Tooth soft dirt model and construction method and application thereof

Abstract

The invention discloses a tooth soft dirt model and a preparation method and application thereof, the tooth soft dirt model comprises a substrate and a soft dirt simulant attached to the substrate, and the soft dirt simulant comprises saliva sediment, plaque sediment and food residue sediment. By using the evaluation model provided by the invention, the oral product can be evaluated more conveniently, scientifically and objectively.

Description

Technical Field

[0001] The present application relates to the technical field of oral cleaning, and in particular to a dental plaque model and a construction method and application thereof. Background Art

[0002] Soft plaque, also known as materia alba, is a soft and sticky deposit attached to the tooth surface, restoration surface, tartar and gingival margin. Soft plaque is mainly composed of microorganisms, desquamated epithelial cells and white blood cells, mucin in saliva, proteins and lipids, and a small amount of food debris. It is mostly attached to the neck of the tooth and is white, light yellow or light gray. It is visible to the naked eye, soft and easy to remove by brushing or rinsing. Soft plaque is one of the important factors leading to many oral diseases. For example, the acidic substances produced by the metabolism of bacteria in soft plaque will erode tooth enamel and form caries; soft plaque will irritate the gums and cause inflammation, leading to gingivitis, periodontitis, etc.; sulfides and other substances produced by bacterial metabolism in soft plaque will cause bad breath.

[0003] With the public's attention to oral health and the development of the oral care industry, a large number of oral care products have appeared on the market. At present, conventional products for cleaning the oral cavity include toothpaste, toothbrushes, dental floss, mouthwash, etc. With the development of science and technology, some oral electronic products have also been developed, such as electric toothbrushes, water flossers, etc., to improve the oral cleaning effect. In view of the impact of soft plaque on oral health, it is also very important to evaluate the cleaning effect of oral care products on soft plaque. At present, the evaluation of the cleaning effect of oral care products on soft plaque is mainly carried out through clinical trials. However, due to the individual differences of the subject population (including differences in dental care conditions and oral cleaning habits, etc.), if there is no sufficient sample size of volunteers participating, the results obtained are difficult to quantify and generate statistical significance. In terms of product testing, this traditional method is affected by large human factors, the test process lacks consistency, and it is difficult to replicate or compare horizontally in different batches of product tests, making it difficult to expand the scale of repeated testing in product efficacy verification, comparison, and optimization, and to have a positive role in promoting product research and development.

[0004] Therefore, there is an urgent need for a dental plaque model for in vitro evaluation and testing of oral care products to improve the scientific nature of the test results. Summary of the invention

[0005] The purpose of the present application is to provide a dental plaque model that can be used to evaluate and test oral care products in vitro, as well as a construction method and application thereof.

[0006] This application adopts the following technical solutions:

[0007] The first aspect of the present application discloses a dental plaque model, comprising: a substrate and a plaque simulant attached to the substrate, wherein the plaque simulant includes saliva deposits, plaque deposits and food residue deposits. It should be noted that the dental plaque model of the present application can simulate the physical and chemical properties of oral plaque, and is very similar to real plaque in terms of texture, viscosity, color, etc., providing a testing environment closer to the actual situation for the evaluation of oral care products. Compared with the clinical trial method for testing oral care products, the dental plaque model of the present application has the advantages of small batch differences, easy quantification of indicators, and more scientific and objective.

[0008] In one implementation of the present application, the salivary deposits and plaque deposits are film-shaped, and the salivary deposits, the plaque deposits and the food residue deposits are cross-linked. It should be noted that the salivary deposits, plaque deposits and food residue deposits in the present application are interrelated as a whole. Specifically, salivary deposits provide conditions for the formation of plaque deposits: after the formation of salivary deposits, bacteria selectively adhere to salivary deposits, promoting the formation of plaques; plaque deposits affect the composition and structure of salivary deposits and food residue deposits: bacteria use carbon sources, nitrogen sources and inorganic ions in salivary deposits and food residue deposits for metabolism, produce acidic substances and other metabolites, affect the composition and structure of salivary deposits and food residue deposits, and further promote the formation and stability of plaque deposits; food residue deposits affect the properties of salivary deposits and plaque deposits: proteins and carbohydrates in food residues can affect the composition and properties of salivary deposits, and affect the formation and growth of plaques.

[0009] In one implementation of the present application, the saliva deposit is formed by contacting the substrate with artificial saliva; the plaque deposit is formed by placing the substrate formed with the saliva deposit in a culture medium containing oral bacteria for culturing; the food residue deposit is formed by placing the substrate formed with the plaque deposit in a culture medium containing food residue for culturing.

[0010] In one implementation of the present application, the pH value of the artificial saliva is 6-6.1, and the composition of the artificial saliva is: 0.5-1.5 g / L NaCl, 0.1-0.3 g / L KCl, 0.1-0.3 g / L CaCl2, 0.1-0.3 g / L Na2HPO4, 0.1-0.5 g / L urea, and 0.01-0.05 g / L Na2S.

[0011] In one implementation of the present application, the culture medium containing oral bacteria is a BHI culture medium containing Streptococcus mutans.

[0012] In one implementation of the present application, the culture medium containing food residues is a BHI culture medium including 1-5 g / L starch, 1-10 g / L soybean powder, 1-10 g / L sugar, 1-5 g / L skim milk powder, and 1-20 g / L dietary fiber.

[0013] In one implementation of the present application, the dental plaque model further comprises a dye combined with the plaque simulant.

[0014] In one implementation of the present application, the dye is fuchsin or crystal violet.

[0015] The second aspect of the present application discloses a method for constructing a dental plaque model, comprising constructing a salivary deposit: placing a substrate in artificial saliva to construct the salivary deposit; constructing a plaque deposit: placing the substrate with the salivary deposit in a culture medium containing oral bacteria for culturing to construct the plaque deposit; constructing a food residue deposit: placing the substrate with the plaque deposit in a culture medium containing food residue for culturing to construct the food residue deposit, thereby obtaining the dental plaque model.

[0016] In one implementation of the present application, the step of constructing the saliva deposit includes: placing the substrate in artificial saliva at 35° C. to 38° C. for 12 h to 36 h.

