An invisible aligner coating for inhibiting periodontal pathogenic bacteria and a preparation method and application thereof

Abstract

The present application relates to the technical field of oral materials, and particularly relates to an invisible aligner coating for inhibiting periodontal pathogenic bacteria and a preparation method and application thereof, the preparation method of the invisible aligner coating comprises the following steps: after cellulose nanofiber is subjected to TEMOF oxidation reaction, the cellulose nanofiber is subjected to amidation reaction with dopamine, then the cellulose nanofiber is mixed with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution; finally, the solution is subjected to film forming treatment to obtain the invisible aligner coating for inhibiting periodontal pathogenic bacteria. In the present application, TOCNF is used as a support, an adhesive coating TOCNF-DA is formed through amidation reaction with DA, PVA is added to increase the dispersibility of TOCNF-DA, and a certain content of Cur is loaded, so that a TOCNF-DA / PVA / Cur coating is constructed on the surface of an invisible aligner base material; the coating has good biological safety, can effectively inhibit the adhesion and multiplication of periodontal pathogenic bacteria Pg, and has low cost, simple manufacturing process, and does not involve toxic reagents in the production process.

Description

Technical Field

[0001] The present invention relates to the technical field of oral materials, and in particular to an invisible orthodontic appliance coating for inhibiting periodontal pathogens, and a preparation method and application thereof. Background Art

[0002] With rising living standards, the demand for and popularity of orthodontic treatment continues to grow. Orthodontic appliances used clinically primarily include fixed appliances and invisible braces. Compared to traditional fixed appliances, invisible braces are increasingly favored by patients and physicians due to their aesthetics, ease of cleaning, and predictability. However, existing invisible braces materials lack inherent antibacterial properties, and the incidence of gingivitis and periodontitis in orthodontic patients remains high.

[0003] In relevant literature reports, the research on antibacterial invisible braces mainly focuses on the prevention of caries and the improvement of enamel demineralization. However, the existing technology mainly targets Streptococcus mutans, which causes caries, and has no antibacterial effect on Pg bacteria, which causes periodontitis. Zhang et al. loaded modified nano-gold particles (AuDAPT) on the surface of invisible braces and found that the antibacterial effect was not significant at a Pg bacteria concentration of 10 5 CFU / mL, it has a certain inhibitory effect, but AuDAPT has high cytotoxicity, and its long-term biosafety for periodontal tissues and mucosa is unknown (see Zhang M, Liu X, Xie Y, Zhang Q, Zhang W, Jiang X, Lin J. Biological safe gold nanoparticle-modified dental aligner prevents the Porphyromonas gingivalis biofilm formation. ACS Omega 2020; 5(30): 18685-92).

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an invisible braces coating that inhibits periodontal pathogens, and its preparation method and application, aiming to solve the problems that the prior art antibacterial invisible braces coating does not have the effect of inhibiting periodontal pathogens and has poor biocompatibility.

[0006] The technical solutions of the present invention are as follows:

[0007] A method for preparing an invisible braces coating for inhibiting periodontal pathogens comprises the following steps:

[0008] Cellulose nanofibers were oxidized using 2,2,6,6-tetramethylpiperidin-1-oxyl to obtain TOCNF;

[0009] The TOCNF is mixed with dopamine, and subjected to an amidation reaction to obtain TOCNF-DA;

[0010] The TOCNF-DA was mixed with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution;

[0011] After the TOCNF-DA / PVA / Cur solution is formed into a film and dried, the invisible brace coating is obtained.

[0012] The method for preparing the invisible braces coating for inhibiting periodontal pathogens, wherein the cellulose nanofibers are cellulose nanofibers derived from plants or cellulose nanofibers derived from bacteria; and the curcumin is natural curcumin or chemically modified curcumin.

[0013] The method for preparing the invisible braces coating that inhibits periodontal pathogens, wherein the step of oxidizing cellulose nanofibers with 2,2,6,6-tetramethylpiperidin-1-oxyl to obtain TOCNF comprises:

[0014] dissolving cellulose nanofibers in deionized water to obtain a cellulose nanofiber solution;

[0015] adding 2,2,6,6-tetramethylpiperidin-1-oxyl, sodium bromide, and sodium hypochlorite to the cellulose nanofiber solution to obtain a reaction system;

[0016] The pH of the reaction system is maintained between 9 and 11 by using an alkali, and TOCNF is obtained by stirring, centrifuging, dialysis, and freeze-drying.

[0017] In the method for preparing the invisible braces coating that inhibits periodontal pathogens, the base is selected from one or more of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0018] The method for preparing the invisible braces coating for inhibiting periodontal pathogens, wherein the step of mixing the TOCNF-DA with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution comprises:

[0019] Dissolving the TOCNF-DA in deionized water to obtain a TOCNF-DA solution;

[0020] The TOCNF-DA solution was mixed with curcumin and stirred to obtain a TOCNF-DA / Cur solution;

[0021] Mixing polyvinyl alcohol with deionized water to obtain a PVA solution;

[0022] The TOCNF-DA / Cur solution and the PVA solution were mixed and stirred to obtain a TOCNF-DA / PVA / Cur solution.

[0023] The method for preparing the invisible braces coating for inhibiting periodontal pathogens, wherein the concentration of the TOCNF-DA solution is 0.1wt%-10wt%.

[0024] The method for preparing the invisible braces coating that inhibits periodontal pathogens, wherein the weight ratio of the TOCNF-DA to the curcumin is 1:1-10:1.

[0025] The method for preparing the invisible braces coating that inhibits periodontal pathogens, wherein the weight ratio of the TOCNF-DA to the polyvinyl alcohol is 1:4-10:1.

[0026] A invisible braces coating for inhibiting periodontal pathogens is prepared by using the method for preparing the invisible braces coating for inhibiting periodontal pathogens.

