CN116515660A - In vitro plaque biofilm model and method for evaluating efficacy of oral products - Google Patents
In vitro plaque biofilm model and method for evaluating efficacy of oral products Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- G01B21/00—Measuring arrangements or details thereof, where the measuring technique is not covered by the other groups of this subclass, unspecified or not relevant
- G01B21/20—Measuring arrangements or details thereof, where the measuring technique is not covered by the other groups of this subclass, unspecified or not relevant for measuring contours or curvatures, e.g. determining profile
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The present invention proposes an in vitro plaque biofilm model and method for evaluating efficacy of an oral product, the plaque biofilm model comprising: a substrate; the dental plaque biomembrane is formed on the surface of the substrate, and microorganisms in the dental plaque biomembrane are oral streptococcus mutans. The dental plaque biomembrane in the isolated dental plaque biomembrane model has moderate viscosity and uniform plaque content, is applied to evaluating the efficacy of oral products, and has the advantages of high accuracy, strong repeatability, good stability and the like, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of efficacy evaluation of oral products. In particular, the invention relates to an ex vivo plaque biofilm model and method for evaluating the efficacy of oral products.
Background
With the development of the consumer electronics industry in China, the competition of oral cavity electronic products in China and internationally is vigorous, and the consumer acceptance of oral cavity cleaning products such as electric toothbrushes, tooth flushers and the like is rapidly growing. Based on the market prospect, a large number of emerging electric toothbrushes and tooth-rinsing devices are on the market, however, due to price competition, the products are uneven, and a unified testing method and a verification method are lacked to evaluate the efficacy and quality of the oral cavity electronic products.
The traditional verification method of the oral electronic product is basically to recruit a certain number of volunteers to conduct actual tooth brushing test through clinical means. However, this method has limitations such as complicated test procedures, long period, high cost, etc., and because it is limited by individual differences among subject population (including differences in dental care conditions, differences in oral cleaning habits, etc.), it is difficult to quantify and make statistical significance for the results obtained if volunteers with a sufficient sample size do not participate. The traditional method is difficult to copy or transversely compare in different batches of product tests due to the fact that the traditional method is influenced by large human factors and lacks consistency among tests, so that the traditional method is difficult to expand in scale to carry out repeated tests on the aspects of efficiency verification and comparison and optimization of products, and positive promotion effects are generated on product research and development. Therefore, there is a lack of effective means for efficient, accurate, stable, repeatable testing of the cleaning efficacy of products in the oral electronics industry.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art at least to some extent. Therefore, the invention provides an in-vitro dental plaque biomembrane model for evaluating the efficacy of an oral product, application of the in-vitro dental plaque biomembrane model in evaluating the efficacy of the oral product and a method for evaluating the dental plaque cleaning efficacy of the oral product. In addition, the method for preparing the isolated dental plaque biomembrane model is simple and convenient to operate, strong in repeatability, good in stability, high in efficiency and suitable for scale production.
In one aspect, the invention provides an in vitro plaque biofilm model for evaluating efficacy of an oral product. According to an embodiment of the present invention, the in vitro plaque biofilm model for evaluating efficacy of oral products comprises: a substrate; the dental plaque biomembrane is formed on the surface of the substrate, and microorganisms in the dental plaque biomembrane are oral streptococcus mutans.
The isolated dental plaque biomembrane model for evaluating the efficacy of the oral product adopts a single strain to form the dental plaque biomembrane, has the advantages that the single strain has outstanding characteristics, can screen culture conditions suitable for stable growth according to the physiological and biochemical characteristics, the structure and the efficacy, and has definite purposes; the method is easy to screen better strains and culture conditions, and the culture conditions are easy to control; the biological membrane is easy to culture and obtain, and the repeatability is strong. However, compared with single strains, if mixed strains are adopted to form dental plaque biomembrane, the strain is complex, the difference of growth characteristics of different strains is strong, and when anaerobic bacteria, facultative anaerobic bacteria and strict anaerobic bacteria coexist, the culture environment is limited, and the screening of the culture conditions is complicated. In addition, the strains contained in the oral cavity belong to a dynamic balance state, if complex bacteria are used for culturing, the proper growth proportion of each strain is more complex to screen, and besides the living habit of the single strain per se, whether the strain is harmful to the growth of individuals or not is considered. If lactobacillus produces a large amount of lactic acid, the growth of acid-fast bacteria is necessarily limited if the bacteria are present in the culture system. In addition, when the bacteria are cultured, the adhesion of the bacterial plaque biological film is easy to be influenced among multiple bacteria and is not uniform in the bacterial plaque forming process, so that the whole environment of the growth of the bacteria is influenced, and the bacterial plaque biological film is not beneficial to forming.
The inventors of the present invention have found that the adhesiveness of dental plaque biofilm in a dental plaque biofilm model can significantly affect its application effects, such as efficacy assessment of oral care products. If the adhesion of the dental plaque biomembrane is weaker, the dental plaque biomembrane is easy to brush or wash and fall off, so that the efficacy of the product cannot be accurately judged, and the efficacy difference between different products cannot be effectively distinguished; if the adhesion of the dental plaque biomembrane is too strong, the dental plaque biomembrane is not easy to remove, the efficacy of the product cannot be accurately judged, the cleaning efficacy of the product can be misjudged, and the product such as a toothbrush is easy to adhere, so that the subsequent treatment cannot be performed, and the evaluation fails.
Furthermore, the inventors conducted a number of optimization screens against numerous bacteria in the oral cavity to obtain the preferred species, streptococci mutans in the oral cavity. Particularly, most of oral bacteria are classified into periodontal pathogenic bacteria and cariogenic bacteria, and early researches find that the two types of bacteria have obviously different adhesive capacities on hydroxyapatite, the adhesive capacities of the periodontal pathogenic bacteria on the surface of the hydroxyapatite are obviously weaker than those of the cariogenic bacteria, the bacteria cannot be like the cariogenic bacteria, the bacteria can be directly adhered to the surface of a carrier, for example, porphyromonas gingivalis cannot form a biological film on the surface of the carrier. The adhesion of bacteria having strong adhesion in periodontal disease, such as Streptococcus sanguis and Actinomyces viscosus, is enhanced by recognition of the corresponding receptor, and the adhesion is reduced when the bacteria are cultured separately.
As described above, the single-cell culture is significantly advantageous over the mixed-cell culture, and thus cariogenic bacteria are preferred. The streptococcus mutans in the cariogenic bacteria has stronger adhesiveness and is easy to culture, and the correspondingly better dental plaque biological film can be obtained by simply changing culture conditions based on different viscosity requirements of the oral care product to be tested. And moreover, the obtained dental plaque biomembrane is good in stability and strong in repeatability, the efficacy of different oral cleaning products can be verified by unified standards, and the efficacy difference of the oral cleaning products can be well screened.