[0017] In one implementation of the present application, the step of constructing the plaque deposit includes: placing the substrate constructed with the saliva deposit in a culture medium containing oral bacteria and culturing for 12 hours to 72 hours under anaerobic conditions at 35° C. to 38° C.

[0018] In one implementation of the present application, the step of constructing the food residue deposit includes: placing the substrate with the bacterial plaque deposit in a food residue mixed culture medium and culturing it for 12 hours to 72 hours under anaerobic conditions at 35° C. to 38° C.

[0019] In one implementation of the present application, after constructing the food residue deposit, the steps of fixation and staining are also included.

[0020] In one implementation of the present application, the fixing and staining steps include: drying at 2°C to 6°C for 15 to 60 minutes, staining with a water-insoluble dye for 0.5 to 1.5 hours after drying, and storing at 2°C to 6°C after washing with phosphate buffer.

[0021] The third aspect of the present application provides a method for evaluating the cleaning efficacy of an oral care product using a dental plaque model, comprising: treating the dental plaque model with an oral care product, and evaluating the cleaning efficacy of the oral care product by qualitatively or quantitatively analyzing the plaque simulant in the dental plaque model.

[0022] In one implementation of the present application, quantifying the plaque simulant in the dental plaque model includes at least one of the following methods: detecting the content of oral bacteria in the dental plaque model by a bacterial culture method or a genetic detection method; obtaining the residual amount of the plaque simulant by performing image analysis on the plaque simulant in the dental plaque model.

[0023] In one implementation of the present application, quantifying the plaque simulant in the dental plaque model includes: collecting images through a microscope and analyzing to obtain the area and optical density of the plaque simulant in the dental plaque model, and calculating by the following formula:

[0024]

[0025] Where Area NC is the total area of ​​the soft scale simulants in the control group, MOD NC is the average optical density of the soft scale simulant in the control group, Area 样品组 is the total area of ​​soft scale simulants in the sample group, MOD 样品组 is the average optical density of the soft plaque simulation of the sample group, the control group refers to the dental plaque model not treated with the oral care product, and the sample group refers to the dental plaque model treated with the oral care product. Thus, a scientific and objective method for calculating the cleaning rate can be provided.

[0026] The beneficial effects of this application are:

[0027] The dental plaque model of the present application can simulate the physical and chemical properties of oral plaque, and is very similar to real plaque in terms of texture, viscosity, color, etc., providing a testing environment closer to the actual situation for the evaluation of oral care products. Compared with the clinical trial method for testing oral care products, the dental plaque model of the present application has the advantages of small batch differences, easy quantification of indicators, and more scientific and objective. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a test result diagram involved in Example 2 of the present application.

[0029] Figure 2 This is a test result diagram involved in Example 3 of the present application.

[0030] Figure 3This is a test result diagram involved in Example 4 of the present application. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and the related operations can be fully understood according to the description in the specification and the general technical knowledge in the art.

[0032] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0033] In the field of oral medicine, soft plaque is one of the important factors leading to a variety of oral diseases. At present, the research on soft plaque mainly focuses on its composition analysis, formation mechanism and impact on oral health. However, the existing research methods have certain limitations, such as the lack of models that can accurately simulate the formation process and characteristics of soft plaque.

[0034] With the continuous advancement of science and technology, the research on oral diseases is also deepening. In order to better understand the formation and development process of soft deposits and explore effective prevention and treatment methods, it is urgent to build a soft deposit model. The development of soft deposit models and their application in the evaluation and application of oral care products mainly include the following aspects:

[0035] 1) Accurately evaluate the cleaning effect of oral care products: Simulate the real situation: Soft scale is a sticky substance formed by a mixture of food residues, bacteria, saliva, etc., and its formation process and distribution in the oral cavity have certain characteristics. By establishing a soft scale model, the generation and accumulation of soft scale in the oral cavity and its attachment to the tooth surface and tooth gaps can be simulated as realistically as possible. In this way, when evaluating oral care products, the product's ability to remove soft scale can be more accurately reflected, such as whether toothpaste can effectively remove soft scale on the tooth surface, whether dental floss can clean soft scale in tooth gaps, etc.

[0036] 2) Quantitative cleaning indicators: The soft scale model can be used to quantitatively evaluate the cleaning effect of oral care products. For example, by comparing the changes in the weight, area, thickness and other indicators of the soft scale on the model before and after using the product, the cleaning efficiency of the product can be determined, providing specific data support for the cleaning performance of the product, which is convenient for comparison and screening between different products.

[0037] 3) Study the preventive effect of oral care products on the formation of soft scale: Test the durability of product efficacy: Oral care products should not only be able to remove existing soft scale, but also have the function of preventing the formation of soft scale. The soft scale model can simulate the long-term changes in the oral environment and observe the inhibitory effect of oral care products on the formation of soft scale over a certain period of time. For example, test whether the formation rate of soft scale on the model slows down after a certain period of time after a certain mouthwash is used, so as to evaluate whether the product's effect of preventing the formation of soft scale is durable.

[0038] 4) Optimize product formula and design: Based on the experimental results of the soft plaque model, we can analyze the effects of oral care products with different ingredients on the formation of soft plaque, and then optimize the product formula and design. For example, if we find that certain additives can effectively prevent bacteria from attaching to the tooth surface to form soft plaque, we can apply them to the product to improve the product's ability to prevent soft plaque.

[0039] 5) Provide a basis for the development of oral care products and screen effective ingredients: When developing new oral care products, the soft scale model can be used as a tool to quickly screen effective ingredients. By testing the effects of various ingredients on soft scale on the model, it is possible to quickly determine which ingredients have a better effect in cleaning or preventing soft scale, thereby providing direction and basis for product development and reducing the blindness and cost of research and development.

[0040] 6) Verify product principle: For some oral care products based on new principles or technologies, the soft deposit model can be used to verify the feasibility of its action principle. For example, some new toothpastes claim to have a special cleaning mechanism. The soft deposit model can simulate the oral environment to verify whether it can remove soft deposits in the expected way, ensuring the theoretical effectiveness of the product.