[0027] Application of an invisible braces coating for inhibiting periodontal pathogens in orthodontic treatment.

[0028] Beneficial effects: The present invention provides an invisible braces coating for inhibiting periodontal pathogens, and a preparation method and application thereof. The preparation method of the invisible braces coating comprises the following steps: cellulose nanofibers are subjected to a 2,2,6,6-tetramethylpiperidine-1-oxyl oxidation reaction, followed by an amidation reaction with dopamine, and then mixed with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution; finally, the solution is subjected to a film-forming treatment to obtain an invisible braces coating for inhibiting periodontal pathogens. The present invention uses TOCNF as a scaffold, forms an adhesive coating TOCNF-DA through an amidation reaction with DA, adds PVA to increase the dispersibility of TOCNF-DA, and loads a certain amount of Cur, thereby constructing a TOCNF-DA / PVA / Cur coating on the surface of the invisible orthodontic appliance base material; the coating has good biosafety and can effectively inhibit the adhesion and proliferation of periodontal pathogens Pg bacteria, and is low in cost and simple in manufacturing process. No toxic reagents are involved in the production process, and it can be widely used in orthodontic correction, periodontal disease treatment and other related oral disease treatments, as well as the treatment of systemic diseases related to periodontal pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a process flow chart of a method for preparing an invisible braces coating that inhibits periodontal pathogens according to the present invention;

[0030] Figure 2 This is the Fourier transform infrared spectrum of the TOBC-DA / PVA / Cur coating prepared in Example 1;

[0031] Figure 3 This is a scanning electron micrograph of the TOBC-DA / PVA / Cur coating prepared in Example 1;

[0032] Figure 4 This is a microscopic image of the thickness of the TOBC-DA / PVA / Cur coating prepared in Example 1;

[0033] Figure 5 This is the experimental data of the contact angle of the TOBC-DA / PVA / Cur coating prepared in Example 1;

[0034] Figure 6 This is a statistical graph of the experimental results of testing the cytotoxicity of the TOBC-DA / PVA / Cur coating by the CCK8 method in Example 1;

[0035] Figure 7 This is a colony growth diagram of the TOBC-DA / PVA / Cur coating prepared in Example 1 co-cultured with Pg bacteria;

[0036] Figure 8 This is a confocal microscopy image of the TOBC-DA / PVA / Cur coating prepared in Example 1 co-cultured with Pg bacteria;

[0037] Figure 9 This is a scanning electron microscope image of the morphology of Pg bacteria on the surface of the TOBC-DA / PVA / Cur coating prepared in Example 1. DETAILED DESCRIPTION

[0038] The present invention provides an invisible braces coating that inhibits periodontal pathogens, as well as a preparation method and application thereof. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0039] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0040] Invisible braces are transparent elastic plastic orthodontic devices designed and manufactured using computer-aided design. They are primarily made by pressing thermoplastic plastic sheets onto dental models using a heating cycle that combines vacuum and pressure molding. The main materials used include polyethylene, polycarbonate, polypropylene, and modified polyethylene terephthalate glycol (PETG) (see Raja TA, Littlewood SJ, Munyombwe TBubb NL. Wear resistance of four types of vacuum-formed retainer materials: alaboratory study. Angle Orthod 2014; 84(4), 656-64). Invisible braces need to be worn for more than 20 hours a day, with the next set replaced every 10-14 days. Treatment lasts for 2-3 years. Since invisible braces cover the buccal and lingual surfaces, occlusal surfaces, and part of the gums for a long time, it increases the difficulty of maintaining oral hygiene. At the same time, plaque accumulates around the invisible braces. Under the action of orthodontic correction force, the movement and remodeling of periodontal tissues cause the accumulation of subgingival plaque, such as Porphyromanas gingivalis (Pg), which in turn causes gingivitis and periodontitis (see Rossini G, Parrini S, Castroflorio T, Deregibus A, Debernardi CL. Periodontal health during clear aligners treatment: a systematic review. Eur J Orthod 2015; 37(5): 539-43). However, existing invisible braces materials themselves do not have antibacterial effects, and the incidence of gingivitis and periodontitis in orthodontic patients remains high.

[0041] In relevant literature reports, the research on antibacterial invisible braces mainly focuses on the prevention of caries and the improvement of enamel demineralization. For example, Park et al. loaded carboxymethyl cellulose (CMC) and chitosan (CHI) on the PETG surface through layer-by-layer assembly technology, and then formed a super-hydrophilic surface of the CMC-CHI coating through chemical cross-linking, thereby reducing the adhesion of Streptococcus mutans by more than 75%, that is, achieving antibacterial effect by inhibiting plaque adhesion (see Park S, Kim HH, Yang SB, Moon JH, Ahn HW, Hong JA polysaccharide-based antibacterial coating with improved durability for clear overlay appliances. ACS Appl Mater Interfaces 2018; 10(21): 17714-21). CN111484641B discloses a long-lasting antibacterial material and its preparation method, a long-lasting antibacterial dental film, and a long-lasting antibacterial invisible braces. The material is made of a polyurethane substrate that has been tertiary ammoniumized and in-situ quaternized. The quaternary ammonium salt antibacterial group is mainly used to inhibit the formation of plaque biofilm in contact with the invisible braces. The antibacterial invisible braces of the above technology cannot release antibacterial substances, cannot inhibit the accumulation of subgingival bacteria, and cannot remove periodontal pathogens free in the periodontal pocket. CN114618023B discloses a hydrogel-coated invisible braces and its preparation method, which specifically includes a thermoplastic polyurethane film and a nanocellulose / nanozinc oxide-casein phosphopolypeptide-amorphous calcium phosphate hydrogel layer covering the inner surface of the thermoplastic polyurethane film. The nanocellulose / nanozinc oxide-casein phosphopolypeptide-amorphous calcium phosphate hydrogel layer acts as a stable calcium and phosphorus ion reservoir, which can reduce the survival rate of Streptococcus mutans and promote the remineralization of demineralized teeth. CN112618802B discloses a fluorine-containing antibacterial invisible orthodontic appliance for improving enamel demineralization and its preparation method. It uses phase-transition lysozyme as the base layer, and fixes lysozyme and fluorine-containing hyaluronic acid on the surface of the base through layer-by-layer stacking and electrostatic force, thereby achieving the antibacterial effect and improving enamel demineralization.