The above-described ex vivo plaque biofilm model for evaluating efficacy of oral products may also have the following additional technical features according to embodiments of the present invention:
according to the embodiment of the invention, the absorbance value of the mixed solution obtained by mixing the dental plaque biomembrane and 1 ml of solvent at the wavelength of 575nm is 3-4; the substrate comprises hydroxyapatite or a cell slide or a glass slide or a cover slip or a collagen-coated resin or enamel slice or plate.
In another aspect of the invention, the invention proposes the use of an ex vivo plaque biofilm model for evaluating the efficacy of an oral product. According to an embodiment of the invention, the isolated dental plaque biofilm model is the isolated dental plaque biofilm model applied to evaluating the efficacy of oral products. The isolated dental plaque biomembrane model can be applied to evaluating the efficacy of oral products, and has the advantages of high accuracy, strong repeatability, good stability and the like, and has good application prospect.
According to an embodiment of the invention, the oral product is selected from oral care electronics, preferably an electric toothbrush or a dental irrigator; the efficacy includes a plaque cleaning efficacy and/or a plaque prevention efficacy.
According to an embodiment of the present invention, the method for preparing an isolated dental plaque biofilm model for evaluating efficacy of an oral product includes: and forming the dental plaque biomembrane on the surface of the substrate to obtain an in-vitro dental plaque biomembrane model for evaluating the efficacy of the oral product.
According to an embodiment of the present invention, the preparation method includes: (1) Carrying out activation culture on the streptococcus mutans in a brain heart infusion culture medium to obtain a bacterial strain enrichment medium; immersing the substrate in artificial saliva for culture to obtain a substrate with a saliva-obtaining film formed on the surface; (2) Co-culturing the activated bacterial liquid, the substrate with the saliva-obtained film formed on the surface and a brain heart infusion culture medium containing sucrose, and forming a dental plaque biomembrane on the surface of the substrate; (3) Collecting the substrate with the dental plaque biomembrane formed on the surface, cleaning and removing excessive plankton and culture medium, and then carrying out fixing treatment on the substrate with the dental plaque biomembrane formed on the surface by using a fixing agent to obtain the isolated dental plaque biomembrane model for evaluating the efficacy of oral products.
According to the embodiment of the invention, the volume ratio of the activated bacteria liquid to the brain heart infusion medium containing sucrose is 1: (8-10).
According to the embodiment of the invention, the concentration of the strain enrichment solution is 10 6 ～10 9 CFU/mL, preferably 10 7 ～10 8 CFU/mL。
According to an embodiment of the invention, the co-cultivation time is 20 to 80 hours.
According to an embodiment of the present invention, the oral care product is a dental irrigator, the co-cultivation time required for the plaque biofilm model is 40 to 80 hours, or the oral care product is an electric toothbrush, and the co-cultivation time required for the plaque biofilm model is 20 to 30 hours.
According to the embodiment of the invention, the washing to remove the superfluous plankton and the culture medium is performed by adopting PBS buffer solution or water; the fixing agent is selected from paraformaldehyde solution; the fixing process includes: and (3) contacting the substrate with the dental plaque biomembrane formed on the surface with 1-8% of paraformaldehyde solution, standing for 5-10 min, and cleaning the obtained substrate to obtain the dental plaque biomembrane model.
According to an embodiment of the present invention, the sucrose content in the brain heart infusion medium containing sucrose is 1 to 5 mass%, preferably 1 mass%.
According to an embodiment of the invention, the cultivation is performed under anaerobic conditions at 35-40 ℃.
In yet another aspect of the invention, the invention provides a method of evaluating the plaque cleaning efficacy of an oral care product. According to an embodiment of the invention, the method comprises: (A) Treating the in vitro dental plaque biofilm model applied to evaluate the efficacy of the oral product with an oral care product to be tested; (B) The efficacy of the oral care product to be tested is evaluated based on the changes in the plaque biofilm before and after treatment.
According to an embodiment of the invention, the method comprises preparing an ex vivo plaque biofilm model for evaluating the efficacy of an oral product, the preparation method comprising a preparation method of an ex vivo plaque biofilm model for evaluating the efficacy of an oral product in use of an ex vivo plaque biofilm model for evaluating the efficacy of an oral product as described previously.
According to an embodiment of the invention, the dental plaque biofilm model is pre-stained prior to the treatment of the ex vivo dental plaque biofilm model for evaluating efficacy of the oral product.
According to an embodiment of the invention, the change in plaque biofilm comprises at least one of:
Morphological changes of the plaque biofilm;
the absorbance value of the plaque biofilm varies;
the surface color difference value of the dental plaque biomembrane changes.
According to an embodiment of the present invention, the oral care product to be tested is a dental rinse, and step (a) comprises: fixing the dental plaque biomembrane model, and adjusting the vertical distance between the nozzle of the dental irrigator and the opposite surface of the dental plaque biomembrane to be 0.1-1 cm; placing a blocking piece between the nozzle of the dental flusher and the dental plaque biomembrane, starting the dental flusher to flush the blocking piece for 3-7 s, taking away the blocking piece, enabling the dental flusher to flush the dental plaque biomembrane for 1-5 s, and airing the dental plaque biomembrane; alternatively, the oral care product to be tested is an electric toothbrush, and step (a) comprises: fixing the dental plaque biomembrane model, simulating a human oral cavity wetting environment, enabling an electric toothbrush to horizontally slide and brush the dental plaque biomembrane for 5-8 s, simulating a human mouth rinsing action after brushing, cleaning the dental plaque biomembrane with water for 1 or 2 times, and airing the dental plaque biomembrane.