[0041] The dental plaque model provided in this application and its construction method and application have the following advantages:

[0042] 1) High Simulation: The soft scale model can accurately simulate the physical and chemical properties of soft scale in the oral cavity. Through in-depth research on the composition of soft scale, the use of specific materials and preparation methods makes the model very similar to real soft scale in terms of texture, viscosity, color, etc., providing a test environment closer to the actual situation for the evaluation of oral care products. Considering the complexity of the oral environment, the model also simulates the formation characteristics of soft scale under different oral environments.

[0043] 2) Adjustability: The parameters of the soft deposit model can be adjusted according to different research needs. For example, the type and number of bacteria in the model, the composition of food residues, the characteristics of saliva, etc. can be changed to simulate the oral conditions of different populations or specific oral disease states. This can meet the diverse evaluation needs of oral care products and provide a wider range of application scenarios for product research and development and improvement. The formation speed and thickness of soft deposits can be controlled to facilitate product testing at different stages. By adjusting the conditions of the model, the formation process of soft deposits can be accelerated or slowed down, thereby studying the effects of oral care products under different degrees of soft deposit accumulation.

[0044] 3) Multi-dimensional evaluation system: A comprehensive oral care product evaluation system was established using the soft scale model. It can not only evaluate the product's ability to remove soft scale, but also examine the product's preventive effect on the formation of soft scale, its impact on the oral microbial community, and its safety on oral tissues. Through a comprehensive analysis of different indicators, a more objective and accurate evaluation result is provided for the performance of oral care products. For example, the weight reduction of soft scale, changes in the number of bacteria, etc., can be combined to comprehensively measure the efficacy of a product.

[0045] 4) Dynamic evaluation method: The dynamic evaluation method is innovatively adopted to simulate the effect of oral care products in actual use. By continuously observing the changes in the soft scale model before and after the use of the product over a certain period of time, the long-term effect and stability of the product can be understood. For example, for mouthwash products, the growth of bacteria and the accumulation rate of soft scale in the soft scale model can be measured at different time points to evaluate the continuous antibacterial and anti-scale effects of the mouthwash.

[0046] 5) Provide guidance for personalized customization and product optimization: Based on the analysis results of the soft deposit model, personalized oral care plans can be customized for different groups of people. According to the individual oral condition differences reflected in the model, such as the tendency of soft deposit formation, the characteristics of the oral microbial community, etc., a suitable oral care product combination and usage method are recommended. For example, for people who are prone to soft deposits, toothpaste and dental floss with stronger cleaning power and anti-scaling effects can be recommended, and guidance on correct brushing and flossing can be provided to improve the effectiveness of oral care. The soft deposit model provides a specific direction and basis for the optimization of oral care products. By analyzing the mechanism of the interaction between soft deposits and products in the model, the shortcomings of the product can be discovered and targeted improvements can be made. For example, if a toothpaste is found to be ineffective in removing a specific type of soft deposit, the formula of the toothpaste can be adjusted to increase ingredients that have a targeted effect on that type of soft deposit to improve the performance of the product.

[0047] In summary, the dental plaque model of the present application is accurate, reliable, efficient and precise in the in vitro efficacy evaluation of oral products. It has many applications, which will help promote the technological progress of oral electronic products, improve the quality and efficiency in the field of oral health, and play a greater role in the future field of oral cleaning.

[0048] The present application relates to a dental plaque model, a method for constructing a dental plaque model, an application of the dental plaque model, and a method for evaluating the cleaning efficacy of an oral care product using the dental plaque model.

[0049] In a specific embodiment, the dental plaque model includes a substrate and a plaque simulant attached to the substrate.

[0050] In a specific embodiment, the substrate may include at least one of an animal enamel block and a tooth-like material tooth film. The animal enamel block may refer to an enamel block sample made of human or other animal teeth, thereby being able to simulate the distribution of dental plaque on the surface of real enamel. The tooth-like material tooth film may refer to a tooth film model made of tooth-like materials (hydroxyapatite, etc.). There are many similarities in the composition and physicochemical properties between hydroxyapatite (HA) and teeth. Hydroxyapatite is stable and biocompatible, has low solubility, is easy to prepare uniformly, and can be used for various applications, such as evaluation of tooth demineralization and remineralization processes, reduction of tooth sensitivity, oral biofilm control, oral tartar prevention, and tooth whitening.

[0051] In a specific embodiment, the substrate may be a full-mouth tooth model, including at least one of a full-mouth animal enamel model and a full-mouth tooth model of a tooth-like material. A full-mouth animal enamel model may refer to the collected teeth of a human or other animal (including incisors, canines, and molars) that are cleaned and then inlaid on a false gum material (which can be made by 3D printing, and the material is resin) according to the actual tooth distribution to simulate the actual tooth distribution and obtain a full-mouth tooth model. A full-mouth tooth model of a tooth-like material may refer to teeth made of tooth-like materials (hydroxyapatite, etc.) through tooth molds (which can be downloaded from a tooth model database to simulate the actual tooth arrangement), and the gum material can be made of resin by 3D printing, and the teeth of the tooth-like material are inlaid into the gums to obtain a full-mouth tooth model of a tooth-like material that simulates the actual tooth distribution. It should be noted that the use of a full-mouth animal enamel model or a full-mouth tooth model made of tooth-like materials for testing can take into account the formation of dental plaque under the actual arrangement of teeth, the gaps between teeth, the depth of the gingival margin, the curvature and grooves of the teeth themselves, etc., and can simulate the distribution of dental plaque under actual conditions; in addition, the use of a full-mouth tooth model for wear testing can also simulate the wear of teeth caused by different brushing methods (including but not limited to the Bass brushing method, the Roche brushing method, the circular brushing method, and the horizontal vibrating brushing method); in addition, different full-mouth tooth models can be customized according to different populations (such as children, teenagers, adults, the elderly, etc.) to obtain dental plaque conditions for different populations.

[0052] In a specific embodiment, the soft deposit simulant may include saliva deposits, plaque deposits, and food residue deposits.

[0053] In a specific embodiment, the saliva deposits and the bacterial plaque deposits are in a film shape. It should be noted that the saliva deposits and the bacterial plaque deposits are in a film shape here means that the overall shape of the saliva deposits and the bacterial plaque deposits is generally in a film shape.