[0042] However, the above technology mainly targets Streptococcus mutans that causes dental caries. The gold nanoparticles modified by Zhang et al. inhibit Pg bacteria, while AuDAPT has high cytotoxicity and its long-term biosafety for periodontal tissues and mucosa is unknown.

[0043] Based on this, Figure 1 As shown, the present invention provides a method for preparing an invisible braces coating for inhibiting periodontal pathogens, comprising the steps of:

[0044] Step S10: oxidizing the cellulose nanofibers using 2,2,6,6-tetramethylpiperidin-1-oxyl to obtain TOCNF;

[0045] Step S20: mixing the TOCNF with dopamine, and subjecting the mixture to an amidation reaction to obtain TOCNF-DA;

[0046] Step S30: mixing the TOCNF-DA with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution;

[0047] Step S40: After the TOCNF-DA / PVA / Cur solution is formed into a film and dried, the invisible braces coating is obtained.

[0048] In this embodiment, the main components of the invisible braces coating obtained by this method include Tempo-oxidized cellulose nanofibers (TOCNF), dopamine (DA), polyvinyl alcohol (PVA) and curcumin (Cur); the invisible braces coating uses TOCNF as a support, and forms an adhesive coating TOCNF-DA through an amidation reaction with DA, and then PVA is added to increase the dispersibility of TOCNF-DA, and a certain content of Cur is loaded, thereby constructing a TOCNF-DA / PVA / Cur coating on the surface of the invisible braces base material.

[0049] Specifically, the cellulose nanofibers and Cur involved in the present invention have good biocompatibility, which overcomes the high cytotoxicity of the previous invisible braces antibacterial coating; the antibacterial property of the invisible braces coating constructed by the present invention comes from the anti-plaque microbial adhesion characteristics of the TOCNF-DA / PVA / Cur coating and the synergistic effect of the slow-release Cur in inhibiting the proliferation of periodontal pathogens Pg bacteria. It also has an inhibitory effect on the crown surface covered by the invisible braces and the cementum surface that cannot be covered, as well as some periodontal pathogens free in the periodontal pocket, thereby achieving the effect of inhibiting the formation of plaque biofilms through dual pathways of antimicrobial adhesion and release of bactericidal substances. In addition, the cost of preparing the invisible braces coating that inhibits periodontal pathogens of the present invention is low, the manufacturing process is simple, and no toxic reagents are involved in the production process. It can be widely used in orthodontic treatment, periodontal disease treatment and other related oral disease treatments, as well as the field of systemic disease treatment related to periodontal pathogens.

[0050] In some embodiments, the cellulose nanofibers are cellulose nanofibers derived from plants or cellulose nanofibers derived from bacteria. The cellulose nanofibers have good biocompatibility, overcoming the high cytotoxicity of the antibacterial coating of previous invisible braces. The cellulose nanofibers (BC) derived from bacteria are oxidized using 2,2,6,6-tetramethylpiperidine-1-oxyl to obtain TOBC; the cellulose nanofibers (CN) derived from plants are oxidized using 2,2,6,6-tetramethylpiperidine-1-oxyl to obtain TOCN. The curcumin is natural curcumin or chemically modified curcumin. The chemically modified curcumin includes but is not limited to 4-phenylaminocarbonylbisdemethoxycurcumin (CMC2.24) and 4-methoxycarbonylcurcumin (CMC 2.5). Curcumin not only has good biocompatibility, but also has the effect of inhibiting periodontal pathogens.

[0051] In some embodiments, in step S10, the step of oxidizing cellulose nanofibers with 2,2,6,6-tetramethylpiperidin-1-oxyl to obtain TOCNF comprises:

[0052] Step S11: dissolving cellulose nanofibers in deionized water to obtain a cellulose nanofiber solution;

[0053] Step S12: adding 2,2,6,6-tetramethylpiperidin-1-oxyl, sodium bromide and sodium hypochlorite to the cellulose nanofiber solution to obtain a reaction system;

[0054] Step S13: using alkali to maintain the pH of the reaction system between 9 and 11, stirring, centrifuging, dialysis, and freeze-drying to obtain TOCNF.

[0055] Specifically, the pH of the reaction system is maintained between 9 and 11 using alkali, and stirred at room temperature for 1 to 24 hours to allow the cellulose nanofibers to undergo an oxidation reaction. Anhydrous ethanol is then added to the reaction system to terminate the reaction. After centrifugation, the system is dialyzed to a pH of 6 to 8, and freeze-dried to obtain Tempo-oxidized cellulose nanofibers (TOCNF).

[0056] In some embodiments, a base is used to maintain the pH of the reaction system between 9 and 11, for example, pH = 9, pH = 10 or pH = 11. Other specific values ​​within the above range can be selected and will not be described here one by one; preferably pH = 10.

[0057] In some embodiments, after centrifugation, dialyzation is performed to a pH of 6-8, such as pH=6, pH=7 or pH=8. Other specific values ​​within the above range can be selected and will not be described in detail here; preferably, pH=7.

[0058] In some embodiments, the base is selected from one or more of sodium hydroxide, potassium hydroxide, and calcium hydroxide; these bases can be used to adjust the pH of the reaction system in step S13 so that the pH of the reaction system is maintained between 9 and 11, which is conducive to the oxidation reaction.