In yet another aspect of the invention, the invention provides a method of evaluating the plaque cleaning efficacy of an oral product. According to an embodiment of the invention, the method comprises:
(1) Activating oral Streptococcus mutans strain, selecting 1-ring activated strain, adding into a test tube containing 10mL brain heart infusion medium, sealing the test tube with sterile cotton plug, anaerobic culturing at 37deg.C for 24 hr to obtain strain multiplication solution, and diluting the strain multiplication solution to 10 with PBS buffer solution 7 ～10 8 CFU/mL, obtaining bacterial suspension;
(2) Soaking the hydroxyapatite sheet in 1mL of artificial saliva, culturing at 37 ℃ for 24 hours to form an artificial acquired membrane, discarding liquid, and flushing redundant artificial saliva on the surface of the hydroxyapatite sheet with sterile PBS;
(3) Co-culturing the bacterial suspension with a brain heart infusion culture medium containing 1 mass% of sucrose and a hydroxyapatite sheet formed with an artificially obtained film, wherein the volume of the bacterial suspension is 9 times that of the bacterial suspension, the co-culture time required by a dental plaque biomembrane model for evaluating the efficacy of a tooth irrigator is 48-72 hours, and the co-culture time required by the dental plaque biomembrane model for evaluating an electric toothbrush is 24 hours;
(4) Transferring the hydroxyapatite sheet with the dental plaque biomembrane to a new 24-hole plate after the co-culture is finished, adding deionized water to the wall, cleaning to remove superfluous plankton and culture medium, fixing for 5-10min by using 4% paraformaldehyde, cleaning superfluous formaldehyde, cleaning crystal violet staining solution for 1min, and cleaning to remove unbound staining solution after the staining is finished; determining one or more of plaque biofilm morphology, absorbance value and surface color difference value of the obtained plaque biofilm model;
(5) Aiming at the evaluation product being a dental irrigator, fixing the dental plaque biomembrane finally obtained in the step (4), and adjusting the vertical distance between a nozzle of the dental irrigator and the opposite surface of the dental plaque biomembrane to be not more than 1cm; placing an acrylic plate between the nozzle of the dental flusher and the dental plaque biomembrane, starting the dental flusher to flush the acrylic plate for 5s, taking away the acrylic plate, enabling the dental flusher to flush the dental plaque biomembrane for 2s, and airing the dental plaque biomembrane; determining one or more of the three types of the obtained dental plaque biofilm morphology, absorbance value and surface color difference value, and comparing with the corresponding parameters determined in the step (4) so as to evaluate the dental plaque cleaning efficacy of the dental irrigator; and/or
Fixing the dental plaque biomembrane model finally obtained in the step (4) aiming at the evaluation product, simulating the moist environment of the oral cavity of a human body, enabling the electric toothbrush to horizontally slide and brush the dental plaque biomembrane for 5-8 s, simulating the mouth rinsing action of the human body after the brushing is finished, cleaning the dental plaque biomembrane model for 1 or 2 times by using water, and airing the dental plaque biomembrane; determining one or more of the three of the resulting plaque biofilm morphology, absorbance values, surface color difference values, and comparing with the corresponding parameters determined in step (4) to evaluate the plaque cleaning efficacy of the electric toothbrush.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows an analysis of absorbance values of dental plaque biofilm at different co-culture times according to example 1 of the invention;
FIG. 2 shows a photograph of plaque biofilm in accordance with example 2 of the present invention;
FIG. 3 shows a schematic view of plaque clearance analysis according to example 2 of the present invention, wherein P-value < 0.01 for sample 2 in each group compared to sample 1;
fig. 4 shows a graph of color difference values according to example 2 of the present invention, wherein P-value < 0.01 for sample 2 compared to sample 1 in 48h group and 72h group;
FIG. 5 shows an analysis of plaque clearance after treatment of isolated bovine teeth by different tooth irrigators according to example 3 of the present invention;
FIG. 6 shows a photograph of plaque biofilm in accordance with example 4 of the present invention;
fig. 7 shows an absorbance analysis schematic of example 4 according to the invention, wherein # indicates that P-value < 0.01 in NC group (negative control group) compared to BC group (blank control group), and x indicates that P-value < 0.01 in electric toothbrush 1 or electric toothbrush 2 compared to NC group;
FIG. 8 shows an analysis of the effect of different sucrose contents on plaque biofilm formation according to example 5 of the present invention;
FIG. 9 shows an analysis of the effect of different species concentrations on plaque biofilm formation according to example 6 of the present invention, representing P-value < 0.01 between the three groups;
fig. 10 shows an analysis of the effect of deionized water and artificial saliva on plaque biofilm formation according to example 7 of the present invention, showing the artificial saliva group versus water ratio P-value < 0.01.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides an in-vitro dental plaque biofilm model for evaluating the efficacy of an oral product, and application of the in-vitro dental plaque biofilm model and a method for evaluating the efficacy of the oral product, and the method is used for evaluating the plaque cleaning efficacy of the oral product, and the in-vitro dental plaque biofilm model and the method are respectively described in detail below.
In-vitro dental plaque biomembrane model applied to evaluation of efficacy of oral product
In one aspect, the invention provides an in vitro plaque biofilm model for evaluating efficacy of an oral product. According to an embodiment of the present invention, the in vitro plaque biofilm model for evaluating efficacy of oral products comprises: a substrate; the dental plaque biomembrane is formed on the surface of the substrate, and microorganisms in the dental plaque biomembrane are oral streptococcus mutans. As described above, the dental plaque biofilm which is stable and moderate in viscosity is easily obtained from the streptococcus mutans, the dental plaque biofilm with better quality can be obtained by simply changing the culture conditions based on the different adhesion requirements of the oral care product to be tested, and the culture conditions are mild and the repeatability is good.
According to the embodiment of the invention, the absorbance value of the mixed solution obtained by mixing the dental plaque biological film with 1 milliliter of solvent at the wavelength of 575nm is 3-4. The absorbance values of the dental plaque biofilm reflect the bacterial content, and the inventor finds that the adhesiveness of the dental plaque biofilm can significantly influence the application effect of the dental plaque biofilm, such as efficacy evaluation of oral products. If the viscosity of the dental plaque biomembrane is weak, the dental plaque biomembrane is easy to brush or wash and fall off, so that the efficacy of the product cannot be accurately judged, and the difference between different products cannot be effectively distinguished; if the viscosity of the dental plaque biomembrane is too high, the dental plaque biomembrane is not easy to remove, the efficacy of the product cannot be accurately judged, the cleaning efficacy of the product can be misjudged, and the product such as a toothbrush is easy to adhere, so that the subsequent treatment cannot be performed, and the evaluation fails. Further, the inventors have found through intensive studies that the viscosity of the dental plaque biofilm having the absorbance value is moderate, so that the efficacy of oral care products can be accurately evaluated, the cleaning efficacy of different oral products can be evaluated with a uniform standard, and the difference in cleaning efficacy of oral products can be well screened.
The type of solvent to be mixed with the dental plaque biofilm is not strictly limited, so long as a uniform bacterial suspension can be formed.