[0054] In a specific embodiment, salivary deposits, plaque deposits and food residue deposits are cross-linked with each other. It should be noted that the salivary deposits, plaque deposits and food residue deposits in the present application are interrelated as a whole. Specifically, salivary deposits provide conditions for the formation of plaque deposits: after the formation of salivary deposits, bacteria selectively adhere to salivary deposits, promoting the formation of plaques; plaque deposits affect the composition and structure of salivary deposits and food residue deposits: bacteria use carbon sources, nitrogen sources and inorganic ions in salivary deposits and food residue deposits for metabolism, produce acidic substances and other metabolites, affect the composition and structure of salivary deposits and food residue deposits, and further promote the formation and stability of plaque deposits; food residue deposits affect the properties of salivary deposits and plaque deposits: proteins and carbohydrates in food residues can affect the composition and properties of salivary deposits, and affect the formation and growth of plaques.

[0055] In a specific embodiment, saliva deposits, plaque deposits and food residue deposits may be formed on the substrate surface in sequence.

[0056] In a specific embodiment, the salivary deposits may include at least one of proteins, polysaccharides, lipids, small molecule organic matter, sulfide metabolites and inorganic ions. Proteins may include at least one of salivary amylase, mucin and proline-rich protein. Small molecule organic matter may include urea. Inorganic ions may include at least one of phosphate ions and calcium ions. It should be noted that the present application can simulate the salivary deposits formed by the contact between teeth and saliva. Specifically, when the tooth surface is in contact with saliva, the phosphate groups on the enamel surface attract opposite charges, so that calcium ions in the saliva are adsorbed to the tooth surface to form a hydration layer. Proteins, sulfides, etc. in the saliva are firmly attached to the tooth surface through electrostatic interactions with the hydration layer, hydrogen bonds and hydrophobic interactions. The adsorbed proteins further attract other saliva components, i.e., salivary deposits. Urea can promote the adsorption of molecules, thereby promoting the formation of salivary deposits. Sulfides can combine with calcium ions in salivary deposits to form insoluble calcium sulfide, further enhancing the adhesion and anti-demineralization ability of the film.

[0057] In a specific embodiment, the components of salivary deposits include at least one of proteins, polysaccharides, lipids and inorganic ions. It should be noted that when the tooth surface comes into contact with saliva, the proteins in the saliva (such as proline-rich proteins, salivary amylase, etc.) will be rapidly adsorbed to the tooth surface, and these proteins are firmly attached to the tooth surface through mechanisms such as electrostatic interaction, hydrogen bonding and hydrophobic interaction. The adsorbed proteins further attract other salivary components, such as polysaccharides, lipids and inorganic ions, and gradually form a film, namely salivary deposits. This film will also affect the formation process of dental plaque and the remineralization process of tooth enamel.

[0058] In a specific embodiment, the saliva deposit may be formed by contacting the substrate with artificial saliva for a predetermined period of time.

[0059] In a specific embodiment, the components of artificial saliva include at least one of protein, polysaccharide, lipid, small molecule organic matter, sulfide metabolite and inorganic ion. Protein includes at least one of salivary amylase, mucin and proline-rich protein. Small molecule organic matter includes urea. Inorganic ions include at least one of phosphate ion, calcium ion, potassium ion and sodium ion.

[0060] In a specific embodiment, the temperature of the artificial saliva can be maintained at 35° C. to 37° C.

[0061] In one embodiment, the pH of the artificial saliva is 6-6.5.

[0062] In a specific embodiment, the artificial saliva may include: 0.1-1 g / L salivary amylase, 0.1-1 g / L mucin, 0.1-1 g / L proline-rich protein, 0.1-1 g / L urea, 0.01-0.1 g / L sodium sulfide, 10-15 mmol / L sodium ions, 5-10 mmol / L potassium ions, 1-2 mmol / L calcium ions, and 1-2 mmol / L phosphate ions.

[0063] In a specific embodiment, the artificial saliva may include: 0.5-1.5 g / L NaCl, 0.1-0.3 g / L KCl, 0.1-0.3 g / L CaCl2, 0.1-0.3 g / L Na2HPO4, 0.1-0.5 g / L urea, 0.01-0.05 g / L Na2S.

[0064] In a specific embodiment, the plaque deposits include oral bacteria and their metabolites. It should be noted that dental plaque deposits are film-like deposits formed by bacterial communities and their metabolites attached to the tooth surface. Due to the saliva deposits on the tooth surface, the bacteria adhere to the surface of the saliva deposits through the bridging effect of calcium ions in the saliva deposits, hydrogen bonds, hydrophobic-related effects, and other mechanisms to grow and reproduce, digest the saliva deposits, and secrete polysaccharides and other extracellular matrices, ultimately forming plaque deposits cross-linked with the saliva deposits.

[0065] In one embodiment, plaque deposits can be formed by culturing a substrate in a culture medium containing oral bacteria.

[0066] In a specific embodiment, the plaque deposits can be formed by placing a substrate with saliva deposits in a culture medium containing oral bacteria for cultivation. Dental plaque deposits are a film formed by bacterial communities and their metabolites attached to the surface of teeth. Teeth form saliva deposits by contacting with saliva and absorbing components in saliva. Subsequently, oral bacteria can attach to the saliva deposits and grow and multiply, digest the saliva deposits, and secrete polysaccharides and other extracellular matrices, ultimately forming plaque deposits.

[0067] In a specific embodiment, the oral bacteria may include but are not limited to at least one of Streptococcus mutans, Streptococcus milleri, Streptococcus sanguinis, Staphylococcus oralis, Actinomyces ilei, Streptococcus salivarius, Porphyromonas, Fusobacterium, Lactobacillus, Bifidobacterium, and Neisseria.

[0068] In a specific embodiment, the oral bacteria may be Streptococcus mutans. Oral Streptococcus mutans has strong adhesion and is easy to culture. Based on the different viscosity requirements of the oral care products to be tested, the corresponding better plaque deposits can be obtained by simply changing the culture conditions. In addition, the obtained plaque deposits have good stability and strong repeatability, and the efficacy of different oral cleaning products can be verified with a unified standard, and the efficacy differences of oral cleaning products can be well identified.