[0059] In some embodiments, in step S20, the step of mixing the TOCNF with dopamine and obtaining TOCNF-DA after amidation reaction comprises: dissolving TOCNF in deionized water, activating the carboxyl group for 0.5-2h, adding dopamine (DA), then stirring at room temperature for 1-48h, removing the supernatant by centrifugation, dialysis for 1-7d, and freeze-drying to obtain TOCNF-DA.

[0060] In some embodiments, in step S30, the step of mixing the TOCNF-DA with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution comprises:

[0061] Step S31: dissolving the TOCNF-DA in deionized water to obtain a TOCNF-DA solution;

[0062] Step S32: mixing the TOCNF-DA solution with curcumin, and stirring to obtain a TOCNF-DA / Cur solution;

[0063] Step S33: mixing polyvinyl alcohol with deionized water to obtain a PVA solution;

[0064] Step S34: The TOCNF-DA / Cur solution and the PVA solution are mixed and stirred to obtain a TOCNF-DA / PVA / Cur solution.

[0065] Specifically, TOCNF-DA was dissolved in deionized water to obtain a TOCNF-DA solution; Cur was then added and stirred at room temperature for 1-48 hours to obtain a TOCNF-DA / Cur solution; the TOCNF-DA / Cur solution was then mixed with a PVA solution and stirred at room temperature for 1-24 hours to obtain a TOCNF-DA / PVA / Cur solution.

[0066] In some embodiments, the molecular weight of the polyvinyl alcohol is 25,000-150,000.

[0067] In some embodiments, the concentration of the TOCNF-DA solution is 0.1 wt%-10 wt% to ensure complete dissolution of TOCNF-DA.

[0068] In a preferred embodiment, the concentration of the TOCNF-DA solution is 0.5 wt%-2.0 wt%.

[0069] In some embodiments, the weight ratio of the TOCNF-DA to the curcumin is 1:1-10:1, ensuring sufficient curcumin loading.

[0070] In a preferred embodiment, the weight ratio of the TOCNF-DA to the curcumin is 1:1-5:1.

[0071] In some embodiments, the weight ratio of the TOCNF-DA to the polyvinyl alcohol is 1:4-10:1.

[0072] In a preferred embodiment, the weight ratio of the TOCNF-DA to the polyvinyl alcohol is 1:1-5:1.

[0073] In some embodiments, the concentration of the PVA solution is 0.1 wt%-25 wt% to ensure that the PVA is completely dissolved.

[0074] In a preferred embodiment, the concentration of the PVA solution is 1 wt%-10 wt%.

[0075] In addition, the present invention also provides an invisible braces coating for inhibiting periodontal pathogens, which is prepared using the preparation method of the invisible braces coating for inhibiting periodontal pathogens.

[0076] In some embodiments, in step S40, the TOCNF-DA / PVA / Cur solution is dried after film formation to obtain the invisible braces coating. Taking a PETG film as an example, the substrate can be: dropping a TOCNF-DA / PVA / Cur solution on the PETG film, centrifuging for 1-10 minutes, or immersing the PETG film in a TOCNF-DA / PVA / Cur solution and placing it at room temperature for 1-7 days to obtain a TOCNF-DA / PVA / Cur coating.

[0077] Specifically, the volume of the TOCNF-DA / PVA / Cur solution added to the PETG membrane is sufficient to cover the surface of the PETG membrane; the volume of the TOCNF-DA / PVA / Cur solution used to soak the PETG membrane is sufficient to completely soak the PETG membrane.

[0078] In addition, the present invention also provides an application of an invisible braces coating for inhibiting periodontal pathogens in orthodontic treatment.

[0079] In this embodiment, the invisible braces coating that inhibits periodontal pathogens has low cost and simple manufacturing process, and does not involve toxic reagents in the production process. It can be widely used in orthodontic correction, periodontal disease treatment and other related oral disease treatments, as well as the treatment of systemic diseases related to periodontal pathogens.

[0080] It should be noted that the invisible braces coating can be used on all existing invisible braces substrates, and it can also inhibit periodontal pathogens.

[0081] The present invention will be described in detail with reference to the following examples. It should also be understood that the following examples are only intended to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above disclosure of the present invention fall within the scope of protection of the present invention.

[0082] Example 1

[0083] This embodiment provides an invisible braces coating that inhibits periodontal pathogens, and the preparation method thereof is as follows:

[0084] Preparation of TOBC: 2.0 g of bacterial cellulose nanofibers (BC) were added to 200 mL of deionized water and treated with a high-speed homogenizer to form a dispersed fiber suspension. 0.032 g of TEMPO (0.1 mmol / g), 2.0 g of NaBr (1 mmol / g), and 3.55 mL of NaClO (6 mmol / g) were added to the suspension. The pH was adjusted to 10 using a 0.5 mol / L NaOH solution. The reaction was stirred at room temperature for 16 hours, during which time the pH of the reaction system was maintained at 10 using a NaOH solution. After completion, the reaction was terminated by adding 10 mL of anhydrous ethanol. The product was centrifuged and dialyzed against deionized water for 3 days to a pH of 7. The resulting TOBC was freeze-dried and stored at 4°C after sealing.

[0085] Preparation of TOBC-DA: TOBC (1.00 g) was dispersed in 300 mL of deionized water. 1.00 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.00 g of N-hydroxysuccinimide (NHS) were added to activate the carboxyl groups for 0.5 h. 0.95 g of DA was then added. The mixture was stirred at room temperature for 24 h and then centrifuged at 5000 rpm for 10 min. The supernatant was removed, the suspension was dialyzed against deionized water for 3 days, and the resulting TOBC-DA was freeze-dried and stored at 4°C in the dark.

