygen generator is configured to generate hydrogen peroxide when the peroxide compound is hydrolyzed or to release oxygen through the oxygen storage material and the microalgae; and

[0029] - nanoparticles encapsulated in the polymer carrier, wherein the nanoparticles are configured to have catalase-like activity and decompose the hydrogen peroxide to produce oxygen and water;

[0030] Wherein, after periodontal treatment, the oxygen generated when the peroxide compound is hydrolyzed and the hydrogen peroxide is decomposed or released by the oxygen storage material and the microalgae is configured to help maintain the oxygen-rich microenvironment around the periodontal pocket where the composition is disposed.

[0031] According to a fourth aspect of the present invention, the polymer carrier comprises a hydrophobic polymer containing the oxygen generating agent.

[0032] According to a fourth aspect of the present invention, the composition further comprises an antibiotic encapsulated by the polymer carrier, wherein the antibiotic is released into the periodontal pocket at a predetermined post-treatment period after periodontal treatment.

[0033] According to the fourth aspect of the present invention, the antibiotic is dissolved or dispersed in water or a hydrophilic polymer, or conjugated to the polymer carrier.

[0034] According to a fourth aspect of the present invention, the antibiotics include fluoroquinolone antibiotics, tetracycline antibiotics, nitroimidazole antibiotics, penicillin antibiotics, glycopeptide antibiotics and / or macrolides, and wherein the antibiotics are dissolved in water or encapsulated in the hydrophilic material, wherein the hydrophilic material includes gelatin, collagen, fibrin, silk protein, hyaluronic acid, cellulose, chitosan, alginate, dextran, polyvinyl alcohol (PVA) and / or polyethylene glycol (PEG).

[0035] According to a fourth aspect of the present invention, the oxygen generating agent comprises CaO2, a peroxide compound of magnesium peroxide, and at least one of an oxygen storage material, wherein the oxygen storage material comprises at least one of sodium percarbonate, manganese dioxide, hemoglobin and microalgae, or a combination thereof, and wherein the peroxide compound is configured to serve as an oxygen generating source.

[0036] According to a fourth aspect of the present invention, the hydrophobic polymer comprises polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyphosphate (PPE) and / or polyorthoester (POE).

[0037] According to the fourth aspect of the present invention, the dosage form of the polymer carrier includes hydrogel, fiber, strip, film or microparticle.

[0038] According to a fourth aspect of the present invention, the hydrogel comprises chitosan hydrogel, poloxamer, poly (N-isopropylacrylamide), PEG and / or cellulose.

[0039] According to a fourth aspect of the present invention, the hydrogel comprises thermosensitive crosslinks.

[0040] According to a fourth aspect of the present invention, the nanoparticles include CeO2, MnO2, Mn3O4, Au, Pd, Pt and / or Prussian blue nanoparticles.

[0041] According to a fourth aspect of the present invention, the composition is further configured to increase the oxygen content of the microenvironment in the periodontal pocket after periodontal treatment.

[0042] According to a fourth aspect of the present invention, the polymer carrier comprises a two-component composite structure having a first polymer layer and a second polymer layer.

[0043] According to a fourth aspect of the present invention, the first polymer layer encapsulates the oxygen generator, and the second polymer layer encapsulates the nanoparticles.

[0044] According to a fourth aspect of the present invention, the first polymer layer comprises an electrospun fiber membrane.

[0045] According to a fourth aspect of the present invention, the electrospun fiber membrane comprises a hydrophobic host polymer such as polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyphosphate (PPE) and / or polyorthoester (POE).

[0046] According to a fourth aspect of the present invention, the second polymer layer comprises a hydrogel.

[0047] According to a fourth aspect of the present invention, the hydrogel comprises gelatin methacryloyl (GelMA), chitosan hydrogel, poloxamer, poly (N-isopropylacrylamide), PEG and / or cellulose.

[0048] According to a fourth aspect of the present invention, the two-component composite structure is a Janus composite material, wherein the first polymer layer and the second polymer layer exhibit different surface properties and / or functions. The first polymer layer contains an oxygen-generating agent and is inserted into the periodontal pocket close to the tooth surface, thereby facilitating the rapid action of hydrogen peroxide and oxygen generated by the oxygen-generating material on the plaque biofilm located on the tooth surface and in the periodontal pocket to exert antibacterial and anti-biofilm effects; while the second polymer layer contains nanoparticles with nanozyme-like activity, and the second polymer layer is an injectable hydrogel with good plasticity, which can be used to fill the space between the first polymer and the gingival tissue through a delivery device. The nanoparticles encapsulated in the hydrogel can fully decompose the hydrogen peroxide produced by the first polymer into oxygen through their catalase-like activity, thereby reducing the oxidative damage of hydrogen peroxide to the gingival tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a diagram showing that a periodontal treatment composition according to an embodiment of the present invention is used.

[0050] Figure 2 It shows Figure 1 Illustration of the mechanism of periodontal treatment.

[0051] Figure 3A is a photograph showing the injectability of CPO-GelMA hydrogel.

[0052] Figure 3B This is a photograph of 15% CPO-GelMA liquid in an inversion test.

[0053] Figure 3C are SEM images of GelMA hydrogels loaded with different concentrations of CPO.

[0054] Figure 3D This is the oxygen release curve of different CPO-GelMA hydrogels within 48 hours.

[0055] Figure 3E This is the oxygen release curve of different CPO-GelMA hydrogels within 7 days.

[0056] Figure 3F is a bar graph showing the residual H2O2 concentration of different CPO-GelMA hydrogels after being immersed in PBS solution with or without catalase for 24 h.

[0057] Figure 3G is a bar graph showing the pH changes caused by different CPO-GelMA hydrogels at 24 h and 48 h.

[0058] Figure 4A This is a bar graph showing the inhibitory effects of different CPO-GelMA hydrogels on the growth of Porphyromonas gingivalis.

[0059] Figure 4B Representative CFU images of Porphyromonas gingivalis after 24 h of treatment with different CPO-GelMA hydrogels.

[0060] Figure 4C is a bar graph of the survival rate of Porphyromonas gingivalis after treatment with different CPO-GelMA hydrogels for 24 hours.

[0061] Figure 4D Representative SEM images of Porphyromonas gingivalis after 3 h of treatment on CPO-GelMA hydrogel.

[0062] Figure 4E Representative SEM images of Porphyromonas gingivalis after 6 h of treatment on CPO-GelMA hydrogel.

[0063] Figure 4F This is a bar graph showing the relative gene expression levels of the main virulence factors of Porphyromonas gingivalis after treatment with 5% CPO-GelMA hydrogel.

[0064] Figure 5A This is a bar graph showing the inhibitory effect of CPO-GelMA hydrogel on the growth of Porphyromonas gingivalis in culture media with different hemin concentrations.

[0065] Figure 5B is a bar graph of the survival rate of Porphyromonas gingivalis after treatment with CPO-GelMA hydrogel at different heme concentrations.

[0066] Figure 5C Representative CFU images of Porphyromonas gingivalis after treatment with CPO-GelMA hydrogels at different hemin concentrations.

[0067] Figure 5D is a bar graph showing the antibacterial effect of CPO-GelMA hydrogel on Porphyromonas gingivalis pretreated with 5% CPO-GelMA.

[0068] Figure 5E OD value bar graph of drug resistance evaluation of Porphyromonas gingivalis under repeated oxygen-producing hydrogel treatment (10 generations).

[0069] Figure 5F gingivalis under repeated oxygen-producing hydrogel treatments (20 generations).

[0070] Figure 5G gingivalis under repeated oxygen-producing hydrogel treatments (30 generations).

[0071] Figure 6A These are live / dead bacteria staining images of single bacterial species biofilms in the control group and treatment group under CLSM.

[0072] Figure 6B It is a bar graph of the thickness of biofilm of single bacterial species under different treatments (inhibition of formation).

[0073] Figure 6C It is a bar graph of the thickness of biofilm of single bacterial species under different treatments (eradication has been formed).

[0074] Figure 6D Representative SEM images of single-species biofilms under different treatments.

[0075] Figure 7A It is a bar graph showing the antibacterial effect of CPO-GelMA hydrogel on different types of planktonic bacteria.

[0076] Figure 7B This is a bar graph showing the inhibitory effect of CPO-GelMA hydrogel on biofilm formation of multiple bacterial species and the degradation and destruction of mature biofilms.

[0077] Figure 7C Representative SEM images of multi-species biofilms under different treatments.

[0078] Figure 8A are the SEM images of PCL and PCA nanofiber membranes.

[0079] Figure 8B It is the EDS spectrum image of PCA nanofibers.

[0080] Figure 8C are SEM images of PCA-GC composites, where the upper layer (a) represents the PCA nanofiber membrane and the lower layer (b) represents the GelMA hydrogel encapsulating CeO2 nanoparticles.

[0081] Figure 8D It is the EDS spectrum image of the cross section of PCA-GC composite material.

[0082] Figure 8E It is a bar graph of the residual H2O2 concentration of PCL-GC and PCA-GC in PBS.

[0083] Figure 8F It is a bar graph of the residual H2O2 concentration of PCL-GC and PCA-GC in α-MEM.

[0084] Figure 8G Figure 2 is the oxygen release curve of PCL-GC and PCA-GC within 24 hours.

[0085] Figure 8H It is the oxygen release curve of PCL-GC and PCA-GC within 7 days.

[0086] Figure 8I Schematic diagram of different effects of PCA-GC double-layer composite structure.