According to an embodiment of the invention, the substrate comprises hydroxyapatite or a cell slide or cover glass or collagen-coated resin or enamel slice or plate. The substrate material plays a supporting role to enable thalli to grow and enrich on the surface to form a biological film, wherein the effect of the hydroxyapatite is better, and the following 3 points are mainly shown: 1) The main component of hydroxyapatite is (Ca 10 (PO 4 ) 6 (OH) 2 ) (HAP) which is consistent with the surface of the oral teeth and can greatly reflect the symbiotic process and the plaque formation trend of the oral cavity and the colony of a human body; 2) The hydroxyapatite surface has small and compact pores, is similar to an enamel surface structure, is easier for colony adhesion, and effectively prevents colony depositionEvaluation errors caused by too little or too much; 3) Synthetic Hydroxyapatite (HA) is a biologically active and non-toxic ceramic material that closely resembles the inorganic parts of human teeth and bones. Various synthetic apatites are used depending on the manufacturing technique used. Hydroxyapatite for biomedical applications is prepared by chemical methods to obtain specific properties such as chemical purity, crystal morphology and crystal size. Specifically, the absorbance value of the aforementioned dental plaque biofilm was calculated as a dental plaque biofilm formed by completely coating a hydroxyapatite surface of 12mm×2mm size.
Application of
In yet another aspect of the invention, the invention provides the use of an ex vivo plaque biofilm model for evaluating the efficacy of an oral product. According to an embodiment of the invention, the isolated dental plaque biofilm model is the isolated dental plaque biofilm model applied to evaluating the efficacy of oral products. Therefore, the dental plaque biomembrane model can be used for evaluating the efficacy of oral products, and has the advantages of high accuracy, strong repeatability, good stability and the like, and has good application prospect.
It should be noted that the type of the oral product is not strictly limited, and the oral product can be an oral care electronic product (such as an electric toothbrush, a tooth washing device and the like), a related personal care product (such as toothpaste, a common toothbrush, mouthwash and the like) and an active ingredient (such as a substance with antibacterial effect and the like), and can be flexibly selected according to actual situations, and the description is omitted here.
According to an embodiment of the invention, the efficacy comprises a plaque cleaning efficacy and/or a plaque prevention efficacy. The plaque cleaning efficacy and/or plaque preventing efficacy of the oral product can be effectively evaluated using the plaque biofilm model as described previously.
According to an embodiment of the present invention, the method for preparing an isolated dental plaque biofilm model for evaluating efficacy of an oral product includes: and forming the dental plaque biomembrane on the surface of the substrate to obtain an in-vitro dental plaque biomembrane model for evaluating the efficacy of the oral product.
According to an embodiment of the present invention, the preparation method includes: (1) Carrying out activation culture on the streptococcus mutans in a brain heart infusion culture medium to obtain a bacterial strain enrichment medium; immersing the substrate in artificial saliva for culture to obtain a substrate with a saliva-obtaining film formed on the surface; (2) Co-culturing the activated bacterial liquid, the substrate with the saliva-obtained film formed on the surface and a brain heart infusion culture medium containing sucrose, and forming a dental plaque biomembrane on the surface of the substrate; (3) Collecting the substrate with the dental plaque biomembrane formed on the surface, cleaning and removing excessive plankton and culture medium, and then carrying out fixing treatment on the substrate with the dental plaque biomembrane formed on the surface by using a fixing agent to obtain the isolated dental plaque biomembrane model for evaluating the efficacy of oral products.
In the step (1), the streptococcus mutans is subjected to activation culture so as to improve the activity and quantity of the streptococcus mutans. The culture of different strains has different proper nutrition components, and the inventor carries out the culture in a trypticase soy agar culture medium (TSA culture medium) and a brain heart infusion culture medium (BHI culture medium) respectively in the earlier stage, and because TSA is a general nutrition culture medium, the culture medium is mainly used for the culture of various microorganisms, but is mainly used for the colony counting culture of microorganisms, and the growth of the streptococcus mutans in the oral cavity is slower on the culture medium. Compared with the method, the BHI culture medium is mainly used for enrichment culture of bacteria, and under the same culture conditions, the oral streptococcus mutans is in a good growth state in the BHI culture medium.
The substrate is used for co-culture with artificial saliva so as to form a saliva-obtaining film on the surface of the substrate, which is helpful for the subsequent formation of dental plaque biomembrane. The artificial saliva is adopted because the artificial saliva has strong controllability and high repeatability, and the saliva of natural people is directly adopted, so that the saliva is easy to have different compositions due to different collected samples, and the whole preparation scheme has low controllability and poor repeatability.
In step (2), during the co-cultivation, bacteria adhere and aggregate on the substrate on which the saliva-obtaining film is formed, and proliferate in large amounts to form mature plaques. Sucrose is added into a BHI culture medium, and the streptococcus mutans in the oral cavity can synthesize a large amount of extracellular polysaccharide by utilizing the sucrose, so that the production of the extracellular polysaccharide, in particular, the dextran which is insoluble in water is the key of the streptococcus mutans caries. The glucosyltransferase synthesizes glucan, which can provide adhesion and binding sites for bacteria and participate in forming a biological film substrate on which the oral mutans streptococcus is adhered and grows, the biological film substrate can accommodate various bacteria, also provides nutrients for adhered bacteria, avoids the forced damage of external factors such as environmental factors, host destruction, the use of antibiotics and the like, and limits the loss of nutrients and acid products in the biological film, so that the adhesion of the oral mutans streptococcus is enhanced by the addition of sucrose so as to facilitate better adhesion. In addition, the substances eaten by human bodies contain a large amount of sugar, which is also the key of oral bacteria adhesion and is also a nutrient substance required by bacterial growth.
According to the embodiment of the invention, the volume ratio of the activated bacteria liquid to the brain heart infusion medium containing sucrose is 1: (8-10). Thus, the streptococcus mutans can absorb nutrient components conveniently, and the enrichment and the improvement of the biological activity are facilitated.
According to the embodiment of the invention, the concentration of the strain enrichment solution is 10 6 ～10 9 CFU/mL, preferably 10 7 ～10 8 CFU/mL. The inventor obtains the above preferred bacterial concentration through a great deal of experiments, so that the structure and the texture of the dental plaque biological film model obtained finally are better, the absorbance value of the mixed solution obtained by mixing the dental plaque biological film and 1 ml of solvent is 15-20 at the wavelength of 575nm, and the accurate evaluation of the oral care product by using the mixed solution is facilitated.
According to the embodiment of the invention, the co-culture time is 20-80 hours, so that the finally formed dental plaque biomembrane is strong in stability and moderate in viscosity, and the dental plaque biomembrane is beneficial to accurately evaluating an oral care product. In some embodiments, the oral care product is a dental rinse and the plaque biofilm model requires a co-incubation time of 40 to 80 hours. In some embodiments, the oral care product is an electric toothbrush and the plaque biofilm model requires a co-cultivation time of 20 to 30 hours.