[0086] Preparation of TOBC-DA / Cur solution: 0.1 g of TOBC-DA was added to 20 mL of deionized water (0.5 wt %) and fully dissolved by high-speed homogenizer at 7000 rpm. 0.02 g of Cur was added and the mixture was stirred at room temperature for 24 h (TOBC-DA:Cur = 5:1).

[0087] Preparation of TOBC-DA / PVA / Cur solution: 1 g of PVA was added to 50 mL of deionized water (2 wt %), sealed and stirred at 90°C for 2 h until it was completely dissolved. Then, 20 mL of TOBC-DA / Cur solution and 5 mL of 2 wt % PVA solution were mixed thoroughly and stirred at room temperature for 2 h to prepare a TOBC-DA / PVA / Cur solution (TOBC-DA:PVA:Cur = 5:5:1).

[0088] Preparation of TOBC-DA / PVA / Cur coating: Prepare a 10mm diameter circular PETG membrane, rinse it twice by immersion in 75% ethanol solution, ultrasonically clean it for 15 minutes, and then rinse it twice with deionized water. Place the cleaned PETG membrane in a centrifuge, and add 200μl of TOBC-DA solution, TOBC-DA / Cur solution, and TOBC-DA / PVA / Cur solution to the surface until the membrane is fully covered. Centrifuge at 3000rpm for 3 minutes and dry overnight in the dark.

[0089] Example 2

[0090] This embodiment provides an invisible braces coating that inhibits periodontal pathogens, and the preparation method thereof is as follows:

[0091] Preparation of TOBC: 2.0 g of bacterial cellulose nanofibers (BC) were added to 200 mL of deionized water and treated with a high-speed homogenizer to form a dispersed fiber suspension. 0.032 g of TEMPO (0.1 mmol / g), 2.0 g of NaBr (1 mmol / g), and 3.55 mL of NaClO (6 mmol / g) were added to the suspension. The pH was adjusted to 11 using a 1 mol / L NaOH solution. The reaction was stirred at room temperature for 24 hours, during which time the pH of the reaction system was maintained at 11 using a NaOH solution. After completion, the reaction was terminated by adding 10 mL of anhydrous ethanol. The product was centrifuged and dialyzed against deionized water for 5 days to a pH of 7. The resulting TOBC was freeze-dried and stored sealed at 4°C.

[0092] Preparation of TOBC-DA: 2.00 g of TOBC was dispersed in 600 mL of deionized water. 2.00 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 2.00 g of N-hydroxysuccinimide (NHS) were added to activate the carboxyl groups for 1 hour. Then, 1.90 g of DA was added. The mixture was stirred at room temperature for 48 hours and then centrifuged at 8000 rpm for 10 minutes. The supernatant was removed, the mixture was dialyzed against deionized water for 5 days, and the resulting TOBC-DA was freeze-dried and stored at 4°C in the dark.

[0093] Preparation of TOBC-DA / Cur solution: 0.2 g of TOBC-DA was added to 20 mL of deionized water (1 wt%) and fully dissolved by a high-speed homogenizer at 8000 rpm. 0.04 g of Cur was added and the mixture was stirred at room temperature for 24 h (TOBC-DA:Cur = 5:1).

[0094] Preparation of TOBC-DA / PVA / Cur solution: 2 g of PVA was added to 50 mL of deionized water (4 wt %), sealed and stirred at 90 ° C for 4 h until it was completely dissolved. Then, 20 mL of TOBC-DA / Cur solution and 5 mL of 4 wt % PVA solution were mixed thoroughly and stirred at room temperature for 4 h to prepare a TOBC-DA / PVA / Cur solution (TOBC-DA:PVA:Cur = 5:5:1).

[0095] Preparation of TOBC-DA / PVA / Cur coating: Prepare a 12mm diameter circular PETG membrane, rinse it twice by immersion in 75% ethanol solution, ultrasonically clean it for 15 minutes, and then rinse it twice with deionized water. Place the cleaned PETG circular membrane in a centrifuge, and add 400μl of TOBC-DA solution, TOBC-DA / Cur solution, and TOBC-DA / PVA / Cur solution to the surface until the membrane is fully covered. Centrifuge at 5000rpm for 5 minutes and dry overnight in the dark.

[0096] Example 3

[0097] This embodiment provides an invisible braces coating that inhibits periodontal pathogens, and the preparation method thereof is as follows:

[0098] Preparation of TOCN: 2.0 g of plant cellulose nanofibers (CN) were added to 200 mL of deionized water and treated with a high-speed homogenizer to form a dispersed fiber suspension. 0.032 g of TEMPO (0.1 mmol / g), 2.0 g of NaBr (1 mmol / g), and 3.55 mL of NaClO (6 mmol / g) were added to the suspension, and the pH was adjusted to 10 using a KOH solution (0.5 mol / L). The reaction was stirred at room temperature for 16 h, during which the pH of the reaction system was maintained at 10 using a KOH solution. After completion of the reaction, 10 mL of anhydrous ethanol was added to terminate the reaction. The product was centrifuged and dialyzed against deionized water for 3 days to a pH of 7. The product, TOCN, was freeze-dried and stored at 4°C after sealing.

[0099] Preparation of TOCN-DA: TOCN (1.00 g) was dispersed in 300 mL of deionized water. 1.00 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.00 g of N-hydroxysuccinimide ester (NHS) were added to activate the carboxyl groups for 2 h. Then, 0.95 g of DA was added. The mixture was stirred at room temperature for 24 h and then centrifuged at 5000 rpm for 10 min. The supernatant was removed, the mixture was dialyzed against deionized water for 3 d, and freeze-dried to obtain TOCN-DA, which was stored at 4°C in the dark.