[0069] In a specific embodiment, the medium containing oral bacteria is a BHI medium containing 1 wt% sucrose. It should be noted that in the medium containing sucrose, sucrose can promote the attachment, proliferation and synthesis of extracellular polysaccharides of Streptococcus mutans, and can more effectively form plaque deposits. Thus, a suitable growth environment for oral bacteria can be provided, which is conducive to the formation of plaque deposits.

[0070] In a specific embodiment, the volume of the culture medium containing oral bacteria is 8 to 12 times the volume of the initial bacterial solution inoculated, and the concentration of the initial bacterial solution is 1.0×10 8 ~9.0×10 8 CFU / mL. For example, the initial bacterial solution volume is 1 mL, and the bacterial content can be 1×10 8 CFU / mL, the culture medium volume can be 8mL to 12mL. Thus, it can provide a suitable growth environment for oral bacteria, which is conducive to the formation of plaque deposits.

[0071] In a specific embodiment, the food residue deposit can be obtained by placing a substrate on which a bacterial plaque deposit is formed in a culture medium containing food residues (food residue mixed culture medium) for culturing.

[0072] In a specific embodiment, food residues that are not completely removed will adhere to the tooth surface or accumulate in plaque deposits, and oral bacteria further metabolize part of the food residues to form food residue deposits, and finally form dental plaque on the tooth surface.

[0073] In a specific embodiment, the food residue mixed culture medium includes food residue and basic culture medium. Thus, the basic culture medium can provide a growth environment for oral bacteria, and the food residue can simulate the food particles remaining in the oral cavity, and the food residue forms food residue deposits by adhering to teeth and plaque deposits under the action of oral bacteria.

[0074] In a specific embodiment, the food waste may include at least one of grains, meat, beans, dairy products, fruits, vegetables, tea and coffee.

[0075] In a specific embodiment, the food residue mixed culture medium can be a BHI culture medium including 2.5g / L corn starch, 2.5g / L lactose, 2.5g / L D-fructose, 2.5g / L skim milk powder, 10g / L pearl powder, 10g / L Euryale powder, 10g / L Poria powder, 10g / L raw malt powder, 10g / L raw coix seed powder, 10g / L soybean powder and 1% sucrose. Among them, the BHI culture medium with 1% sucrose can better support the growth and reproduction of oral bacteria, and an appropriate amount of corn starch, lactose, etc. can simulate food residues and can be better adsorbed on the substrate surface and / or plaque deposits, and further digested under the action of oral bacteria, and finally form food residue deposits.

[0076] In a specific embodiment, salivary deposits, plaque deposits and food residue deposits are cross-linked. It should be noted that the soft scale simulant in the present application includes salivary deposits, plaque deposits and food residue deposits, and the salivary deposits, plaque deposits and food residue deposits are interrelated to form a soft scale simulant as a whole. Specifically, salivary deposits provide attachment points for plaque deposits: salivary proteins and the like form acquired films on the tooth surface, provide attachment points for bacteria, and promote the formation of plaque; plaque deposits utilize salivary deposits and food residue deposits for metabolism: bacteria utilize carbon sources and nitrogen sources in salivary deposits and food residue deposits for metabolism, produce acidic substances and other metabolites, and further promote the formation and stability of biofilms; food residue deposits affect the properties of salivary deposits: proteins and carbohydrates in food residues can affect the composition and properties of salivary deposits, and thus affect the formation and growth of plaque.

[0077] In a specific embodiment, the dental plaque model may further include a dye. The dye may be used to dye the plaque simulant, and the dye is a water-insoluble dye. Through dyeing, the cleaning effect of a toothbrush or other cleaning tools can be intuitively evaluated. For example, the dyed area can be observed with the naked eye or a microscope, and the uncleaned stained area can be recorded for quantitative analysis; the cleaning effect of oral care products in different parts, such as the gum margin, tooth gap, etc., can be evaluated for qualitative analysis. In summary, dyes can help to better characterize plaque simulants and conduct qualitative or quantitative detection of plaque simulants.

[0078] In a specific embodiment, the dye can be a 5% basic fuchsin staining solution or a 5% crystal violet staining solution. Basic fuchsin and crystal violet staining solutions can dye dental plaque and soft scale into obvious colors (red or purple), making these deposits more obvious on the tooth surface; and the contrast between the dyed dental plaque and soft scale and the substrate surface is high, which is convenient for observation and recording. By dyeing bacteria and soft scale simulants, it is helpful to observe and quantify bacteria or soft scale simulants, and then it is helpful for the efficacy evaluation of oral care products.

[0079] As mentioned above, the present application also relates to a method for constructing a dental plaque model.

[0080] In a specific embodiment, the method for constructing a dental plaque model may include the following steps: constructing saliva deposits, constructing plaque deposits, constructing food residue deposits, fixing, and staining.

[0081] In a specific embodiment, constructing the saliva deposit may include placing the substrate in artificial saliva for a predetermined time. The relevant contents of the substrate and the artificial saliva can refer to the above content and are not repeated here. The predetermined time may be 12h to 36h, and the temperature of the artificial saliva may be 35°C to 38°C. In this way, the temperature environment of the human oral cavity is simulated to better form the saliva deposit.

[0082] In a specific embodiment, constructing the plaque deposit may include placing the substrate after constructing the saliva deposit in a culture medium containing oral bacteria. The oral bacteria and the culture medium may refer to the above content and are not described in detail here. The culture conditions may include: culturing at a temperature of 35°C to 38°C under anaerobic conditions for 12h to 72h.

[0083] In a specific embodiment, constructing food residue deposits may include: placing the substrate after constructing the bacterial plaque deposits in a food residue mixed culture medium for further cultivation. The food residue mixed culture medium can refer to the above content and is not described in detail here. The culture conditions may include: culturing at a temperature of 35°C to 38°C under anaerobic conditions for 12h to 72h.

[0084] In a specific embodiment, the staining and fixing may include: drying at 2°C to 6°C for 15 minutes to 60 minutes to fix the plaque simulant. It should be noted that drying at 2°C to 6°C is conducive to stabilizing the activity of oral bacteria and inhibiting their continued growth and reproduction, so as to improve the consistency between different dental plaque models.