According to the embodiment of the invention, the washing to remove the superfluous plankton and the culture medium is performed by adopting PBS buffer solution or water; the fixing agent is selected from paraformaldehyde solution; the fixing process includes: and (3) contacting the substrate with the dental plaque biomembrane formed on the surface with 1-8% of paraformaldehyde solution, standing for 5-10 min, and cleaning the obtained substrate to obtain the dental plaque biomembrane model.
The PBS buffer solution or water is adopted for cleaning, so that not only can the superfluous plankton and culture medium which do not form the biological film be effectively removed, but also the structure of the biological film can be prevented from being damaged. The dental plaque biomembrane structure can be effectively fixed by adopting the fixing treatment conditions and the fixing agent, so that the dental plaque biomembrane structure can exist stably without damaging the structure.
According to an embodiment of the present invention, the sucrose content in the brain heart infusion medium containing sucrose is 1 to 5 mass%, preferably 1 mass%. The inventor obtains the better sucrose content through a large number of experiments, so that the dental plaque biomembrane is formed, the obtained dental plaque biomembrane has moderate viscosity, the cleaning efficacy of oral products is evaluated, the cleaning efficacy of different oral products can be verified by a unified standard, and the difference of the cleaning efficacy of the oral products can be well screened.
According to an embodiment of the invention, the cultivation is performed under anaerobic conditions at 35-40 ℃. Thereby facilitating the growth and reproduction of the oral cavity mutans streptococcus.
It should be noted that the features and advantages described above for the plaque biofilm model are equally applicable to this application and are not described here in detail.
Method for evaluating plaque cleaning efficacy of oral products
In yet another aspect of the invention, the invention provides a method of evaluating the plaque cleaning efficacy of an oral product. According to an embodiment of the invention, the method comprises: (A) Treating the dental plaque biofilm of the isolated dental plaque biofilm model previously described for evaluating efficacy of an oral product with an oral care product to be tested; (B) The efficacy of the oral care product to be tested is evaluated based on the changes in the plaque biofilm before and after treatment. Therefore, the dental plaque biomembrane model can be used for evaluating the dental plaque cleaning effect of an oral product, and has the advantages of high accuracy, strong repeatability, good stability and the like, and has good application prospect.
According to an embodiment of the invention, the method comprises preparing an ex vivo plaque biofilm model for evaluating the efficacy of an oral product, the preparation method comprising a preparation method of an ex vivo plaque biofilm model for evaluating the efficacy of an oral product as described in the application of an ex vivo plaque biofilm model for evaluating the efficacy of an oral product. The specific preparation method is consistent with the features and advantages of the aforementioned "application of the isolated dental plaque biofilm model in evaluating oral product efficacy" regarding the "preparation method of an isolated dental plaque biofilm model for evaluating oral product efficacy", and will not be described in detail herein.
According to an embodiment of the invention, the dental plaque biofilm model is pre-stained prior to the treatment of the ex vivo dental plaque biofilm model for evaluating efficacy of the oral product. Thereby facilitating the observation of the morphology of the plaque biofilm.
According to an embodiment of the invention, the change in plaque biofilm comprises at least one of: the morphological change of the dental plaque biomembrane, the absorbance value change of the dental plaque biomembrane and the surface color difference value change of the dental plaque biomembrane.
The morphology and surface color differences of plaque biofilm before and after treatment of the oral care product can be detected using known methods. If bacterial suspension is directly prepared by dental plaque biological film to be treated to measure absorbance value, the subsequent experiment of oral care product treatment cannot be performed. Therefore, a dental plaque biofilm model obtained by an experiment parallel to the preparation of the dental plaque biofilm model to be treated is used as a test object, and is prepared into a bacterial suspension, and the absorbance value of the bacterial suspension is measured and is recorded as the absorbance value of the dental plaque biofilm before treatment. The treated dental plaque biofilm can be directly prepared into bacterial suspension, and the absorbance value of the bacterial suspension can be measured and recorded as the absorbance value of the treated dental plaque biofilm.
According to embodiments of the present invention, the oral care product comprises an electric toothbrush or a dental rinse.
According to an embodiment of the present invention, the oral care product to be tested is a dental rinse, and step (a) comprises: fixing the dental plaque biomembrane model, and adjusting the vertical distance between the nozzle of the dental irrigator and the opposite surface of the dental plaque biomembrane to be 0.1-1 cm; placing a blocking piece between the nozzle of the dental flusher and the dental plaque biomembrane, starting the dental flusher to flush the blocking piece for 3-7 s, taking away the blocking piece, enabling the dental flusher to flush the dental plaque biomembrane for 1-5 s, and airing the dental plaque biomembrane. The front 3-7 s after the tooth flusher is started can not reach the set pressure value, so that the blocking piece is firstly adopted for blocking, and after the stable pressure is reached, the blocking piece is taken away, so that the tooth flusher can directly flush dental plaque.
According to an embodiment of the present invention, the oral care product to be tested is an electric toothbrush, and step (a) comprises: fixing the dental plaque biomembrane model, simulating a human oral cavity wetting environment, enabling an electric toothbrush to horizontally slide and brush the dental plaque biomembrane for 5-8 s, simulating a human mouth rinsing action after brushing, cleaning the dental plaque biomembrane with water for 1 or 2 times, and airing the dental plaque biomembrane. By adopting the method, the tooth brushing process of a person can be simulated, and the accuracy of the evaluation result is improved.