[0100] Preparation of TOCN-DA / Cur solution: 0.1 g of TOCN-DA was added to 20 mL of deionized water (0.5 wt %) and fully dissolved by a high-speed homogenizer at 7000 rpm. 0.02 g of Cur was added and the mixture was stirred at room temperature for 24 h (TOCN-DA:Cur = 5:1).

[0101] Preparation of TOCN-DA / PVA / Cur solution: 1 g of PVA was added to 50 mL of deionized water (2 wt %), sealed and stirred at 90 ° C for 2 h until it was completely dissolved, then 20 mL of TOCN-DA / Cur solution and 5 mL of 2 wt % PVA solution were taken and mixed thoroughly, and stirred at room temperature for 2 h to prepare TOCN-DA / PVA / Cur solution (TOCN-DA:PVA:Cur=5:5:1).

[0102] Preparation of TOCN-DA / PVA / Cur coating: 10 mm diameter circular PETG membranes were prepared and rinsed twice by immersion in 75% ethanol, ultrasonically cleaned for 15 minutes, and then rinsed twice with deionized water. The cleaned PETG membranes were placed in a centrifuge, and 200 μl of TOCN-DA solution, TOCN-DA / Cur solution, and TOCN-DA / PVA / Cur solution were added dropwise to thoroughly cover the membranes. The membranes were then centrifuged at 3000 rpm for 3 minutes and dried overnight in the dark.

[0103] Example 4

[0104] This embodiment provides an invisible braces coating that inhibits periodontal pathogens, and the preparation method thereof is as follows:

[0105] Preparation of TOBC: 2.0 g of bacterial cellulose nanofibers (BC) were added to 200 mL of deionized water and treated with a high-speed homogenizer to form a dispersed fiber suspension. 0.032 g of TEMPO (0.1 mmol / g), 2.0 g of NaBr (1 mmol / g), and 3.55 mL of NaClO (6 mmol / g) were added to the suspension. The pH was adjusted to 10 using a 0.5 mol / L NaOH solution. The reaction was stirred at room temperature for 16 hours, during which time the pH of the reaction system was maintained at 10 using a NaOH solution. After completion, the reaction was terminated by adding 10 mL of anhydrous ethanol. The product was centrifuged and dialyzed against deionized water for 3 days to a pH of 7. The resulting TOBC was freeze-dried and stored at 4°C after sealing.

[0106] Preparation of TOBC-DA: TOBC (1.00 g) was dispersed in 300 mL of deionized water. 1.00 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1.00 g of N-hydroxysuccinimide (NHS) were added to activate the carboxyl groups for 0.5 h. 0.95 g of DA was then added. The mixture was stirred at room temperature for 24 h and then centrifuged at 5000 rpm for 10 min. The supernatant was removed, the suspension was dialyzed against deionized water for 3 days, and the resulting TOBC-DA was freeze-dried and stored at 4°C in the dark.

[0107] Preparation of TOBC-DA / Cur solution: 0.1 g of TOBC-DA was added to 20 mL of deionized water (0.5 wt %) and fully dissolved by high-speed homogenizer at 7000 rpm. 0.02 g of Cur was added and the mixture was stirred at room temperature for 24 h (TOBC-DA:Cur = 5:1).

[0108] Preparation of TOBC-DA / PVA / Cur solution: 1 g of PVA was added to 50 mL of deionized water (2 wt %), sealed and stirred at 90°C for 2 h until it was completely dissolved. Then, 20 mL of TOBC-DA / Cur solution and 5 mL of 2 wt % PVA solution were mixed thoroughly and stirred at room temperature for 2 h to prepare a TOBC-DA / PVA / Cur solution (TOBC-DA:PVA:Cur = 5:5:1).

[0109] Preparation of TOBC-DA / PVA / Cur coating: 10 mm diameter circular PETG membranes were prepared and rinsed twice in 75% ethanol solution by immersion, ultrasonically cleaned for 15 minutes, and then rinsed twice with deionized water. The cleaned PETG membranes were then immersed in 10 mL of TOBC-DA solution, TOBC-DA / Cur solution, and TOBC-DA / PVA / Cur solution for 48 hours, then removed and dried overnight in the dark.

[0110] Example 5

[0111] Physical and chemical properties testing of TOBC-DA / PVA / Cur coating

[0112] The TOBC-DA / PVA / Cur coating prepared in Example 1 was subjected to Fourier transform infrared spectroscopy (FT-IR) acquisition using a Bruker Vertex 70v instrument. The scanning wavenumber range was 500-4000 / cm, and 32 scans were accumulated at a resolution of 4 / cm. The acquired data were processed using Omnic, and the results were as follows: Figure 2 As shown, it shows that the TOBC-DA / PVA / Cur coating was successfully synthesized.

[0113] Then, the PETG films with no coating (Bare), TOBC-DA / Cur coating, and TOBC-DA / PVA / Cur coating in Example 1 were electroplated with platinum to improve conductivity. The surface morphology of the coating was observed using a scanning electron microscope (SEMTESCAN MIRA3) at an operating voltage of 10 kV and a resolution of 5 nm. The results are as follows: Figure 3 and Figure 4 The characterization results show that the TOBC-DA / PVA / Cur coating has a more uniform surface than the TOBC-DA / Cur coating. The thickness of the coating does not exceed 5 μm. The thickness of the PETG film used in clinical invisible braces is generally between 0.7 and 1.0 mm. Therefore, the thickness of the coating has little effect on the overall thickness of the invisible braces.

[0114] Example 6

[0115] Hydrophilicity and hydrophobicity testing of TOBC-DA / PVA / Cur coating

[0116] The contact angles of the PETG films without coating, with TOBC-DA coating, and with TOBC-DA / PVA / Cur coating in Example 1 were measured using a contact angle meter. Three parallel samples were measured in each group. The results are shown in Figure 2. Figure 5 As shown, the contact angle of the uncoated PETG membrane surface is 82.43±1.54°, the contact angle of the TOBC-DA coated PETG membrane surface is 81.03±0.68°, and the contact angle of the TOBC-DA / PVA / Cur coated PETG membrane surface is 19.35±5.04°, with statistically significant differences. The characterization results show that the uncoated PETG membrane and the TOBC-DA coated PETG membrane are hydrophobic, while the TOBC-DA / PVA / Cur coated PETG membrane is significantly hydrophilic.