[0085] In a specific embodiment, the staining and fixing may include: staining with a dye for 0.5 h to 1.5 h after drying, and storing at 2° C. to 6° C. after washing with a phosphate buffer.

[0086] As mentioned above, the present application also relates to the application of a dental plaque model.

[0087] In a specific embodiment, the dental plaque model can be applied to the efficacy evaluation of oral care products, including the evaluation of the cleaning effect and the evaluation of the usage method (the treatment site and treatment time of the oral care product, etc.).

[0088] In a specific embodiment, the oral care product may include at least one of toothpaste, mouthwash, toothpick, dental floss, tooth powder, toothbrush, electric toothbrush and oral irrigator.

[0089] As mentioned above, the present application also relates to a method for evaluating the cleaning efficacy of an oral care product using a dental plaque model (hereinafter sometimes referred to as "evaluation method").

[0090] The evaluation method may include: treating a dental plaque model with an oral care product, and evaluating the cleaning efficacy of the oral care product by qualitatively or quantitatively analyzing the plaque simulant in the dental plaque model.

[0091] The method for qualitatively characterizing the soft plaque simulant in the dental plaque model may be: observing the area or thickness of the treated soft plaque simulant with the naked eye or a microscope.

[0092] The method for quantifying the plaque simulant in the dental plaque model can be: detecting the content of oral bacteria in the dental plaque model by bacterial culture method or gene detection method; and / or obtaining the residual amount of the plaque simulant by image analysis of the plaque simulant in the dental plaque model.

[0093] The method for quantifying the plaque simulants in the dental plaque model may include at least one of the following methods: 1) Collecting images under a microscope, using Image-Pro Plus image analysis software to analyze the residual area and surface optical density mean of the dental plaque simulants in all test groups, and characterizing the residual amount of the dental plaque simulants by multiplying the area by the optical density value. 2) Using the bacterial culture method, collect the treated dental plaque model, scrape the substrate surface material for culture, and detect the types and number of bacteria related to dental plaque. 3) Using molecular biological techniques such as polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR to detect the number of gene sequences of specific bacteria in the substrate surface material. This method has higher sensitivity and specificity, and can quickly and accurately detect low concentrations of bacteria. 4) Using optical coherence tomography (OCT), it can clearly show the adhesion and thickness of the plaque on the substrate surface and edge. By comparing the OCT images before and after the removal of the plaque, the removal effect can be intuitively evaluated.

[0094] In a specific embodiment, the calculation method of the soft scale removal effect can be as follows:

[0095]

[0096] Where Area NC is the total area of ​​the soft scale simulants in the control group, MOD NC is the average optical density of the soft scale simulant in the control group, Area 样品组 is the total area of ​​soft scale simulants in the sample group, MOD 样品组 is the mean optical density of the soft plaque simulation of the sample group. The control group refers to the dental plaque model not treated with oral care products, and the sample group refers to the dental plaque model treated with oral care products.

[0097] The present application is further described in detail below by specific examples. The following examples are only further described for the present application and should not be construed as limiting the present application. In the present example, unless otherwise specified, the reagents and instruments used are all commercially available, and the experimental operations are carried out in accordance with the product instructions and conventional experimental specifications.

[0098] Example 1: Construction of dental plaque model

[0099] 1) Base: Add hydroxyapatite powder into the mold groove of the tooth, filling it as much as possible without leaving any gaps; use a powder tablet press to press the mold filled with powder together, and then carefully demold it; arrange a batch of hydroxyapatite sheets pressed after demolding neatly, put them into a high-temperature sintering furnace and sinter them at 1100℃ to convert the material into ceramics; after cooling, take out the model from the high-temperature sintering furnace, measure the size, and select the size with a diameter of 12mm and a thickness of 2mm.

[0100] 2) Construction of saliva sediment: According to the test groups, the hydroxyapatite sheet carriers of each group were immersed in 1 mL of artificial saliva (preparation method: take NaCl 0.67 g, KCl 0.2 g, CaCl2 0.2 g, Na2HPO4 0.2 g, urea 0.1 g, Na2S 0.01 g, add distilled water to 1 L, and adjust the pH to 6.0-6.1), and the hydroxyapatite carriers of the blank control group were immersed in 1 mL of sterile PBS and cultured at 37°C for 24 h to form artificial saliva sediment, and the liquid was discarded.

[0101] 3) Construction of plaque deposits:

[0102] 1 μL of fresh culture of the third generation Streptococcus mutans (ATCC 25175) was selected and added to 10 mL of 1% sucrose BHI medium for activation culture for 24 h to obtain the strain enrichment solution with a concentration of 1.0-9.0×10 8 CFU / mL bacterial solution is reserved for later use.

[0103] After the prepared bacterial solution was diluted 10 times with BHI medium containing 1% sucrose, the hydroxyapatite sheet carrier treated with the above artificial saliva was placed in 2 mL of the bacterial solution and cultured under anaerobic conditions at 37°C for 12 h, 24 h, 48 h, and 72 h (constructing plaque deposits under different time treatments according to test requirements) to form plaque deposits.

[0104] 4) Construction of food residue deposits: The constructed hydroxyapatite sheets with dental plaque deposits of the negative control group and the sample group were further placed in 2 mL of food residue mixed culture medium, wherein the food residue mixed culture medium was obtained by mixing 2.5 g / L corn starch, 2.5 g / L lactose, 2.5 g / L D-fructose, 2.5 g / L skim milk powder, 10 g / L pearl powder (edible), 10 g / L Euryale powder, 10 g / L Poria powder, 10 g / L raw malt powder, 10 g / L raw coix seed powder, 10 g / L soybean powder and 1% sucrose in BHI culture medium, and cultured at 37°C under anaerobic conditions for 12 h, 24 h, 48 h, and 72 h to form soft scale, and fresh food residue culture medium was replaced every 12 h.

[0105] 5) Fixation and staining: After the culture was completed, the food residue mixed culture medium was discarded, and each group of hydroxyapatite sheet carriers were placed in a new 24-well plate and dried at 4°C for 30 minutes. After drying, 1 mL of 5% alkaline fuchsin staining solution or crystal violet was used for staining for 60 minutes. After staining, PBS was added to the wall for washing three times to remove excess stain, 2 mL / time. After washing, the plates were allowed to stand at 4°C for 120 minutes, and the stained soft scale was photographed and recorded.