[0087] Figure 8J The effect of PCL-GC and PCA-GC on the cell activity of macrophages (RAW 264.7).

[0088] Figure 8K The effect of PCL-GC and PCA-GC on the cell activity of gingival fibroblasts.

[0089] Figure 8L The effect of PCL-GC and PCA-GC on the cell activity of periodontal ligament cells.

[0090] Figure 8M This is the peak graph of fluorescence intensity detected by flow cytometry after macrophages were infected with fluorescently labeled Porphyromonas gingivalis under different conditions.

[0091] Figure 8N It is the content of viable bacteria in macrophages infected with Porphyromonas gingivalis after different treatments.

[0092] Figure 8O It is the content of nitric oxide in the cell culture supernatant of macrophages infected with Porphyromonas gingivalis after different treatments.

[0093] Figure 9A It is a bar graph showing the inhibitory effects of different materials on Porphyromonas gingivalis.

[0094] Figure 9B It is a bar graph showing the CFU count results of Porphyromonas gingivalis using different materials.

[0095] Figure 9C Representative CFU images of Porphyromonas gingivalis after treatment with different materials.

[0096] Figure 9D It is a bar graph showing the inhibitory effects of different materials on Fusobacterium nucleatum.

[0097] Figure 9E It is a bar graph showing the CFU count results of Fusobacterium nucleatum using different materials.

[0098] Figure 9F Representative CFU images of Fusobacterium nucleatum after treatment with different materials.

[0099] Figure 9G It is a bar graph showing the inhibitory effects of different materials on Streptococcus gordonii.

[0100] Figure 9H It is a bar graph showing the CFU count results of Streptococcus gordonii using different materials.

[0101] Figure 9I Representative CFU images of Streptococcus gordonii after treatment with different materials.

[0102] Figure 10A Representative fluorescence images showing the hypoxic conditions of multi-species biofilms after treatment with different materials for 3 h.

[0103] Figure 10B yes Figure 10A The histograms showing the quantification of fluorescence intensity for different groups are shown in .

[0104] Figure 10C Representative fluorescence images showing the hypoxic conditions of multi-species biofilms after treatment with different materials for 6 h.

[0105] Figure 10D yes Figure 10C The histograms showing the quantification of fluorescence intensity for different groups are shown in .

[0106] Figure 11A It is a bar graph showing the number of Streptococcus gordonii remaining in multi-species biofilms after treatment with different materials for 24 hours.

[0107] Figure 11B This is a bar graph showing the number of Porphyromonas gingivalis remaining in multi-species biofilms after 24 hours of treatment with different materials.

[0108] Figure 11C It is a bar graph showing the residual number of Fusobacterium nucleatum in multi-species biofilms after treatment with different materials for 24 hours.

[0109] Figure 11D Pie chart showing the bacterial proportions of Streptococcus gordonii, Porphyromonas gingivalis, and Fusobacterium nucleatum remaining in a multispecies biofilm.

[0110] Figure 11E is a bar graph showing IL-6 expression in PDLCs treated with different biofilms.

[0111] Figure 11Fis a bar graph showing IL-8 expression in PDLCs treated with different biofilms.

[0112] Figure 11G is a bar graph showing the concentration of IL-6 secreted by PDLCs treated with different biofilms.

[0113] Figure 11H is a bar graph showing the concentration of IL-8 secreted by PDLCs treated with different biofilms.

[0114] Figure 12A Micro-CT images of the effects of different materials on experimental periodontitis in rats.

[0115] Figure 12B is a bar graph of the distance of alveolar bone loss assessed by Micro-CT.

[0116] Figure 13 is a flow chart showing the preparation of an oxygen-antibiotic sequential release composition for periodontal treatment according to an embodiment of the present invention

[0117] Figure 14 OD value bar graph showing the synergistic inhibitory effect of the oxygen-generating material (Example 1) and ciprofloxacin on Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans.

[0118] Figure 15 1 is a bar graph showing the synergistic bactericidal effects of the combination of the oxygen-generating material (Example 1) and ciprofloxacin at different concentrations on Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans.

[0119] Figure 16 Schematic diagram of the core-shell structure of the microspheres.

[0120] Figure 17A This is the oxygen release curve of microspheres containing different amounts of CPO within 24 hours.

[0121] Figure 17B This is a graph showing the oxygen release curves of microspheres containing different amounts of CPO within 7 days.

[0122] Figure 17C is a schematic diagram of different microsphere diameters.

[0123] Figure 17D This is the oxygen release curve of different microspheres within 24 hours.

[0124] Figure 17E This is the oxygen release curve of different microspheres within 7 days.

[0125] Figure 18A is the SEM image of the PCA-CIP microsphere surface.

[0126] Figure 18B is the SEM image of the cross section of PCA-CIP microspheres.

[0127] Figure 19A This is a standard calibration curve graph of ciprofloxacin concentration.

[0128] Figure 19B It is a table showing the loading capacity and encapsulation efficiency of ciprofloxacin in microspheres.

[0129] Figure 20A It is a bar graph showing the CFU count results of Porphyromonas gingivalis using different materials.

[0130] Figure 20B It is a bar graph showing the CFU count results of Fusobacterium nucleatum using different materials.

[0131] Figure 20C It is a bar graph showing the CFU count results of Aggregatibacter actinomycetemcomitans using different materials.

[0132] Figure 20D It is a bar graph showing the CFU count results of Streptococcus gordonii using different materials.

[0133] Figure 21 It is the release curve of oxygen produced by CeO2 or natural catalase decomposing H2O2 under different conditions.

[0134] Figure 22A These are Micro-CT images of the effects of different materials on experimental periodontitis in rats in Example 3.

[0135] Figure 22B is a bar graph of the alveolar bone resorption distance in Example 3 evaluated by Micro-CT.

[0136] Figure 22C 3 is a bar graph of the probing depths of periodontal pockets in rats of different treatment groups in Example 3.

[0137] Figure 23A It is a bar graph of the body weights of rats in different treatment groups in Example 3.

[0138] Figure 23B It is a bar graph of the number of white blood cells in the blood of rats in different treatment groups in Example 3.

[0139] Figure 23C It is a bar graph of the number of red blood cells in the blood of rats in different treatment groups in Example 3.

[0140] Figure 23D 3 is the histopathological HE staining of the visceral sections of rats in different treatment groups in Example 3. DETAILED DESCRIPTION

[0141] The local application of oxygen-generating materials is a promising strategy for the adjuvant treatment of periodontitis. In addition, compositions with the dual release capabilities of oxygen and antibiotics may have better therapeutic effects in some cases of severe periodontal inflammation because the oxygen-rich environment formed by oxygen release can promote the bactericidal and anti-biofilm efficiency of the antibiotics and reduce potential antibiotic resistance.

[0142] refer to Figure 1 , shows an embodiment of providing periodontal treatment by applying a CPO-based oxygen-generating hydrogel 102 in a periodontal pocket 100. The hydrogel composition comprises: a polymer 102 encapsulating a peroxide compound 104, wherein the peroxide compound 104 is configured to generate hydrogen peroxide upon hydrolysis; and catalase 106 or nanoparticles having catalase-like activity encapsulated in the polymer 102, wherein the catalase 106 or the nanoparticles are configured to self-decompose the hydrogen peroxide to generate oxygen and water.

[0143] This embodiment can be used to treat periodontitis. For example, the treatment may involve injecting an oxygen-producing hydrogel into the periodontal pocket 100, so that bacteria, particularly anaerobic bacteria, can be reduced in the oxygen-rich microenvironment, thereby effectively improving the dysbiosis and preventing the re-formation of biofilm that is highly pathogenic to periodontitis.

[0144] In this example, a dentist can apply the polymer 102 encapsulating a selected peroxide compound (e.g., calcium peroxide or other metal peroxides) to a periodontal pocket 100 in periodontal treatment. The polymer 102 can be cured so that the polymer 102 remains intact and in the periodontal pocket 100 in need of periodontal treatment.

[0145] Preferably, after periodontal treatment, the oxygen generated when the peroxide compound is hydrolyzed and the hydrogen peroxide is decomposed is configured to help maintain an oxygen-rich microenvironment within the periodontal pocket 100 where the polymer 102 is placed.

[0146] For example, also refer to Figure 2 Calcium peroxide can be hydrolyzed, that is, it can react with water. Once the hydrogen peroxide produced by the reaction of CaO2 with water is further decomposed into water and oxygen, oxygen is released to the surrounding of the polymer 102.

[0147] In this example, a polymer carrier such as chitosan can be used to encapsulate a plurality of microspheres having a core-shell structure, wherein each microsphere has an outer shell of a peroxide compound surrounding an inner core of an antibiotic.

[0148] After alleviating the hypoxic microenvironment, the antibiotic in the core will be released when the peroxide shell is hydrolyzed. Since the oxygen-rich microenvironment increases the sensitivity of biofilms and drug-resistant pathogens, the effect of the antibiotic on biofilms or drug-resistant pathogens will be enhanced.

[0149] The hypoxic environment of the periodontal pocket 100 is conducive to the growth of anaerobic pathogens, biofilm formation, and recurrence of inflammation after periodontal treatment. In contrast, oxygen is harmful to anaerobic pathogens such as Porphyromonas gingivalis (P. gingivalis) because they lack a complete antioxidant mechanism to relieve the toxicity of oxygen challenge. Therefore, continuously providing sufficient oxygen to anaerobic pathogens is an effective countermeasure.