In addition, the present invention provides another method for evaluating the plaque cleaning efficacy of an oral product. According to an embodiment of the invention, the method comprises:
(1) Activating oral Streptococcus mutans strain, selecting 1-ring activated strain, adding into a test tube containing 10mL brain heart infusion medium, sealing the test tube with sterile cotton plug, anaerobic culturing at 37deg.C for 24 hr to obtain strain multiplication solution, and diluting the strain multiplication solution to 10 with PBS buffer solution 7 ～10 8 CFU/mL, obtaining bacterial suspension;
(2) Soaking the hydroxyapatite sheet in 1mL of artificial saliva, culturing at 37 ℃ for 24 hours to form an artificial acquired membrane, discarding liquid, and flushing redundant artificial saliva on the surface of the hydroxyapatite sheet with sterile PBS;
(3) Co-culturing the bacterial suspension with a brain heart infusion culture medium containing 1 mass% of sucrose and a hydroxyapatite sheet formed with an artificially obtained film, wherein the volume of the bacterial suspension is 9 times that of the bacterial suspension, the co-culture time required by a dental plaque biomembrane model for evaluating the efficacy of a tooth irrigator is 48-72 hours, and the co-culture time required by the dental plaque biomembrane model for evaluating an electric toothbrush is 24 hours;
(4) Transferring the hydroxyapatite sheet with the dental plaque biomembrane to a new 24-hole plate after the co-culture is finished, adding deionized water to the wall, cleaning to remove superfluous plankton and culture medium, fixing for 5-10min by using 4% paraformaldehyde, cleaning superfluous formaldehyde, cleaning crystal violet staining solution for 1min, and cleaning to remove unbound staining solution after the staining is finished; determining one or more of plaque biofilm morphology, absorbance value and surface color difference value of the obtained plaque biofilm model;
(5) Aiming at the evaluation product being a dental irrigator, fixing the dental plaque biomembrane finally obtained in the step (4), and adjusting the vertical distance between a nozzle of the dental irrigator and the opposite surface of the dental plaque biomembrane to be not more than 1cm; placing an acrylic plate between the nozzle of the dental flusher and the dental plaque biomembrane, starting the dental flusher to flush the acrylic plate for 5s, taking away the acrylic plate, enabling the dental flusher to flush the dental plaque biomembrane for 2s, and airing the dental plaque biomembrane; determining one or more of the three types of the obtained dental plaque biofilm morphology, absorbance value and surface color difference value, and comparing with the corresponding parameters determined in the step (4) so as to evaluate the dental plaque cleaning efficacy of the dental irrigator; and/or
Fixing the dental plaque biomembrane model finally obtained in the step (4) aiming at the evaluation product, simulating the moist environment of the oral cavity of a human body, enabling the electric toothbrush to horizontally slide and brush the dental plaque biomembrane for 5-8 s, simulating the mouth rinsing action of the human body after the brushing is finished, cleaning the dental plaque biomembrane model for 1 or 2 times by using water, and airing the dental plaque biomembrane; determining one or more of the three of the resulting plaque biofilm morphology, absorbance values, surface color difference values, and comparing with the corresponding parameters determined in step (4) to evaluate the plaque cleaning efficacy of the electric toothbrush. Therefore, the method provided by the embodiment of the invention can be used for evaluating the plaque cleaning effect of the oral care product, and has the advantages of high accuracy, strong repeatability, good stability and the like, and has a good application prospect.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products available commercially, without the manufacturer's knowledge.
Example 1
1. Preparation of dental plaque biofilm model
S1, taking streptococcus mutans (Streptococcus mutans) in the oral cavity as a strain, activating for 24 hours, picking 1-ring strain from the activated strain by using an inoculating loop, dissolving the strain in a test tube filled with 10mL of BHI-containing liquid culture medium, plugging the test tube by using a sterile cotton plug, performing anaerobic culture for 24 hours at 37 ℃ to obtain a strain multiplication liquid, and adjusting the concentration of the strain multiplication liquid to 10 5 CFU/mL。
The hydroxyl apatite tablet (-. Gamma12 mm. Times.2 mm) was immersed in 1mL of artificial saliva (Shanghai remote mu organism, 1206A 21) and incubated at 37℃for 24h to form an artificially obtained membrane, the liquid was discarded, and the surface of the hydroxyapatite carrier was rinsed with sterile PBS for excess artificial saliva.
Mixing the bacterial strain enrichment solution with BHI-containing culture medium (1% sucrose) at a volume ratio of 1:9, wherein the total volume is 2mL, and placing the hydroxyapatite sheet with the artificially obtained membrane in the mixed culture medium, and co-culturing for 24h, 48h and 72h in an anaerobic incubator at 37 ℃.
S2, transferring the hydroxyapatite sheet with the bacterial plaque biomembrane to a new 24-hole plate, adding deionized water to the wall, cleaning to remove superfluous plankton and culture medium, fixing for 5-10min by using 4% paraformaldehyde, cleaning superfluous formaldehyde, cleaning crystal violet staining solution for 1min, cleaning to remove unbound staining solution after the staining is finished, adding 1mL of 33% glacial acetic acid into each hole, placing the hole plate on an oscillator, oscillating for 30min at room temperature, sucking 200 mu L of glacial acetic acid-crystal violet solution from each hole to a new 96-hole plate after the oscillating is finished, and measuring absorbance value at 575nm for detection.
As a result, as shown in FIG. 1, the absorbance value for the measurement of the amount of the plaque biofilm at 24 hours of co-culture was about 3 to 3.5, and the average value was 3.24. When the culture is prolonged to 48 hours, the absorbance value is continuously increased, the range is about 3.2-3.6, the average value is 3.51, the absorbance value is higher than 24 hours and is 1.08 times of 24 hours. When the culture is prolonged to 72 hours, the result is consistent with the culture for 24 hours and 48 hours, the absorbance value is continuously increased, the range is about 3.6-3.9, the average value is 3.73, and the average value is 1.06 times of 48 hours. Therefore, under the condition, the 24h bacterial plaque biomembrane can be formed, and the bacterial plaque biomembrane is more and more compact and the absorbance value is higher and higher along with the prolonged culture time. By comparison, under the same culture conditions, the amount of plaque biofilm was maintained substantially within a certain range between different batches. Therefore, the culture conditions of the invention show that the bacterial plaque biomembrane is stable to form and has strong repeatability.
Example 2 evaluation of the cleaning force of a dental irrigator based on plaque model at different culture times
1. Preparation of dental plaque biofilm model
S1, taking streptococcus mutans (Streptococcus mutans) in the oral cavity as a strain, activating for 24 hours, picking 1-ring strain from the activated strain by using an inoculating loop, dissolving the strain in a test tube filled with 10mL of BHI-containing liquid culture medium, plugging the test tube by using a sterile cotton plug, performing anaerobic culture for 24 hours at 37 ℃ to obtain a strain multiplication liquid, and adjusting the concentration of the strain multiplication liquid to 10 5 CFU/mL。
According to the test group, each experimental group of hydroxyapatite tablets (the following steps are carried out12mm×2mm) were respectively immersed in 1mL of artificial saliva, and the blank control hydroxyapatite sheet was immersed in 1mL of sterile PBS, and incubated at 37℃for 24 hours to formThe artificially obtained membrane was discarded, and the surface of the hydroxyapatite carrier was rinsed with sterile PBS for excess artificial saliva.
Mixing the strain enrichment solution with BHI-containing culture medium (1% sucrose) at a volume ratio of 1:9 according to the above concentration adjustment, and placing the above hydroxyapatite sheet with artificially obtained membrane in the mixed culture medium, and co-culturing in anaerobic incubator at 37deg.C for 24 hr.