[0117] Example 7

[0118] Cytotoxicity assay of TOBC-DA / PVA / Cur coating

[0119] The cytotoxicity of the TOBC-DA / PVA / Cur coating was determined using the CCK8 method, and the cytotoxicity of the PETG membranes without coating, with TOBC-DA coating, and with TOBC-DA / PVA / Cur coating in Example 1 was tested.

[0120] The specific operation steps are as follows: add a density of 2*10 4 hPDLC cells were cultured at a density of 100 cells / mL in αMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% dual-antibody at 37°C and 5% CO2 for 6 hours. PETG membranes (uncoated, TOBC-DA-coated, and TOBC-DA / PVA / Cur-coated) that had been UV-sterilized for 2 hours were then placed in 12-well plates and incubated at 37°C and 5% CO2 for 1 and 3 days, respectively. After incubation, the membranes were removed, the culture medium discarded, and 1 mL of fresh αMEM medium and 100 μL of CCK8 reagent were added. The cells were incubated at 37°C in the dark for 4 hours. 100 μL of the solution was then aliquoted from each well of a 96-well plate and the absorbance at 450 nm was measured using a microplate reader. Triplicate data were measured and plotted.

[0121] The results are as follows Figure 6 As shown in the data, there was no statistically significant difference in the co-cultured 1d and 3d between the PETG membranes constructed with TOBC-DA coating and TOBC-DA / PVA / Cur coating and the uncoated PETG membrane, indicating that both TOBC-DA coating and TOBC-DA / PVA / Cur coating have good biocompatibility, which is more conducive to the application of this coating material in invisible orthodontics.

[0122] Example 8

[0123] Performance testing of TOBC-DA / PVA / Cur coating in inhibiting the adhesion and proliferation of Pg bacteria

[0124] The performance of the TOBC-DA / PVA / Cur coating in inhibiting the adhesion and proliferation of Pg bacteria was detected by counting the number of colonies, staining live and dead bacteria, and observing the morphology of Pg bacteria. The performance of the PETG membranes with no coating, TOBC-DA coating, and TOBC-DA / PVA / Cur coating in Example 1 in inhibiting the adhesion and proliferation of Pg bacteria was tested.

[0125] The specific steps are:

[0126] 1) Prepare BHI liquid medium: Weigh 7.4 g of bovine brain histamine broth (BHI powder) and dilute to 200 mL with deionized water. Sterilize by autoclaving at 121°C for 20 min. After cooling, add 2 mL of a hemin-vitamin K1 mixture in a laminar flow hood, mix thoroughly, and store at 4°C.

[0127] 2) Prepare BHI solid medium: Weigh 7.4 g of BHI powder and 4 g of agar, dilute to 200 mL with deionized water, and sterilize at 122°C and 15 MPa for 20 min. After cooling until cool to the touch, add 2 mL of hemin-vitamin K1 mixture and 10 mL of sheep blood in a laminar flow hood. Mix thoroughly, then add approximately 20 mL per dish to 90 mm bacterial culture plates. Cool to solidify, and store at 4°C.

[0128] 3) Place Pg bacteria in BHI liquid medium, resuscitate under anaerobic conditions at 37°C for 24 hours, and dilute to a concentration of 106 CFU / mL. Add PETG membranes (uncoated, TOBC-DA-coated, or TOBC-DA / PVA / Cur-coated) that have been UV-sterilized for 2 hours, along with 1 mL of Pg bacterial solution, to a 24-well plate. Incubate under anaerobic conditions at 37°C for 24 hours, and then dilute to 102 CFU / mL. Apply 0.1 mL of the bacterial solution to BHI solid medium, spread two circles, and incubate under anaerobic conditions at 37°C for 5-7 days. Observe and count colonies. Perform three parallel measurements and plot the data.

[0129] The results are as follows Figure 7 As shown in the figure, the number of Pg bacterial colonies in the culture medium of PETG membranes with TOBC-DA coating and TOBC-DA / PVA / Cur coating was significantly reduced compared with that without coating, and the difference was statistically significant.

[0130] 4) Place Pg bacteria in BHI liquid medium, resuscitate under anaerobic conditions at 37°C for 24 hours, and dilute to a concentration of 107 CFU / mL. Add uncoated, TOBC-DA-coated, and TOBC-DA / PVA / Cur-coated PETG membranes sterilized by ultraviolet light for 2 hours, and 1 mL of Pg bacterial solution to a 24-well plate and incubate under anaerobic conditions at 37°C for 24 hours. Discard the bacterial solution, fix with 4% paraformaldehyde for 1 hour, then wash three times with 0.9% NaCl solution, add 500 μL of 0.9% NaCl solution and 1.5 μL of bacterial viability detection reagent, and let it stand for 15 minutes. Remove the membrane and place it in a confocal dish for observation using a confocal microscope (STELLARIS 5).

[0131] The results are as follows Figure 8As shown in the figure, the number of live Pg bacteria (green fluorescence) on the surface of PETG membranes with TOBC-DA coating and TOBC-DA / PVA / Cur coating is less than that of the uncoated one. At the same time, the number of dead Pg bacteria (red fluorescence) on the surface of PETG membranes with TOBC-DA / PVA / Cur coating increases, and the ratio of live to dead bacteria decreases.