[0150] In one exemplary embodiment, an injectable oxygen-generating hydrogel can be used as a local oxygen source to alleviate the hypoxic microenvironment in the periodontal pocket and eliminate periodontal pathogens.

[0151] Calcium peroxide (CaO2)-loaded gelatin methacryloylation (GelMA) hydrogels have excellent injectability and exhibit a burst release of oxygen within 24 hours, with a peak oxygen partial pressure of 40%. This composition, also referred to as "CPO-GelMA hydrogel" in this disclosure, reduced the survival rate of Porphyromonas gingivalis by 60%, 99%, and 89.9% at CPO concentrations of 5%, 10%, and 15% (w / v%), respectively.

[0152] Furthermore, the hydrogel may also promote a slight increase in pH, which is beneficial for periodontal treatment, as will be further explained in a subsequent section of this disclosure.

[0153] Example 1

[0154] First, to demonstrate the effectiveness of oxygen-generating materials for the treatment of periodontitis, the inventors conducted a series of in vitro studies on CPO-GelMA hydrogels. The results showed that CPO-GelMA hydrogels not only had significant antibacterial effects against periodontal pathogens but also downregulated the expression levels of the virulence factors gingipain and fimA in Porphyromonas gingivalis without inducing significant tolerance in the pathogens. Furthermore, the CPO-GelMA hydrogels significantly inhibited biofilm formation and effectively eliminated both single-species and multi-species bacterial biofilms. These results suggest that oxygen-generating materials are a promising new strategy for the treatment of periodontitis.

[0155] Hydrogels with good injectability can be formulated as drug delivery vehicles, particularly for hard-to-reach areas in periodontal pockets. For example, gelatin methacryloyl (GelMA) hydrogels are suitable drug delivery vehicles due to their excellent biocompatibility and tunability. They can maintain the release of proteins and drugs throughout their degradation process.

[0156] Advantageously, CPO-loaded GelMA hydrogels can be injected into periodontal pockets and readily cross-linked in situ using a dental curing light. This hydrogel system can maintain dissolved oxygen concentrations above 20% for 24 hours. CPO loaded into GelMA hydrogels may be a viable carrier for topical delivery into periodontal pockets. Therefore, the inventors prepared oxygen-producing CPO-GelMA hydrogels and evaluated their antibacterial and antibiofilm efficacy against periodontal pathogens.

[0157] GelMA can be synthesized as follows. Briefly, porcine skin type A gelatin (10%) (Sigma-Aldrich, St. Louis, MO, USA) can be completely dissolved in 100 mL of Dulbecco's phosphate buffered saline (DPBS, Sigma-Aldrich) and kept at 55°C for 1 hour. Then, 8 mL of methacrylic anhydride (MA) (Sigma-Aldrich) is added dropwise and stirred at 50°C for 2 hours. The reaction of gelatin and MA is stopped by adding the same volume of DPBS. GelMA is dialyzed against deionized water at 50°C for 1 week using dialysis tubing with a molecular weight cutoff of 12-14 kDa (Merck, Darmstadt, Germany). Finally, the solution is freeze-dried at -80°C for 1 week using a freeze dryer (Labconco, Kansas, USA).

[0158] Freeze-dried GelMA foam (10% w / v%) was completely dissolved in DPBS containing lithium acylphosphinate (LAP, 0.075 w / v%, Sigma-Aldrich) at 50°C. Then, 1 mg / mL bovine liver catalase (Sigma-Aldrich) was added to the GelMA solution. Commercial CPO (75% purity, 200 mesh; Sigma-Aldrich) was dispersed into the sterilized GelMA solution at different ratios (1, 5, 10, and 15, CPO:GelMA wt%) under ultrasonic vibration. The homogeneous mixture was cross-linked by a dental curing light (Ultradent, South Jordan, Utah, USA) to obtain 1%, 5%, 10%, and 15% CPO-GelMA hydrogels.

[0159] Preferably, CeO2 can be selected to have catalase-like activity, antioxidant effect, anti-inflammatory effect and oxygen storage capacity. Alternatively, other nanoenzymes 106 with catalase-like activity can also be used, including Au, Pt, MnO2, Mn3O4 and Fe3O4. Advantageously, nanoparticles with catalase-like activity can be used instead of natural catalase because of their better stability. For example, CeO2 with catalase-like activity can be provided in the form of nanoparticles encapsulated in a polymer carrier so that the nanoparticles can decompose the hydrogen peroxide like catalase to produce oxygen and water, thereby inhibiting the accumulation of hydrogen peroxide produced when the peroxide compound is hydrolyzed.

[0160] The prepared CPO-GelMA hydrogels were freeze-dried overnight, and their cross-sectional microstructure and morphology were characterized by scanning electron microscopy (SEM; Hitachi, Ibaraki, Japan) after gold coating using a magnetron sputtering coater (MSP-2S System, Hitachi). O₂ tension was measured using an optical oxygen microsensor (PreSens, Regensburg, Germany) under hypoxic conditions (1% O₂).

[0161] The inventors immersed GelMA hydrogels without or with CPO (1, 5, 10, and 15 wt%) in 1 mL of culture medium and placed them in an anoxic incubator (Baker, Sanford, Maine, USA). O2 levels were measured every day for 7 days. In addition, Amplex TM The H2O2 concentration in DPBS in which CPO-GelMA (with or without catalase hydrogels) was immersed for 24 hours was measured using a Red hydrogen peroxide / peroxidase detection kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). After the GelMA hydrogels were immersed in DPBS for 24 and 48 hours, the pH value was measured using a pH meter (ISE710A, Orion Research Inc., Boston, MA, USA).

[0162] In an exemplary experiment, the injectability of the CPO-GelMA hydrogel was visually observed by loading the CPO-GelMA solution into a 1 mL syringe and manually injecting. A 1 mL syringe with a 21-gauge needle (Therumo, Shibuya-ku, Tokyo, Japan) was filled with 15% CPO-GelMA hydrogel and manually injected to form the desired shape. An inverted tube test was performed to evaluate the transformation of the GelMA hydrogel after irradiation with a dental curing light (wavelength: 385-515 nm).

[0163] Streptococcus gordonii (ATCC 10558), Actinomyces naeslundii (ATCC 12104), Fusobacterium nucleatum (ATCC 10953), Porphyromonas gingivalis (ATCC 33277), and Aggregatibacter actinomycetemcomitans (ATCC 33384) were obtained from the Central Research Laboratory, Faculty of Dentistry, The University of Hong Kong. Porphyromonas gingivalis and Fusobacterium nucleatum were cultured in tryptic soy broth (TSB, Sigma-Aldrich) supplemented with yeast extract (5 mg / mL, Sigma-Aldrich), hemin (5 mg / mL, Sigma-Aldrich), and menadione (1 mg / mL, Sigma-Aldrich). Streptococcus gordonii, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum were cultured in brain heart infusion (BHI) broth (Sigma-Aldrich). Cultivation was performed in an anaerobic chamber (N2 80%, H2 10%, CO2 10%, Baker) at 37°C.

[0164] A certain volume of Porphyromonas gingivalis (10 7 Colony forming units (CFU / mL) were pipetted into microcentrifuge tubes containing GelMA and CPO-GelMA hydrogels (1%, 5%, 10% and 15%) and incubated under anaerobic conditions for 24 hours. The turbidity of the bacterial suspension was then determined by measuring the optical density (OD) value at 660 nm to evaluate the growth of bacteria under different conditions. At the same time, the Porphyromonas gingivalis suspension was gradiently diluted and spread on Columbia blood agar plates. After 7 days, the colonies on the blood agar plates were calculated as a percentage (CFU of different groups / CFU% of blank group) to evaluate bacterial viability.

[0165] The inventors added heme (0.5, 2.5, 5, 25 μg / mL) to the culture medium and studied the bactericidal effect of CPO-GelMA hydrogel on planktonic Porphyromonas gingivalis.

[0166] The inventors evaluated the resistance of P. gingivalis to oxygen-producing hydrogels under high-dose and long-term treatment. In the experiment, the inventors selected a 5% CPO-GelMA hydrogel, which exhibits significant bactericidal activity, for high-dose treatment. Surviving bacteria 24 hours after treatment were subcultured for additional antibacterial testing. Long-term resistance was also assessed based on previous research.

[0167] First, the inventors treated a suspension of P. gingivalis with 1% CPO-GelMA hydrogel for 24 hours, which was considered the first generation. The bacteria were then serially subcultured in fresh culture medium containing the 1% CPO-GelMA hydrogel for 30 generations. Antibacterial efficacy was measured at the 10th, 20th, and 30th generations.

[0168] The inventors believe that multiple virulence factors are associated with the survival and pathogenicity of P. gingivalis. Virulence factors, including gingipain and fimA, were measured by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). In an exemplary experiment, 5×10 8 gingivalis at 500 CFU / mL for 3, 6, and 24 hours. The specimens were then collected by centrifugation, and mRNA was extracted using RNAfast 200 (Fastagen, Shanghai, China). Reverse transcription was then performed using SuperScript VILO Master Mix (Life Technologies, Grand Island, NY, USA). RT-qPCR analysis was performed using the StepOne Real-Time PCR System (Applied Biosystems, Grand Island, NY, USA) and SYBR Premix Ex TaqII (Cat No. RR820A, Takara Bio, Shiga, Japan). Primer sequences for 16S rRNA, rgpA, rgpB, kgp, and fimA are listed in Table 1.