S2, transferring the hydroxyapatite sheet with the 24-hour bacterial film to a new 24-hole plate after the culture is finished, adding deionized water to the wall, cleaning to remove superfluous plankton and culture medium, cleaning for 3 times, 2 mL/time, and standing for 1 min/time at room temperature.
S3, discarding the cleaning solution in the 24 pore plates, adding 4% paraformaldehyde into each pore of the 24 pore plates for fixing for 5min, and after fixing, adding deionized water into the wall for cleaning, cleaning to remove 4% formaldehyde solution, cleaning for 3 times, 2 mL/time, and standing for 1 min/time at room temperature.
2. Evaluation of plaque cleaning efficacy of the dental irrigator
S4, after fixation, using crystal violet staining solution for 1min for each hydroxyapatite sheet with dental plaque biological membranes, after staining, adding deionized water to the wall for cleaning, cleaning to remove unbound staining solution, cleaning for 3 times, 2 mL/time, standing for 1 min/time at room temperature, naturally airing for 10min at room temperature, placing the aired hydroxyapatite sheets in a studio, and recording the apparent state of the dental plaque biological membranes of the hydroxyapatite sheets after staining. The L x a x b x values of the stained plaque biofilm were measured using a color difference meter. Placing the dental plaque biomembrane model in an experimental group parallel to the preparation process of the dental plaque biomembrane model to be treated in an orifice plate, adding 1mL of 33% glacial acetic acid-crystal violet solution into the orifice plate, placing the orifice plate on an oscillator, oscillating at room temperature for 30min, sucking 200 mu L of bacteria-containing glacial acetic acid-crystal violet solution from each orifice to a new 96 orifice plate after the oscillation is finished, and measuring absorbance value at 575nm for detection.
S5, fixing the hydroxyapatite sheets after the fixing in the S4 on a stainless steel fixer, respectively fixing the sample 1 tooth washer and the sample 2 tooth washer on an adjustable stainless steel frame, and adjusting the vertical distance between each hydroxyapatite sheet and a corresponding tooth washer nozzle (water outlet) to be 0.1-1 cm. After the position is adjusted, firstly separating the hydroxyapatite sheet from the nozzle of the tooth washer by using a Asian plate, opening a switch according to the set gear (fixed pressure value) of the tooth washer, removing the Asian plate after water is discharged for 5s stably, flushing for 2s, closing the switch, taking down the flushed hydroxyapatite sheet, naturally airing at room temperature for 10min, and guaranteeing the edge dryness of the hydroxyapatite sheet. Meanwhile, a negative control group is arranged, and dental plaque biomembrane is not treated by adopting any tooth flusher.
And (3) placing the dried hydroxyapatite sheet in a studio, taking a photo at the same light source, the same illumination intensity and the same position as in the step (4), recording the value of Lx a x b x of the apparent form sum of the residual dental plaque biomembrane on the surface after the hydroxyapatite sheet is washed by a tooth washer, and calculating a color difference value (delta E value). And placing the hydroxyapatite sheet with the measured value of Lab in a new 24-well plate, adding 95% ethanol-crystal violet solution into each well, placing the well plate on an oscillator for 30min at room temperature, sucking 200 mu L of ethanol-crystal violet solution from each well to a new 96-well plate after the oscillation is finished, and measuring absorbance value at 575 nm.
The results are shown in FIGS. 2 to 4. The dental plaque cultured for 24 hours can not be sufficiently adhered on the hydroxyapatite sheet due to low adhesive force, and can be directly removed from the hydroxyapatite once the water flow of the dental irrigator flows through, so that some parts are not cleaned in practice, and the error is large. The growth of the dental plaque cultivated in 48 hours and 72 hours is more sufficient, the adhesion of the dental plaque is stronger, and the functional requirements of the dental irrigator can be met. From the practical point of view analysis, the dental irrigator is mainly used for cleaning dental plaque of periodontal tissues, deep layers of the teeth and other difficult-to-clean parts, the growth time of the dental plaque at the position is longer, the dental plaque is more consistent with the dental plaque cultured for 48 hours and 72 hours theoretically, and a certain mapping can be obtained in the result of the embodiment 3. Thus, 72 hours of incubation was selected as the preferred incubation time.
Example 3
The electric tooth brushing plaque cleaning efficacy is evaluated by using isolated bovine teeth, and the specific steps are as follows:
s1, selecting whole mouth cattle teeth with bacterial plaques by using isolated cattle teeth (adult cattle) as bacterial plaque carriers, performing soft tissue pretreatment, and cleaning food residues remained on the surfaces of the cattle teeth by using normal saline.
S2, uniformly smearing the pretreated dental plaque staining indicator on the surface of the teeth (with the upper part of the gingiva within 10 mm), staining for 10min, cleaning the unnecessary non-combined dental plaque indicator, and photographing and recording the dental plaque staining area on the surface of the teeth.
S3, washing four bovine teeth on the left by using the sample 1 in the embodiment 2, washing four bovine teeth on the right by using the sample 2 in the embodiment 2, washing each tooth for 2S, washing for 16S in total, respectively counting the residual plaque staining area after washing, calculating the plaque clearance, using an oral cavity product to be tested, photographing the plaque staining area after the test is finished, and counting the clearance by using the plaque flusher which does not use the sample to be tested and the residual plaque area after using the sample to be tested in the S3, thereby evaluating the effectiveness of the sample to be tested.
Results as shown in fig. 5, using isolated bovine teeth (adult cattle) as plaque carriers to evaluate plaque cleaning efficacy of oral care products is a common means in the industry and can be considered an industry standard. The plaque cleaning efficacy results of samples 1 and 2 were evaluated using this method to be consistent with example 2, indicating that the evaluation results of example 2 were accurate.
Example 4 evaluation of electric toothbrush cleaning force based on plaque model at different culture times
1. Preparation of dental plaque biofilm model
S1 to S3 are the same as those of example 2.
2. Evaluation of plaque cleaning efficacy of electric toothbrushing
S4, S4 of the embodiment 2.
S5, simulating the moist environment of the oral cavity of a human body by using the hydroxyapatite sheet after the fixing in S4, enabling the electric toothbrushes 1 and 2 to respectively treat corresponding dental plaque biological films, specifically, brushing the dental plaque biological films horizontally for 5-8 seconds, simulating the mouthwash action of the human body after the brushing is finished, cleaning the dental plaque biological film model after the brushing for 1 or 2 times by using water, and airing the dental plaque biological films. Meanwhile, a negative control group is arranged, and dental plaque biomembrane is not processed by adopting any electric toothbrush.