[0132] 5) Pg bacteria were placed in BHI liquid medium and revived under anaerobic conditions at 37°C for 24 hours, then diluted to a concentration of 106 CFU / mL. Uncoated, TOBC-DA-coated, and TOBC-DA / PVA / Cur-coated PETG membranes, sterilized by UV for 2 hours, along with 1 mL of Pg bacterial solution, were added to a 24-well plate and incubated under anaerobic conditions at 37°C for 24 hours. The bacterial solution was discarded, and the membranes were fixed with 4% paraformaldehyde for 1 hour and then washed three times with 0.9% NaCl solution. Subsequently, dehydration was performed using a gradient of ethanol concentrations (30%, 50%, 70%, 85%, 95%, and 100%), with each concentration treated for 15 minutes. The membranes were electroplated with platinum to increase conductivity. The morphology of Pg bacteria was observed using a scanning electron microscope (SEMTESCAN MIRA3) at a voltage of 10 kV and a resolution of 5 nm.

[0133] The results are as follows Figure 9 As shown, the integrity of the Pg cell membrane on the surface of the PETG film coated with TOBC-DA / PVA / Cur was destroyed. These results indicate that the construction of a TOBC-DA coating on the surface of the invisible aligner base material PETG can reduce the adhesion of Pg bacteria, while the construction of a TOBC-DA / PVA / Cur coating can not only reduce the adhesion of Pg bacteria but also inhibit the proliferation of Pg bacteria by destroying the integrity of the Pg cell membrane.

[0134] In summary, the present invention provides an invisible braces coating for inhibiting periodontal pathogens, and a preparation method and application thereof. The preparation method of the invisible braces coating includes the following steps: cellulose nanofibers are subjected to a 2,2,6,6-tetramethylpiperidine-1-oxyl oxidation reaction, followed by an amidation reaction with dopamine, and then mixed with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution; finally, the solution is subjected to a film-forming treatment to obtain an invisible braces coating for inhibiting periodontal pathogens. The present invention uses TOCNF as a scaffold, forms an adhesive coating TOCNF-DA through an amidation reaction with DA, adds PVA to increase the dispersibility of TOCNF-DA, and loads a certain amount of Cur, thereby constructing a TOCNF-DA / PVA / Cur coating on the surface of the invisible orthodontic appliance base material; the coating has good biosafety and can effectively inhibit the adhesion and proliferation of periodontal pathogens Pg bacteria, and is low in cost and simple in manufacturing process. No toxic reagents are involved in the production process, and it can be widely used in orthodontic correction, periodontal disease treatment and other related oral disease treatments, as well as the treatment of systemic diseases related to periodontal pathogens.

[0135] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing an invisible braces coating that inhibits periodontal pathogens, characterized in that: Including steps: Cellulose nanofibers were oxidized using 2,2,6,6-tetramethylpiperidin-1-oxyl to obtain TOCNF; The TOCNF is mixed with dopamine, and subjected to an amidation reaction to obtain TOCNF-DA; The TOCNF-DA was mixed with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution; After the TOCNF-DA / PVA / Cur solution is formed into a film and dried, the invisible brace coating is obtained.

2. The method for preparing an invisible braces coating for inhibiting periodontal pathogens according to claim 1, characterized in that: The cellulose nanofibers are plant-derived cellulose nanofibers or bacteria-derived cellulose nanofibers; and the curcumin is natural curcumin or chemically modified curcumin.

3. The method for preparing an invisible braces coating for inhibiting periodontal pathogens according to claim 1, characterized in that: The step of oxidizing cellulose nanofibers using 2,2,6,6-tetramethylpiperidin-1-oxyl to obtain TOCNF comprises: dissolving cellulose nanofibers in deionized water to obtain a cellulose nanofiber solution; adding 2,2,6,6-tetramethylpiperidin-1-oxyl, sodium bromide, and sodium hypochlorite to the cellulose nanofiber solution to obtain a reaction system; The pH of the reaction system is maintained between 9 and 11 by using an alkali, and TOCNF is obtained by stirring, centrifuging, dialysis, and freeze-drying.

4. The method for preparing an invisible braces coating for inhibiting periodontal pathogens according to claim 3, characterized in that: The alkali is selected from one or more of sodium hydroxide, potassium hydroxide and calcium hydroxide.

5. The method for preparing an invisible braces coating for inhibiting periodontal pathogens according to claim 1, characterized in that: The step of mixing the TOCNF-DA with curcumin and polyvinyl alcohol to obtain a TOCNF-DA / PVA / Cur solution comprises: Dissolving the TOCNF-DA in deionized water to obtain a TOCNF-DA solution; The TOCNF-DA solution was mixed with curcumin and stirred to obtain a TOCNF-DA / Cur solution; Mixing polyvinyl alcohol with deionized water to obtain a PVA solution; The TOCNF-DA / Cur solution and the PVA solution were mixed and stirred to obtain a TOCNF-DA / PVA / Cur solution.

6. The method for preparing an invisible braces coating for inhibiting periodontal pathogens according to claim 5, characterized in that: The concentration of the TOCNF-DA solution is 0.1 wt%-10 wt%.

7. The method for preparing an invisible braces coating for inhibiting periodontal pathogens according to claim 1, characterized in that: The weight ratio of the TOCNF-DA to the curcumin is 1:1-10:

1.

8. The method for preparing an invisible braces coating for inhibiting periodontal pathogens according to claim 1, characterized in that: The weight ratio of the TOCNF-DA to the polyvinyl alcohol is 1:4-10:

1.

9. An invisible braces coating for inhibiting periodontal pathogens, characterized in that: The invisible braces coating for inhibiting periodontal pathogens is prepared using the method for preparing the invisible braces coating for inhibiting periodontal pathogens as described in any one of claims 1 to 8.

10. Use of the invisible braces coating for inhibiting periodontal pathogens as claimed in claim 9 in orthodontic treatment.

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