[0169] Table 1 RT-qPCR primer sequences

[0170]

[0171] In the experiment, first, Porphyromonas gingivalis (1×10 8Biofilm formation was performed in confocal microplates (SPL LifeScience, Gyeonggi-do, Korea) using different hydrogels (0, 5, 10, and 15%) of 1% CPO-GelMA for 48 hours. The biofilms were then stained using the BacLight Live / Dead viability kit (Thermo Fisher Scientific, Waltham, MA). Live bacteria were stained with SYTO9 (green), and dead bacteria were stained with propidium iodide (PI). Bacterial viability and biofilm thickness were assessed using confocal laser scanning microscopy (CLSM), and biofilm morphology was examined using scanning electron microscopy (SEM).

[0172] At the same time, in the biofilm eradication experiment, Porphyromonas gingivalis (1×10 8 CFU / mL) were cultured on confocal plates for 48 h to form single-species biofilms, and then treated with different hydrogels (0, 5, 10, and 15% CPO-GelMA) for another 48 h. Similarly, the morphology and thickness of the biofilms were evaluated under CLSM and SEM.

[0173] Periodontitis is a polymicrobial infectious disease that requires therapeutic strategies to overcome multi-species infection. Therefore, the inventors evaluated the effect of CPO-GelMA hydrogel on different periodontal pathogens, including Streptococcus gordonii, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. 5 CFU / mL of Streptococcus gordonii or Actinomyces naeslundii and a concentration of 10 7 CFU / mL of Aggregatibacter actinomycetemcomitans or Fusobacterium nucleatum.

[0174] The inventors established a multi-species biofilm. In the experiment, the concentration of 10 5 CFU / mL of Streptococcus gordonii and Actinomyces naeslundii and the concentration of 10 8 To form a multispecies biofilm, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum were added to the confocal microtubules at 500 CFU / mL. These different bacterial suspensions were mixed at a ratio of 1:1:1:1:1, and 2 mL of the mixture was added to the confocal microtubules.

[0175] Statistical analyses were performed using GraphPad Prism software (version 9.0, GraphPad Inc., San Diego, CA, United States). Data are presented as mean ± SD. One-way analysis of variance was performed to determine the significance of differences. P < 0.05 was considered statistically significant.

[0176] GelMA hydrogels exhibited excellent fluidity and cross-linking ability. Figure 3A It is shown that the CPO-GelMA liquid 302 can be formed into various desired shapes by a syringe and has flexibility. In order to study whether the addition of CPO affects the photopolymerization of GelMA hydrogel, Figure 3B Figure 2 shows an inversion test of 15% CPO-GelMA after cross-linking using a dental curing light (wavelength: 385-515 nm). Figure 3C The pore size of the porous microstructures shown increases corresponding to the encapsulated CPO concentration.

[0177] refer to Figure 3D As well as 3E encapsulated with CPO-GelMA hydrogels of different concentrations, it showed a burst release of O2 within 30 minutes, followed by sustained oxygen release for up to 48 hours. After loading with 10% or 15% CPO, the GelMA hydrogels showed a similar O2 release curve, initially reaching a maximum release of approximately 40% and maintaining the oxygen pressure within 2 hours, and then gradually decreasing to approximately 30% within 12 hours. Except for the 15% CPO-GelMA hydrogel, the oxygen partial pressure of all CPO-GelMA hydrogels dropped below 20% after 24 hours. No H2O2 residue was detected in the group exposed to catalase. At the same time, as Figure 3F As shown, the group without catalase showed an increase in H2O2 concentration with increasing CPO.

[0178] The CCK8 results also demonstrated that 1%, 5%, and 10% CPO-GelMA hydrogels had no cytotoxicity to HGFs at 24 and 48 hours. In contrast, 15% CPO-GelMA hydrogels inhibited the proliferation of HGFs at 24 hours, and the inhibitory effect was reversed at 48 hours. The pH value increased slightly with CPO concentration, reaching a maximum of pH 9 in 10% and 15% CPO-GelMA hydrogels. Figure 3G shown.

[0179] like Figure 4A As shown in Figure 3, 5%, 10%, and 15% CPO-GelMA hydrogels significantly inhibited the growth of P. gingivalis for up to 48 hours. Figure 4B As shown in Figure 2, the colonies on the plates of the 5%, 10% or 15% CPO-GelMA hydrogel-treated groups were drastically reduced compared to the other groups. Figure 4C As shown in Figure 2, 1% CPO-GelMA hydrogel reduced the number of viable bacteria by 60%. In contrast, 5%, 10%, and 15% CPO-GelMA hydrogels reduced the number of viable bacteria by 96%, 99%, and 98.9%, respectively. In addition, the morphology of Porphyromonas gingivalis on the surface of 5% CPO-GelMA hydrogel was observed under SEM. Figure 4DAs shown in Figure 4E, some typical short rod-shaped bacterial cells with intact structures still remained after 3 hours of treatment; however, only shrunken and broken cells were observed after 6 hours of treatment.

[0180] The inventors evaluated the relative expression levels of rgpA, rgpB, kgp and fimA. Figure 4F As shown, all virulence factors decreased after treatment with 5% CPO-GelMA hydrogel. Gene expression of rgpB was significantly reduced 3 and 6 hours after treatment, while fimA gene expression was downregulated 6 and 24 hours after treatment. Expression of kgp was significantly reduced throughout the 24 hours. However, rgpA gene expression did not decrease significantly.

[0181] like Figure 5A As shown in Figure 2, CPO-GelMA hydrogels significantly inhibited the growth of P. gingivalis regardless of the heme concentration. Figure 5B gingivalis, except for 5-fold hemin (25 μg / mL), which is toxic to P. gingivalis, the colonies on the plates treated with 5% CPO-GelMA hydrogels were reduced compared with the control group at the same hemin concentration. Figure 5C As shown in the figure, the survival rate of P. gingivalis increased with the increase of hemin concentration. Although high concentration of hemin (5 times, 25 μg / mL) inhibited the growth of P. gingivalis without CPO-GelMA hydrogel treatment, it increased the viability of P. gingivalis to 30% when treated with CPO-GelMA hydrogel.

[0182] Then, refer to Figure 5D , the surviving bacteria treated with 5% CPO-GelMA hydrogel were subcultured to evaluate their tolerance to CPO-GelMA hydrogel. Compared with the control group, the OD value of the 1% CPO-GelMA hydrogel group increased, while the OD values ​​of the 5%, 10% and 15% CPO-GelMA hydrogel groups decreased significantly. At the same time, Porphyromonas gingivalis in suspension was continuously treated with 1% CPO-GelMA hydrogel for 30 generations to induce drug resistance. Figures 5E to 5G As shown in the results, after treatment with CPO-GelMA hydrogel, the OD value of the P. gingivalis suspension was significantly reduced even after 10 to 30 passages. These findings suggest that P. gingivalis has limited tolerance to high oxygen levels (5% CPO-GelMA treatment) or prolonged exposure to low oxygen levels (1% CPO-GelMA treatment).

[0183] like Figure 6AAs shown in the figure, in both the control and GelMA-treated groups, the P. gingivalis biofilm was stained green fluorescent and had a highly dense structure. After treatment with 10% and 15% CPO-GelMA hydrogels, the thickness of the bacterial layer was significantly reduced. The distribution of the biofilm became more dispersed, and the number of dead bacteria with red fluorescence increased. Figure 6B As shown in Figure 6C , in the 10% and 15% CPO-GelMA hydrogel-treated groups, the average biofilm thickness was less than 4 μm, whether inhibiting biofilm formation or eradicating the formed biofilm, which was reduced by about 50% compared with the control group. Figure 6D The SEM images in the figure also showed that P. gingivalis formed a dense biofilm structure, while in the CPO-GelMA-treated group, the bacteria were scattered compared to the control group. Similar results were found in CPO-GelMA hydrogels that eradicated established biofilms.

[0184] like Figure 7A As shown, CPO hydrogels at 10% and 15% concentrations significantly inhibited the growth of planktonic Fusobacterium nucleatum. In contrast, when CPO-GelMA hydrogels were applied to planktonic Streptococcus gordonii and Aggregatibacter actinomycetemcomitans, no significant differences were observed compared to the control group. Furthermore, CPO encapsulation had no effective antibacterial activity against Actinomyces naeslundii.

[0185] To simulate the complex structure of dental plaque with inter- and intra-species coaggregation and co-adhesion interactions, a multi-species biofilm was established, including Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Figure 7B CLSM images showed the formation of a multispecies biofilm with a dense structure, with an average biofilm thickness of 9 μm during biofilm formation and 12 μm during biofilm eradication. Treatment with 5%, 10%, and 15% CPO-GelMA hydrogels significantly reduced the thickness and biomass of live bacteria and multispecies biofilms compared to the control group.

[0186] like Figure 7C As shown, SEM images show that the biofilm structure contains rod-shaped, short rod-shaped, and spherical bacteria. In the biofilm formation or eradication experiments, after treatment with CPO-GelMA, fewer bacteria attached to the surface than the control group and pure GelMA group.

[0187] The key pathogens causing periodontal infections are primarily anaerobic bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum. Complete elimination of these infectious microorganisms and prevention of their recolonization and reinfection are crucial to ensure long-term successful treatment. However, the efficacy of current antimicrobial treatment modalities is often short-lived and requires repeated application, which can lead to adverse reactions such as antibiotic resistance and loss of taste (chlorhexidine). Therefore, one of the main goals of many studies is to develop innovative antimicrobial strategies against infectious microorganisms.