[0106] Example 2: Dental plaque model for evaluation of the efficacy of water flossers and other types of products

[0107] The testing methods for different products are as follows:

[0108] Water flosser: Fix the dental plaque model prepared in Example 1 on a laboratory-made stainless steel fixture, and also fix the water flosser on a laboratory-made adjustable stainless steel frame, and adjust the vertical distance between the hydroxyapatite sheet and the water flosser nozzle (water outlet) to be less than or equal to 1 cm. Rinse the entire surface in a circular motion for 10 seconds. The water flossers tested include the usmile C10 portable water flosser and two commercially available water flossers (water flosser 1 and water flosser 2).

[0109] Toothpicks and dental floss sticks: The dental plaque model prepared in Example 1 was fixed on a flat surface, and the dental plaque on the surface of the hydroxyapatite sheet was scraped with a toothpick and a dental floss stick. Each hydroxyapatite sheet with soft plaque was scraped 3 times. The toothpicks and dental floss sticks used in the test were purchased from the market.

[0110] Mouthwash: The dental plaque model prepared in Example 1 was placed in a 24-well plate, 2 mL of mouthwash was added to each well, and a pipette was used to simulate the human mouthwashing action, and the solution was rinsed and blown 3 times. The solution was discarded after each blowing, and the mouthwash solution was replaced 3 times in total. The mouthwash used in the test was a commercially available mouthwash.

[0111] After all the test groups were completed, they were naturally dried at room temperature for 5 minutes. Then images were collected under a microscope, and the Image-Pro Plus image analysis software was used to analyze the soft scale area and surface optical density average of all test groups.

[0112] Figure 1 1 is a test result diagram related to Example 2 of the present application, and the following table shows the results of using a water flosser and other products to treat a dental plaque model:

[0113]

[0114] Note: Blank control (BC) refers to hydroxyapatite sheets without saliva deposits, plaque deposits or food residue deposits. Negative control (NC) refers to hydroxyapatite sheets with 24-hour saliva deposits, 24-hour in vitro dental plaque deposits and 24-hour food residue deposits. Statistical analysis was performed using the t-test method, compared with the NC group; significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0115] The results showed that compared with the negative control group (NC), the soft plaque removal rate of C10 portable water flosser (refreshing mode) was 98.553%, the soft plaque removal rate of C1 portable water flosser (refreshing mode) was 96.661%, the soft plaque removal rate of water flosser 1 (powerful mode) was 86.864%, the soft plaque removal rate of water flosser 2 (refreshing mode) was 33.030%, the soft plaque removal rate of toothpick was 3.153%, the soft plaque removal rate of dental floss stick was 18.276%, and the soft plaque removal rate of mouthwash was 12.743%.

[0116] Example 3: Evaluation of the efficacy of electric toothbrushes using a dental plaque model

[0117] The dental plaque model prepared in Example 1 is fixed in a straight line inside the groove of the 3D printed part with the surface of the hydroxyapatite sheet facing upward. A blank hydroxyapatite sheet needs to be added to both sides of the three parallel hydroxyapatite sheets to buffer the deformation caused by the bristles; the electric toothbrush is fixed to the brushing action simulation device by adjusting the toothbrush fixing assembly to ensure that the toothbrush does not move in the horizontal and vertical directions when it is turned on and the brush head should be placed in the middle of the hydroxyapatite sheet carrier; when the electric toothbrush is not turned on, the contact surface of the hydroxyapatite sheet and the electric toothbrush is adjusted to stabilize the pressure (the pressure of the electric toothbrush head is adjusted to 100±20g for testing); before preparing the test, the toothbrush head is completely immersed in ultrapure water for 10s, and after immersion, the brush head is wiped with a non-woven fabric to ensure that the brush head brush wire does not hang water droplets. Then the image is collected under a microscope, and the image analysis software Image-Pro Plus is used to analyze the mean of the soft scale area and surface optical density of all test groups.

[0118] Figure 2 1 is a test result diagram related to Example 3 of the present application, and the following table shows the results of brushing the soft plaque model with different models of electric toothbrushes:

[0119]

[0120] Note: Blank control (BC) refers to hydroxyapatite sheets without saliva deposits, plaque deposits or food residue deposits. Negative control (NC) refers to hydroxyapatite sheets with 24-hour saliva deposits, 24-hour in vitro dental plaque deposits and 24-hour food residue deposits. Statistical analysis was performed using the t-test method, compared with the NC group; significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0121] The results showed that compared with the negative control group (NC), the soft plaque removal rate of the usmile sonic electric toothbrush (model: Y10) (whitening mode) was 59.43%, and the plaque deposit removal rate of the usmile sonic electric toothbrush (model: Y10 Pro Max) (whitening mode) was 64.00%.

[0122] Example 4: Testing of dental plaque models with different treatment times

[0123] In this embodiment, hydroxyapatite sheets constructed with 24h saliva deposit, 24h in vitro dental plaque deposit and 12h, 24h, 48h and 72h in vitro food residue deposit were used as dental plaque models, and brushing was performed with a manual toothbrush at a certain pressure. Each group included 3 hydroxyapatite sheets. The 3 hydroxyapatite sheets were placed under the same conditions, and a brushing action simulation device was turned on. The toothbrush movement speed was 4mm / s, and the toothbrush running path was a single linear motion. A complete test process was completed from the start of brushing the first blank hydroxyapatite sheet to the last blank hydroxyapatite sheet. After the brushing test, the hydroxyapatite sheet after the brushing test was transferred to 2mL PBS for temporary storage. After all tests were completed, the hydroxyapatite sheet was placed in a 24-well plate, adhered to the wall and added with water for cleaning to simulate the human brushing process. The plate was cleaned 3 times, 2mL / time. After the cleaning was completed, all hydroxyapatite sheets were placed at 4°C to dry for about 30min. Then, images were collected under a microscope, and the mean values ​​of soft scale area and surface optical density of all test groups were analyzed using Image-Pro Plus image analysis software.