[0188] Due to their obligate anaerobic nature, periodontal pathogens are sensitive to local oxygen levels. An oxygen-rich environment can inhibit or even destroy these bacteria. If the inventors could alter the local periodontal environment from a hypoxic to an oxygen-rich environment, for example by increasing O₂ levels to as high as 20%, the basis for the survival and pathogenicity of pathogenic anaerobic bacteria would be destroyed. In addition to combating anaerobic infection, oxygen improves vascularization and reduces edema, thereby enhancing wound healing and regenerative responses within the periodontal pocket wound environment. Hyperbaric oxygen therapy has been reported to be therapeutically effective in patients with aggressive periodontitis, significantly reducing gingival index, probing depth, and attachment loss by inhibiting anaerobic bacterial growth and enhancing connective tissue healing. However, high O₂ levels, such as those achieved with 3% H₂O₂ for periodontal pocket irrigation, are short-lived; once O₂ levels drop, anaerobic recurrence and reinfection may occur, compromising therapeutic efficacy.

[0189] On the other hand, the irregularity of the periodontal pocket makes it difficult to deliver drugs to the affected area. Therefore, there is an urgent need to develop materials with long-lasting O2 production capacity to achieve O2 enrichment, suitable injectability for delivery to deep and narrow spaces, and good fluidity to adapt to irregular periodontal pockets.

[0190] Advantageously, the present invention provides an injectable hydrogel containing CPO to change the oxygen level in periodontitis pockets. CPO, MPO and H2O2 have been shown to be oxygen sources that can be used in tissue engineering, all of which can produce O2 within a few days. CPO is preferred because it is a safe biomaterial that produces oxygen and has high purity. Advantageously, GelMA hydrogel can be used as a carrier for growth factors and oxygen-producing materials, can be injected into hard-to-reach areas, and can fill irregular periodontal pockets, and can be rapidly cross-linked using dental curing lights, with the potential for clinical application. Therefore, it is also preferred to select GelMA hydrogel as a carrier for encapsulating CPO.

[0191] Scanning electron microscopy (SEM) results confirmed that increasing the CPO concentration in the GelMA hydrogel increased porosity. Furthermore, the results confirmed that a higher CPO concentration in the GelMA hydrogel generated more oxygen. The oxygen partial pressure remained above 20% for 24 hours, which is sufficient to kill any remaining bacteria.

[0192] To address the potential presence of residual reactive oxygen species (ROS), since hydrogen peroxide is an intermediate product in the decomposition of calcium peroxide, catalase can be added to the hydrogel to ensure efficient and rapid decomposition of H2O2. High concentrations of H2O2 have bactericidal effects and can be harmful to periodontal cells. Therefore, by comparing H2O2 levels in solutions with and without catalase, the inventors confirmed that in the presence of catalase, H2O2 can be completely hydrolyzed to produce O2. In the presence of catalase, hydrogen peroxide is virtually undetectable in the solution.

[0193] The inventors also measured changes in pH, which showed a slight increase. The inventors believe that the saliva pH of patients with severe periodontitis is lower, suggesting that an increase in pH would create a favorable environment for wound healing. Advantageously, the CPO-GelMA hydrogel can alleviate the acidic microenvironment caused by hypoxia and acute inflammation in severe periodontitis by producing oxygen and increasing pH.

[0194] Subsequently, the inventors confirmed the inhibitory effect of CPO-GelMA hydrogel on Porphyromonas gingivalis. High concentrations of CPO, including 5%, 10%, and 15% loaded GelMA hydrogels, showed excellent antibacterial effects against Porphyromonas gingivalis after 24 and 48 hours of treatment. Notably, 10% and 15% CPO-GelMA hydrogel treatment significantly reduced bacterial survival to below 1.1%. This is because increasing the concentration of CPO in the GelMA hydrogel enables it to release higher amounts of oxygen over a longer period of time to kill Porphyromonas gingivalis, as it can only tolerate low levels of oxygen.

[0195] Furthermore, the inventors studied the tolerance of P. gingivalis to oxygen at different heme concentrations, and found that increasing heme concentration enhanced P. gingivalis's tolerance to oxygen. However, CPO-GelMA hydrogel maintained its bactericidal efficacy at 0.5-5 μg / mL heme. Unlike other antibiotics, oxygen as a therapeutic agent rarely causes bacterial resistance. Even after 30 generations of long-term treatment or high-concentration oxygen treatment, P. gingivalis did not rapidly develop tolerance to oxygen.

[0196] To fully understand how oxygen affects the pathogenicity of P. gingivalis, the inventors further measured key virulence factors, including rgpA, rgpB, kgp, and fimA, using RT-qPCR. Oxygen generated by the CPO-GelMA hydrogel significantly inhibited the expression of rgpB, kgp, and fimA, which are responsible for promoting co-aggregation with other bacteria, oral colonization, and dental plaque formation. This suggests that oxygen can attenuate the gene expression of key virulence factors, bacterial colonization, and dental plaque formation, thereby downregulating the pathogenicity of P. gingivalis. The inventors further evaluated the effects of CPO-GelMA hydrogel on P. gingivalis biofilm formation and eradication. CPO-GelMA hydrogels loaded with more than 10% CPO showed significant efficacy in inhibiting new biofilm formation and disrupting mature biofilms.

[0197] Subgingival biofilms may contain a variety of bacteria. In addition to P. gingivalis, Streptococcus gordonii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Actinomyces naeslundii are also involved in biofilm formation and gingival inflammation. The inventors also confirmed the effects of CPO-GelMA hydrogel on other bacterial cells. Although 5% CPO-GelMA hydrogel killed most P. gingivalis, it was ineffective against F. nucleatum. On the other hand, 10% and 15% CPO-GelMA hydrogels showed antibacterial effects against F. nucleatum, indicating that F. nucleatum is more tolerant of oxygen than P. gingivalis.

[0198] Furthermore, oxygen enrichment had no effective antibacterial activity against Streptococcus gordonii, Actinomyces naeslundii, and Aggregatibacter actinomycetemcomitans. CPO-GelMA hydrogel even promoted the growth of Aggregatibacter actinomycetemcomitans, which may be because Streptococcus gordonii, Actinomyces naeslundii, and Aggregatibacter actinomycetemcomitans are facultative anaerobes or microaerophiles.

[0199] Example 2

[0200] In a preferred embodiment, the polymer support comprises a layered composite structure comprising a first polymer layer and a second polymer layer. Preferably, the first polymer layer encapsulates the oxygen generator, and the second polymer layer encapsulates the nanoparticles. The layered composite structure is preferably a Janus composite, wherein the first polymer layer and the second polymer layer exhibit different surface properties and / or functionalities.

[0201] The oxygen-generating material, composed of CPO and cerium oxide (CeO2), was shown to have sustainable oxygen-release properties and selective antibacterial effects against periodontal pathogens, further inducing a shift in the composition of the subgingival flora. Although the composition of this example does not contain antibiotics, the results indicate that the composition also exhibits significant antibacterial effects against anaerobic pathogens.

[0202] Preferably, the first polymer layer comprises an electrospun fiber membrane, such as polycaprolactone (PCL), while the second polymer layer comprises a hydrogel, such as gelatin methacrylate (GelMA). The inventors further verified the efficacy of different embodiments through experiments. Pristine polycaprolactone (PCL) and PCL-encapsulated calcium peroxide (CPO) membranes were prepared by electrospinning and divided into PCL and PCA groups. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were used to characterize the structure and composition of the membranes.

[0203] The preparation method of Example 2 is as follows:

[0204] -First, the synthesized CPO nanoparticles (0, 20 mg / mL) were added to a DCM and DMF solution (DCM:DMF=5:5),

[0205] Ultrasonication was performed for 10 minutes to uniformly disperse the particles, and then 0.1 g / mL PCL particles were added to the above solution. After stirring for 5-6 hours to fully dissolve and obtain a homogeneous solution, the solution was loaded into a 5 mL syringe with an 18G blunt needle. The solution was output at a flow rate of 1.5 mL / h using a syringe pump, and a voltage of 13-15 kV was applied to the needle using a high-voltage generator to spray out nanofibers. The nanofibers were collected on a collector with a rotation speed of 300 rpm. Pure PCL was prepared according to the same procedure without adding CPO nanoparticles.

[0206] Nanofiber membrane.

[0207] -GelMA was synthesized as described in Example 1, and then 20 mg / mL cerium oxide (CeO2) nanoparticles (used as nanozymes) were encapsulated into

[0208] Methacrylated gelatin (GelMA) hydrogel was coated onto the surface of the membrane (PCL-GC / PCA-GC) to decompose the generated hydrogen peroxide (H2O2) into oxygen and reduce cell damage.

[0209] like Figure 8A As shown in Figure 8B, both fiber membranes showed uniform fibers with a diameter of approximately 1 μm, and EDS results showed that CPO was uniformly distributed in the PCA membrane. The cross-section of the prepared Janus composite (PCA-GC) was further studied under SEM; Figure 8C As shown, the two compositions are tightly attached on both sides of the contact interface, and the structures on both sides of the interface are completely different.

[0210] exist Figure 8D The distribution of Ca and Ce elements in the EDS analysis shown in Figure 3 revealed the presence of CPO and CeO2 nanoparticles, with CPO mainly incorporated into the fiber membrane and CeO2 mainly encapsulated in the GelMA hydrogel. Figure 8EAs shown in Figure 8F, when different composite materials were immersed in a liquid environment, the residual concentration of H2O2 in PBS or culture medium was significantly reduced and lower than 100 μM in the presence of CeO2.