[0124] Figure 3 1 is a test result diagram related to Example 4 of the present application, and the following table shows the results of manually brushing the dental plaque model with different treatment times using a toothbrush:

[0125]

[0126] Note: Negative control (NC) refers to hydroxyapatite sheets that underwent 24-hour saliva deposit construction, 24-hour in vitro dental plaque deposit construction, and 24-hour food residue deposit construction. The group "negative control (NC)" refers to hydroxyapatite sheets that underwent 24-hour saliva deposit construction, 24-hour in vitro dental plaque deposit construction, and 24-hour food residue deposit construction; the group "food residue deposit construction treatment time is 12 hours" refers to hydroxyapatite sheets that underwent 24-hour saliva deposit construction, 24-hour in vitro dental plaque deposit construction, and 12-hour food residue deposit construction; the group "food residue deposit construction treatment time is 24 hours" refers to hydroxyapatite sheets that underwent 24-hour saliva deposit construction, 24-hour in vitro dental plaque deposit construction, and 24-hour food residue deposit construction. The meanings of other groups are similar to those described above and will not be repeated here. Statistical analysis was performed using the t-test method, and the results were compared with the NC group; significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0127] As can be seen from the table above, the results show that compared with the negative control group (NC), the manual toothbrush has a soft plaque removal rate of 92.96% for 12 hours of construction, a soft plaque removal rate of 78.25% for 24 hours of construction, a soft plaque removal rate of 74.93% for 48 hours of construction, and a soft plaque removal rate of 61.58% for 72 hours of construction. As the soft plaque formation time increases, it fits more closely with the tooth-like material, and the cleaning ability of the manual toothbrush tends to decrease.

[0128] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A dental plaque model, characterized in that: include: A substrate and a soft dirt simulant attached to the substrate, wherein the soft dirt simulant comprises saliva deposits, plaque deposits and food residue deposits.

2. The dental plaque model according to claim 1, characterized in that: The saliva deposits and the bacterial plaque deposits are in a film-like form, and the saliva deposits, the bacterial plaque deposits and the food residue deposits are cross-linked with each other.

3. The dental plaque model according to any one of claims 1 to 3, characterized in that: The saliva deposit is formed by contacting the substrate with artificial saliva; the bacterial plaque deposit is formed by placing the substrate with the saliva deposit in a culture medium containing oral bacteria for culturing; and the food residue deposit is formed by placing the substrate with the bacterial plaque deposit in a culture medium containing food residue for culturing.

4. The dental plaque model according to claim 3, characterized in that: The pH value of the artificial saliva is 6-6.1, and the artificial saliva comprises: 0.5-1.5 g / L NaCl, 0.1-0.3 g / L KCl, 0.1-0.3 g / L CaCl2, 0.1-0.3 g / L Na2HPO4, 0.1-0.5 g / L urea, and 0.01-0.05 g / L Na2S.

5. The dental plaque model according to claim 4, characterized in that: The culture medium containing oral bacteria is a BHI culture medium containing Streptococcus mutans.

6. The dental plaque model according to claim 5, characterized in that: The culture medium containing food residues is a BHI culture medium comprising 1-5 g / L starch, 1-10 g / L soybean powder, 1-10 g / L sugar, 1-5 g / L skim milk powder and 1-20 g / L dietary fiber.

7. The dental plaque model according to claim 1, characterized in that: The dental plaque model further includes a dye bound to the plaque simulant.

8. A method for constructing a dental plaque model according to any one of claims 1 to 7, characterized in that: include: Constructing a saliva deposit: placing a substrate in artificial saliva to construct the saliva deposit; Constructing bacterial plaque deposits: placing the substrate with the saliva deposits constructed in a culture medium containing oral bacteria for cultivation to construct the bacterial plaque deposits; Constructing food residue deposits: placing the substrate with the bacterial plaque deposits in a culture medium containing food residues for culturing to construct the food residue deposits, that is, to obtain the dental plaque model.

9. The construction method according to claim 8, characterized in that: The step of constructing the saliva deposit comprises: placing the substrate in artificial saliva at 35° C. to 38° C. for 12 h to 36 h; The step of constructing the bacterial plaque deposit comprises: placing the substrate constructed with the saliva deposit in a culture medium containing oral bacteria and culturing for 12 hours to 72 hours under anaerobic conditions at 35° C. to 38° C.; The step of constructing the food residue deposit comprises: placing the substrate with the bacterial plaque deposit in a food residue mixed culture medium and culturing it for 12 hours to 72 hours under anaerobic conditions at 35° C. to 38° C.

10. The construction method according to claim 8, characterized in that: After constructing the food residue sediment, the method further includes fixing and dyeing steps; the fixing and dyeing steps include: drying at 2°C to 6°C for 15 to 60 minutes, dyeing with a water-insoluble dye for 0.5 to 1.5 hours after drying, and storing at 2°C to 6°C after washing with a phosphate buffer.

11. A method for evaluating the cleaning efficacy of an oral care product using the dental plaque model according to any one of claims 1 to 7, characterized in that: include: The dental plaque model is treated with an oral care product, and the cleaning efficacy of the oral care product is evaluated by qualitatively or quantitatively measuring the plaque simulant in the dental plaque model.

12. The method according to claim 11, characterized in that The quantification of the plaque simulant in the dental plaque model comprises at least one of the following methods: detecting the content of oral bacteria in the dental plaque model by a bacterial culture method or a gene detection method; obtaining the residual amount of the plaque simulant by performing image analysis on the plaque simulant in the dental plaque model; Wherein, quantifying the plaque simulant in the dental plaque model comprises: collecting images through a microscope and analyzing to obtain the area and optical density of the plaque simulant in the dental plaque model, and calculating by the following formula: Where Area NC is the total area of ​​the soft scale simulants in the control group, MOD NC is the average optical density of the soft scale simulant in the control group, Area 样品组 is the total area of ​​soft scale simulants in the sample group, MOD 样品组 is the mean optical density of the soft plaque simulation of the sample group, the control group refers to the dental plaque model not treated with the oral care product, and the sample group refers to the dental plaque model treated with the oral care product.

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