[0211] like Figure 8G Figure 8H shows the oxygen release curve under hypoxic conditions measured using an oxygen sensor over 7 days. The oxygen release curves for PCL-GC / PCA-GC demonstrate that PCA-GC produces significant amounts of oxygen within 24 hours and continuously releases oxygen for up to 5 days, making it suitable for restoring hypoxic conditions in periodontal pockets to a hyperoxic microenvironment. This sustained release allows for weekly follow-up of periodontal treatment, making it suitable for clinical application.

[0212] The purpose of designing two layers of different polymers is as follows Figure 8I As shown, when used, first insert the PCL fiber membrane containing CPO into the periodontal pocket close to the tooth surface. When CPO comes into contact with water, a hydrolysis reaction occurs to produce hydrogen peroxide and oxygen, which quickly act on the plaque biofilm on the tooth surface and in the periodontal pocket to exert antibacterial and anti-biofilm effects; and the injectable hydrogel containing CeO2 nanoparticles with nanozyme-like activity can be injected into the periodontal pocket through a drug delivery device and fill the gap between the fiber membrane and the gingival tissue to isolate the direct contact between the CPO-containing fiber membrane and the gingival soft tissue. The nanoparticles encapsulated in the hydrogel can fully decompose the hydrogen peroxide produced by CPO into oxygen through its catalase-like activity, reducing the oxidative damage of hydrogen peroxide to the gingival tissue. At the same time, the oxygen-rich environment can promote the phagocytosis and bactericidal effects of immune cells on pathogens. Figures 8J to 8L As shown in the cell activity experiments, the cell activity of macrophages (RAW 264.7), gingival fibroblasts, and periodontal ligament cells in the different material treatment groups was above 80% within 3 days, indicating good cell compatibility. In addition, macrophages were infected with fluorescently labeled Porphyromonas gingivalis at a multiplicity of infection of 100 under different conditions, and the cell fluorescence intensity was then detected by flow cytometry, as shown in the figure below. Figure 8M , the peak fluorescence intensity of the hypoxic group shifted to the left relative to the normoxic group, indicating that the phagocytic ability of macrophages to Porphyromonas gingivalis was reduced under hypoxic conditions. When PCA-GC was applied under hypoxic conditions, the peak fluorescence intensity shifted to the right relative to that under simple hypoxic conditions, indicating that the application of PCA-GC could reverse the inhibition of hypoxia on the ability of macrophages to phagocytose bacteria. After Porphyromonas gingivalis infected macrophages, the infected macrophages were given different treatments, and the number of live bacteria in the cells was detected by swelling the cells and smearing the plates for colony counting, as shown in the following figure: Figure 8N As shown in the results, the number of intracellular viable bacteria in the PCA-GC group under hypoxic conditions was significantly higher than that in the simple hypoxia group. The results showed that the application of PCA-GC can promote the bactericidal effect of macrophages under hypoxic conditions. Then the nitric oxide content, which is closely related to the cell bactericidal effect, was detected. Figure 8OAs shown, the application of PCA-GC under hypoxic conditions significantly increased nitric oxide levels in the supernatant of infected macrophage cultures. This is likely because nitric oxide production requires the presence of oxygen, and the oxygen-rich environment created by PCA-GC exerts a bactericidal effect by increasing nitric oxide production. These results demonstrate that PCA-GC, with its bilayer structure, not only possesses excellent biocompatibility but also significantly promotes the bactericidal effects of immune cells under hypoxic conditions, preventing further imbalance in local immune function and worsening periodontal inflammation.

[0213] Periodontitis is an infectious disease whose pathogenesis is associated with dysbiosis of the subgingival microbiota, with a high relative abundance of Gram-negative anaerobic bacteria. Therefore, two periodontitis-associated pathogens, Porphyromonas gingivalis and Fusobacterium nucleatum, were selected to validate the antimicrobial activity of PCA-GC. Porphyromonas gingivalis (W83, 5 x 10^7 CFU / mL) and Fusobacterium nucleatum (CCUG 9126, 5 x 10^7 CFU / mL) were used for antimicrobial activity evaluation. The antimicrobial activity of PCA-GC and PCL-GC was evaluated by growth inhibition assay in liquid culture medium and colony forming unit (CFU) counting method.

[0214] PCA-GC is effective in the control of planktonic periodontal pathogens including Porphyromonas gingivalis ( Figures 9A-9C ) and Fusobacterium nucleatum ( Figures 9D-9F ) showed excellent bactericidal efficacy. Furthermore, the inventors selected Streptococcus gordonii (ATCC35105, 1 x 10^7 CFU / mL), a commensal bacterium that has been shown to be more abundant in healthy dental plaque than in the plaque of periodontitis patients, to verify the selective antibacterial effect of PCA-GC.

[0215] like Figure 9G As shown in Figure 2, both PCL-GC and PCA-GC slightly increased the proliferation of S. gordonii. Figure 9I The CFU count results also show that neither PCL-GC nor PCA-GC affects the viability of S. gordonii. The selective antibacterial effect of the present invention makes it superior to current treatment strategies of topical or systemic antibiotics, as the broad-spectrum antibacterial properties of antibiotics may exacerbate oral dysbiosis.

[0216] To test the hypothesis that oxygen-generating materials can increase the oxygen content in periodontal biofilms, the hypoxia status of multi-species biofilms was assessed by hypoxia probe labeling after 3 and 6 hours of PCL / PCA-GC treatment. Figure 10AAs shown in Figure 10B, after 3 hours of treatment, the biofilms in the PCA-GC group had lower fluorescence intensity (higher oxygen tension) than the control group. The biofilms in the PCL-GC group also showed slightly less hypoxia, even though there was no statistical difference, which may be attributed to the oxygen storage capacity of CeO2. After 6 hours, the biofilms in the PCA-GC group still did not show obvious hypoxia, while the biofilms in the PCL-GC group showed fluorescence intensity similar to that of the control group, indicating that the oxygen release capacity of CeO2 alone is weak ( Figure 10C To investigate the changes in the microbial composition of subgingival biofilms in the presence of oxygen, the established three-species biofilms were treated with PCL-GC or PCA-GC for 24 h, and the abundance of various bacteria was determined by qRT-PCR.

[0217] like Figure 11A As shown in , the number of periodontal pathogen Porphyromonas gingivalis was significantly reduced after treatment with PCA-GC. Figure 11B As well as 11C, the numbers of Fusobacterium nucleatum and Streptococcus gordonii in the PCL-GC group were similar to those in the control group. Notably, after treatment with PCA-GC, the proportion of Porphyromonas gingivalis decreased from 78.86% (control group) to 0.06%, while the commensal bacteria Streptococcus gordonii increased from 19.22% (control group) to 98.57% ( Figure 11D ), indicating that PCA-GC induced a significant shift in the microbial composition of multi-species biofilms.

[0218] It should be emphasized that existing treatment strategies such as scaling and root planing are usually temporary in improving clinical indicators because the changes in the subgingival flora structure are temporary. In contrast, the special properties of the present invention that promote the transformation of the subgingival plaque microbial composition help to resolve the dysbiosis associated with periodontitis, promote the restoration of oral microecological balance, and prevent the recurrence of periodontitis in the long term. In addition, the effect of changes in microbial composition structure on the inflammatory ability of biofilms was evaluated by detecting the inflammatory response of PDLCs stimulated by biofilms with different treatments. The results of qRT-PCR and ELISA showed that under the stimulation of untreated three-species biofilms, proinflammatory cytokines (IL-6 and IL-8) were significantly increased, while PDLCs stimulated with biofilms treated with PCA-GC had lower expression and secretion of IL-6 and IL-8 than cells stimulated with untreated biofilms, such as Figures 11E to 11H The above results further prove that our invention can reduce the pathogenicity of biofilm and reduce inflammatory response. The results of animal studies also prove that the oxygen-releasing material (PCA-GC) has an enhanced therapeutic effect compared with Periodontitis (current local adjuvant medication for periodontitis - Palio / minocycline hydrochloride), which is manifested in reduced alveolar bone resorption. Figure 12A and 12B.

[0219] Example 3

[0220] Preferably, if Figure 13 As shown, the composition further comprises an antibiotic 108 encapsulated by the polymer 102, wherein the antibiotic 108 is released into the periodontal pocket after the oxygen-generating agent in the microsphere shell hydrolyzes to create an oxygen-rich environment. Preferably, the composition comprises a microsphere structure having ciprofloxacin in the core and PCL / CaO2 as the shell (i.e., the peroxide compound 104).

[0221] Hydrophilic polymers, including PVA, GelMA, and alginate, can be used to encapsulate the antibiotic 108 to form the inner layer. Alternatively, a double emulsion method can be employed, with the inner layer consisting of the antibiotic 108 dissolved in distilled water. Microspheres coated with the antibiotic 108 can also be prepared using the same method as in Example 1, with the polymer 102 loaded with the oxide compound 104 and the catalase 106.

[0222] Ciprofloxacin is preferably selected in this embodiment because its antimicrobial activity is related to oxidative stress, and therefore hypoxic conditions can weaken its antimicrobial activity. Supplemental oxygen significantly enhances its antimicrobial activity. Therefore, the drug delivery system can be designed so that the outer layer of the microspheres can generate sufficient oxygen to alleviate hypoxia, thereby increasing the sensitivity of pathogens to the effects of ciprofloxacin.

[0223] Although ciprofloxacin has a weak antibacterial effect against anaerobic pathogens, the combination of oxygen-releasing materials and ciprofloxacin will address this issue. As in the two examples above, the oxygen-generating material alone exhibits a strong antibacterial effect against anaerobic pathogens. Preferably, to prepare core-shell microspheres with a sequential oxygen-antibiotic release effect, PCL-CaO2 microspheres loaded with ciprofloxacin (Cipro) can be prepared using a W / O / W double emulsion solvent evaporation technique. In this method, Cipro is dissolved in 2 mL of distilled water to form an aqueous Cipro solution; PCL polymer dissolved in a DCM solution serves as the oil phase; 2 mL of the prepared aqueous Cipro solution is added to 10 mL of the DCM solution and sonicated for 2 minutes using a probe ultrasonic processor to form a stable W / O emulsion. This stable W / O emulsion is then slowly added to 120 mL of an aqueous solution containing 0.5% PVA and emulsified at ambient temperature using a mechanical stirrer or ultrasonic processor to form a W / O / W emulsion.

[0224] Furthermore, the solvent removal and microsphere hardening can be achieved by continuous stirring for 11-2 hours. Subsequently, the microspheres are separated by filtration and washed several times with distilled water to remove the PVA. The microspheres thus produced are vacuum dried at 25°C for 24 hours to remove residual solvent.

[0225] The inventors discovered that oxygen-generating hydrogels exhibit significant antibacterial effects against periodontal pathogens, including Porphyromonas gingivalis and Fusobacterium nucleatum, without affecting the viability of commensal bacteria such as Streptococcus gordonii. However, in aggressive periodontitis, anaerobic bacteria only account for approximately 65% ​​of the subgingival plaque and are often accompanied by specific bacterial infections, such as Aggregatibacter actinomycetemcomitans. These bacteria can produce leukotoxins, inducing leukocyte destruction and helping bacteria evade the immune system. As facultative anaerobes, they are more resistant to oxygen. Therefore, for periodontitis characterized by severe inflammation and rapid progression, a potent oxygen-generating material encapsulating antibiotics is needed. Ciprofloxacin, the only antibiotic used in periodontal therapy that is sensitive to all Aggregatibacter actinomycetemcomitans strains while having minimal effect on Streptococci, has been incorporated into the hydrogel system. The addition of ciprofloxacin will enhance the antibacterial capacity of oxygen-generating hydrogels and expand their application.

[0226] The inventors first conducted an antibacterial experiment on three representative periodontal pathogens by combining oxygen-generating materials (Example 1) with different concentrations of ciprofloxacin to demonstrate the synergistic antibacterial effect of oxygen and antibiotics. Figures 14 to 15 As shown in Figure 2, ciprofloxacin enhanced the antibacterial effect of an oxygen-generating material (5% CPO-GelMA) against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. Furthermore, the combination of oxygen-generating materials reduced the minimum bactericidal concentration of ciprofloxacin against Aggregatibacter actinomycetemcomitans from 0.8 μg / mL to 0.2 μg / mL. Since antibiotics are generally sensitive to bacteria with active metabolism, the presence of oxygen can avoid potential antibiotic resistance issues.

[0227] Increased oxygen levels will increase the low metabolic level that bacteria maintain under hypoxic conditions, making them more sensitive to the effects of antibiotics. Figure 16 As shown, the potent oxygen-generating material is designed into a core-shell structure with sequential release properties, generating oxygen from the shell structure and releasing antibiotics from the inner core space, thereby enhancing the antibacterial effect of the oxygen-generating material and avoiding potential antibiotic resistance. Furthermore, our invention can address the difficulties of current periodontal drug treatments (primarily topical or systemic antibiotic application), namely, the limited oxygen supply in the biofilm leads to a low metabolic state of bacteria, thereby causing antibiotic resistance in the biofilm.

[0228] First, in order to determine the appropriate concentration of CaO2 / CPO encapsulated in PCL microspheres, the inventors prepared PCL microspheres containing different ratios of CPO (PCA microspheres, CPO:PCL mass ratio of 0.5 / 1:1) and tested their oxygen release curves. Figures 17A to 17BAs shown in the figure, PCA microspheres with a CPO to PCL mass ratio of 1:1 have more sustainable oxygen release, lasting up to 7 days, which is more suitable for clinical applications due to weekly follow-up of periodontal treatment. Therefore, in the following study, we chose to prepare PCA microspheres by adding CPO at a ratio of 1:1. In addition, we also prepared microspheres with different particle sizes by adjusting the concentration of PCL in the oily solution (10%, 20%) and the speed of the homogenizer in the emulsification reaction (5000rpm, 10000rpm), and tested the oxygen release curves of different microspheres. ... Figure 17C As shown in the figure, the particle size of the microspheres in the 20% PCL (1:1) 5000rpm group was the largest, with an average diameter of 488.25μm, and the average diameters of the others were all below 200μm, among which the particle size of the 10% PCL (1:1) 10000rpm group was the smallest, with an average diameter of 85.27μm. Figures 17D to 17E As shown in the results, the oxygen release in the 20% PCL (1:1) 5000rpm group was the most sustained, with a certain amount of oxygen released within 7 days, while the oxygen release period in the 10% PCL (1:1) 10000rpm group was the shortest, with no obvious release after 24 hours. Therefore, the larger the microsphere diameter, the more conducive it is to the long-term release of oxygen.

[0229] like Figure 18A as well as Figure 18B As shown in FIG, microspheres (PCA-ciprofloxacin) of different sizes with relatively regular structures can be observed under SEM, and the cross-sectional image shows a core-shell structure. Figure 19A The loading capacity and encapsulation efficiency of Cipro were determined based on the standard calibration curve, which showed that 100 mg microspheres contained 60 μg Cipro, with a drug loading rate of 0.06% and an encapsulation efficiency of 3%. Figure 19B The above results indicate that microspheres with sequential release of oxygen and ciprofloxacin can be prepared.

[0230] The percentage of drug loading in the microspheres was calculated using the following formula:

[0231] L=Qm / Wm×100

[0232] Where L is the drug loading percentage, Qm is the drug loaded into the microspheres, and Wm is the weight of the microspheres. The percentage of encapsulation efficiency is determined by the following formula:

[0233] EE=Qa / Qt×100

[0234] Where EE is the encapsulation efficiency percentage, Qa is the actual drug content, and Qt is the theoretical drug content.

[0235] Next, we prepared PCL microspheres without CPO or ciprofloxacin using the above procedure and incorporated the different microspheres into chitosan hydrogels to compare their antimicrobial effects against periodontal pathogens. PCL-Ciprofloxacin-CC represents a hydrogel that releases only ciprofloxacin; PCA-CC represents a hydrogel that releases only oxygen; and PCA-Ciprofloxacin-CC represents a hydrogel that releases both ciprofloxacin and oxygen.

[0236] like Figures 20A to 20C As shown, compared with hydrogels releasing oxygen or ciprofloxacin alone, PCA-ciprofloxacin-CC had enhanced bactericidal effects against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans at the same concentration, indicating that oxygen and ciprofloxacin have synergistic antimicrobial effects against different periodontal pathogens. Surprisingly, none of the microspheres showed significant bactericidal effects against Streptococcus gordonii, one of the commensal bacteria. These results indicate that compared with microspheres releasing oxygen or ciprofloxacin alone, microspheres with sequential release of oxygen and ciprofloxacin have enhanced antimicrobial effects against periodontal pathogens, while having no significant effect on the commensal bacteria Streptococcus gordonii. This will be a potential treatment strategy for periodontitis with severe inflammation and rapid progression.

[0237] Furthermore, the catalase activities of CeO2 and natural enzyme in different solutions were compared, since the microspheres and catalase were to be encapsulated in chitosan hydrogel for topical application, and the hydrogel was dissolved in 0.1 M acetic acid. Figure 21 As shown in the figure, the activity of natural catalase was completely destroyed in an acidic environment compared to its activity in PBS. In contrast, the catalase-like activity of CeO2 was significantly enhanced compared to that in PBS. The above results further indicate that CeO2, as one of the nanozymes, has good stability and can maintain its catalase-like activity under different environments. It will be a suitable candidate for encapsulation in chitosan hydrogels to promote the decomposition of generated hydrogen peroxide (H2O2).

[0238] The therapeutic effects of different materials were further studied by constructing an experimental periodontitis model in rats, such as Figures 22A to 22C As shown in the results, the PCA-ciprofloxacin group with the sequential release of oxygen and ciprofloxacin significantly reduced the amount of alveolar bone resorption and the probing depth of periodontal pockets compared with the other groups, indicating that it has a significant therapeutic effect on periodontitis, reduces the inflammatory response of periodontal tissues, and avoids further damage to periodontal tissues. Figures 23A to 23D As shown, the application of different materials had no significant effect on rat body weight, blood cells and internal organs, indicating that they all had good biosafety.

[0239] These embodiments are advantageous because they provide new uses for oxygen-generating hydrogels, improve oxygen-generating hydrogels, and provide a new method for periodontal therapy.

[0240] Advantageously, topically applied hydrogels with prolonged oxygen release have high translational potential. In addition, many periodontal pathogens that are not obligate anaerobes are also involved in the progression of periodontitis, such as Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans.

[0241] In addition, hypoxia can accelerate antibiotic resistance; therefore, given the antibiotic-releasing capacity of oxygen-generating hydrogels, the synergistic effect of increasing oxygen levels and antibiotics will promote bactericidal effects against periodontal pathogens and reduce the possibility of antibiotic resistance.

[0242] In order to facilitate the preservation of the composition for periodontal treatment, components containing oxygen-generating agents such as calcium peroxide (CPO), magnesium peroxide and other peroxide compounds can be mixed into a hydrophobic polymer or matrix such as glycerol, mineral oil, vaseline, wax, etc., or a combination of the components. If the antibiotic has poor long-term stability in a hydrophilic matrix, it can also be stored in the hydrophobic polymer or matrix to enhance its stability and facilitate long-term storage.

[0243] The terms "a" or "an" used herein to describe components and elements of the present invention are for convenience only and to provide a basic understanding of the present invention. Such descriptions should be understood to include one or at least one, unless expressly specified otherwise, and the singular also includes the plural. The term "or" herein is synonymous with "and / or."

[0244] It should be noted that although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and adjustments may be made to the present invention without departing from the scope or principles of the present invention. Therefore, the present invention is intended to cover all modifications and adjustments that fall within the scope of the appended claims and their equivalents.

Claims

1. A composition for periodontal treatment, characterized in that Include: - a polymer carrier encapsulating microspheres comprising an oxygen generator shell and an antibiotic core, wherein the oxygen generator is configured to generate hydrogen peroxide when the peroxide compound is hydrolyzed or release oxygen through the oxygen storage material and the microalgae, and the antibiotic is subsequently released under the oxygen-rich microenvironment; and - nanoparticles also encapsulated in the polymer carrier, wherein the nanoparticles are configured to have catalase-like activity and decompose the hydrogen peroxide to produce oxygen and water; In which, after periodontal treatment, the oxygen generated when the peroxide compound is hydrolyzed and the hydrogen peroxide is decomposed or released by the oxygen storage material and the microalgae helps to maintain the oxygen-rich microenvironment in the periodontal pocket where the composition is placed, and the released antibiotics further exhibit enhanced antibacterial and anti-biofilm effects in the oxygen-rich microenvironment.

2. The composition according to claim 1, characterized in that The shell of the microsphere is a hydrophobic polymer containing the oxygen generator, and the core of the microsphere includes the antibiotic dissolved or dispersed in water or a hydrophilic polymer.

3. The composition according to claim 2, characterized in that The antibiotics located in the core of the microspheres are released through the pores formed after peroxide hydrolysis.

4. The composition according to claim 2, characterized in that The oxygen generating agent comprises calcium peroxide, magnesium peroxide, and / or a peroxide compound, at least one of an oxygen storage material and microalgae, or a combination thereof, which acts as the source of oxygen generation, wherein the oxygen storage material comprises sodium percarbonate, manganese dioxide and / or hemoglobin.

5. The composition according to claim 2, characterized in that The hydrophobic polymer comprises polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyphosphate (PPE) and / or polyorthoester (POE).

6. The composition according to claim 2, characterized in that The antibiotics include fluoroquinolone antibiotics, tetracycline antibiotics, nitroimidazole antibiotics, penicillin antibiotics, glycopeptide antibiotics and / or macrolides, and the hydrophilic material includes at least one of gelatin, collagen, fibrin, silk protein, hyaluronic acid, cellulose, chitosan, alginate, dextran, polyvinyl alcohol (PVA) and / or polyethylene glycol (PEG).

7. The composition according to claim 1, characterized in that The dosage forms of the polymer carrier include hydrogels, fibers, strips, films and / or microparticles.

8. The composition according to claim 7, characterized in that The hydrogel comprises chitosan hydrogel, poloxamer, poly (N-isopropylacrylamide), PEG and / or cellulose.

9. The composition according to claim 1, characterized in that The nanoparticles include CeO2, MnO2, Mn3O4, Au, Pd, Pt and / or Prussian blue nanoparticles.

10. The composition according to claim 1, characterized in that wherein the composition is further configured to increase the oxygen content of the microenvironment in the periodontal pocket after periodontal treatment.

11. A method for preparing core-shell structured microspheres of the composition according to claim 2, characterized in that: The following steps are involved: -Microspheres with a ciprofloxacin core and a PCL / calcium peroxide (CPO) shell were prepared using a double emulsion method (w / o / w); - Dissolve 10 mg / mL to less than 20 mg / mL of ciprofloxacin hydrochloride (Cipro) in 1 mL of distilled water to form a Cipro aqueous solution (w1); dissolve a predetermined amount of PCL with an upper limit of 50% or 60% w / v% in 5 mL of DCM, and add CPO in a 1:1 ratio to form a polymer oily solution (o); add the prepared Cipro aqueous solution (w1) to the polymer oily solution (o); - Use a homogenizer at 15000 rpm to emulsify to form a stable w1 / o emulsion; - The stable w1 / o emulsion was slowly added to 60 mL of 0.5% PVA (w2) and homogenized at less than 10,000 rpm for 1 minute to disperse the emulsion into microspheres (PCA-CIP) with Cipro as the core and PCL / CPO as the shell. The final microsphere dispersion was stirred for 1-2 hours to evaporate the solvent and harden the microspheres, which were then collected, washed, and dried.

12. A method for preparing the composition according to claim 8, characterized in that: The following steps are involved: - Chitosan was dissolved in 0.1 M acetic acid solvent and sodium β-glycerophosphate (β-GP) was dissolved in pure water and then stored in an ice bath; - Dispersing the microspheres and the CeO2 nanoparticles contained in the composition in the β-GP solution, and respectively loading them into a double-tube applicator, wherein the double-tube applicator is suitable for mixing the composition and injecting it into the periodontal pocket.

13. A composition for periodontal treatment, characterized in that: Include: - a polymer carrier encapsulating an oxygen generator comprising a peroxide compound, wherein the oxygen generator is configured to generate hydrogen peroxide when the peroxide compound is hydrolyzed or to release oxygen through the oxygen storage material and the microalgae; as well as - nanoparticles encapsulated in the polymer carrier, wherein the nanoparticles are configured to have catalase-like activity and decompose the hydrogen peroxide to produce oxygen and water; Wherein, after periodontal treatment, the oxygen generated when the peroxide compound is hydrolyzed and the hydrogen peroxide is decomposed or released by the oxygen storage material and the microalgae is configured to help maintain the oxygen-rich microenvironment around the periodontal pocket where the composition is disposed.

14. The composition according to claim 13, characterized in that The polymer carrier comprises a hydrophobic polymer containing the oxygen generating agent.

15. The composition according to claim 14, characterized in that Further comprising an antibiotic encapsulated by the polymer carrier, wherein the antibiotic is released into the periodontal pocket at a predetermined post-treatment period after periodontal treatment.

16. The composition according to claim 15, characterized in that The antibiotic is dissolved or dispersed in water or a hydrophilic polymer, or conjugated to the polymer carrier.

17. The composition according to claim 16, characterized in that The antibiotics include fluoroquinolone antibiotics, tetracycline antibiotics, nitroimidazole antibiotics, penicillin antibiotics, glycopeptide antibiotics and / or macrolides, and the antibiotics are dissolved in water or encapsulated in the hydrophilic material, wherein the hydrophilic material includes gelatin, collagen, fibrin, silk protein, hyaluronic acid, cellulose, chitosan, alginate, dextran, polyvinyl alcohol (PVA) and / or polyethylene glycol (PEG).

18. The composition according to claim 13, characterized in that The oxygen generating agent comprises calcium peroxide, magnesium peroxide, and / or a peroxide compound, and at least one of an oxygen storage material and microalgae, or a combination thereof, serving as the source of oxygen generation, wherein the oxygen storage material comprises sodium percarbonate, manganese dioxide and / or hemoglobin.

19. The composition according to claim 14, characterized in that The hydrophobic polymer comprises polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyphosphate (PPE) and / or polyorthoester (POE).

20. The composition according to claim 13, characterized in that The dosage forms of the polymer carrier include hydrogels, fibers, strips, films or microparticles.

21. The composition according to claim 20, characterized in that The hydrogel comprises chitosan hydrogel, poloxamer, poly (N-isopropylacrylamide), PEG and / or cellulose.

22. The composition according to claim 21, characterized in that wherein the hydrogel comprises thermosensitive crosslinks.

23. The composition according to claim 13, characterized in that The nanoparticles include CeO2, MnO2, Mn3O4, Au, Pd, Pt and / or Prussian blue nanoparticles.

24. The composition according to claim 13, characterized in that wherein the composition is further configured to increase the oxygen content of the microenvironment in the periodontal pocket after periodontal treatment.

25. The composition according to claim 13, characterized in that The polymer support comprises a two-component composite structure having a first polymer layer and a second polymer layer.

26. The composition according to claim 25, characterized in that The first polymer layer encapsulates the oxygen generator, and the second polymer layer encapsulates the nanoparticles.

27. The composition according to claim 26, characterized in that The first polymer layer comprises an electrospun fibrous membrane.

28. The composition according to claim 27, characterized in that The electrospun fiber membrane comprises a hydrophobic host polymer such as polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyphosphate (PPE) and / or polyorthoester (POE).

29. The composition according to claim 26, characterized in that The second polymer layer comprises a hydrogel.

30. The composition according to claim 29, characterized in that The hydrogel comprises gelatin methacrylate (GelMA), chitosan hydrogel, poloxamer, poly (N-isopropylacrylamide), PEG and / or cellulose.

31. The composition of claim 25, wherein the two-component composite structure is a Janus composite material, and the first polymer layer and the second polymer layer exhibit different surface properties and / or functions.

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