And (3) placing the dried hydroxyapatite sheet in a studio, taking a photo at the same light source, the same illumination intensity and the same position as in the step (4), recording the value of Lx a x b x of the apparent form sum of the residual dental plaque biomembrane on the surface after the hydroxyapatite sheet is washed by a tooth washer, and calculating a color difference value (delta E value). And placing the hydroxyapatite sheet with the measured value of Lab in a new 24-well plate, adding 95% ethanol-crystal violet solution into each well, placing the well plate on an oscillator for 30min at room temperature, sucking 200 mu L of ethanol-crystal violet solution from each well to a new 96-well plate after the oscillation is finished, and measuring absorbance value at 575 nm.
The results are shown in fig. 6 and 7. The dental plaque biomembrane formed after 48h and 72h of co-culture is too compact and high in viscosity, so that the dental plaque biomembrane is stuck on an electric toothbrush, and cannot be brushed later, and evaluation fails. The dental plaque biomembrane formed after 24 hours of co-culture has moderate viscosity, can be used for evaluating the dental plaque cleaning efficacy of the electric toothbrush, and can distinguish the efficacy of different tooth flushers. The experimental 24h results are consistent with the clinical plaque cleaning effects performed for electric toothbrushes 1 and 2.
Example 5
In this example, the method for preparing a dental plaque biofilm model of example 1 was performed, wherein the co-cultivation time of step 1 was 24 hours, and the sucrose addition concentration in the bhi medium was 0%, 0.5%, 1%, 2%, 3%, 4%, 5% in this order. Thus, the effect of different sucrose content in BHI medium on plaque biofilm formation was compared.
As shown in fig. 8, when no sucrose is added, no plaque biofilm is formed on the surface of the hydroxyapatite sheet, the average absorbance between the repetition of the plaque biofilm is 0.895, and when sucrose is added in the culture medium, the surface of the hydroxyapatite sheet forms a visible plaque biofilm, and the adhesion is better, compared with the case that no sucrose is added, the plaque biofilm is increased after the sucrose is added, but the plaque biofilm amount is not increased along with the increase of the concentration of the sucrose, and when the concentration of the sucrose is 1%, the plaque biofilm amount is almost maintained in a certain range and is between about 3.0 and 3.5, thereby indicating that the formation of the plaque biofilm depends on the addition of the concentration of the sucrose, and the effect of 1% of the sucrose is better than that of the sucrose without the addition of the sucrose and 0.5%; the addition of 2%, 3% and 5% sucrose has no significant effect on the formation of bacterial plaque. Finally, a sucrose content of 0.1% was selected as the preferred additive amount.
Example 6
In this example, the method for producing a dental plaque biofilm model according to example 1 was carried out, wherein the co-cultivation time of step 1 was 24 hours, and the concentration of the strain-increasing liquid was adjusted to be set to three concentration ranges of low concentration, medium concentration, high concentration, and the like in this order, and the low concentration was 10 6- 10 7 CFU/mL, medium concentration of 10 7- 10 8 CFU/mL, high concentration of 10 8- 10 9 CFU/mL. Thus, the effect of different bacterial concentrations on plaque biofilm formation was compared.
As shown in FIG. 9, the concentration of the strain was closely related to the adhesion of the plaque biofilm, and the low concentration range was 10 6- 10 7 CFU/mL plaque biofilm has poor adhesion, and the plaque biofilm is formed sparsely, while the medium concentration range is 10 7- 10 8 CFU/mL, the bacterial plaque biomembrane is compact, the bacterial plaque biomembrane quantity is about 1.6 times of the low concentration, and the high concentration range is 10 8- 10 9 CFU/mL, the formed bacterial plaque biomembrane is also compact, and compared with the medium concentration, the bacterial plaque biomembrane quantity is about 1 time of the medium concentration, and although the bacterial plaque biomembrane quantity is statistically different, the bacterial plaque biomembrane quantity is slowly increased. Finally, select 10 7 ～10 8 CFU/mL was used as the preferred bacteria concentration.
Example 7
In this example, the method of preparing a plaque biofilm model according to example 1 was performed, wherein the artificial saliva in step 1 was replaced with deionized water. Thus, the effect of artificial saliva on plaque biofilm formation was compared.
As a result, as shown in fig. 10, when the hydroxyapatite sheet was immersed for 24 hours in deionized water, a plaque biofilm was formed on the surface, but it was sparse, quantified by the plaque biofilm, the average absorbance between repetitions was 1.73, and immersed for 24 hours in artificial saliva, the plaque biofilm was formed on the surface, relatively dense, quantified by the plaque biofilm, and the average absorbance was 3.24, thereby demonstrating that the amount of plaque biofilm in the artificial saliva group was significantly increased compared to the deionized water immersed group, thereby demonstrating the importance of the presence of artificial saliva.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in the present specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (19)
1. An isolated dental plaque biofilm model for evaluating efficacy of an oral product, comprising:
a substrate;
the dental plaque biomembrane is formed on the surface of the substrate, and microorganisms in the dental plaque biomembrane are oral streptococcus mutans.
2. The isolated dental plaque biofilm model for evaluating the efficacy of an oral product according to claim 1, wherein the absorbance value of the mixed solution obtained by mixing the dental plaque biofilm with 1 ml of solvent is 3-4 at a wavelength of 575 nm.
3. The isolated dental plaque biofilm model for use in assessing the efficacy of an oral product according to claim 1 wherein said substrate comprises hydroxyapatite or a cell slide or a glass slide or a cover slip or a collagen coated resin or enamel slice or plate.
4. Use of an isolated dental plaque biofilm model for evaluating the efficacy of an oral product according to any of claims 1 to 3.
5. The use according to claim 4, wherein the oral product is selected from oral care electronics, preferably an electric toothbrush or a dental irrigator;
the efficacy includes a plaque cleaning efficacy and/or a plaque prevention efficacy.
6. A method of evaluating the plaque cleaning efficacy of an oral care product comprising:
(A) Treating the ex vivo plaque biofilm model of any one of claims 1-3 with an oral care product to be tested for evaluating efficacy of the oral product;
(B) The efficacy of the oral care product to be tested is evaluated based on the changes in the plaque biofilm before and after treatment.
7. The method of claim 6, wherein the method of preparing an ex vivo plaque biofilm model for evaluating efficacy of an oral product comprises:
and forming the dental plaque biomembrane on the surface of the substrate to obtain an in-vitro dental plaque biomembrane model for evaluating the efficacy of the oral product.
8. The method according to claim 7, wherein the preparation method comprises:
(1) Carrying out activation culture on the streptococcus mutans in a brain heart infusion culture medium to obtain a bacterial strain enrichment medium; immersing the substrate in artificial saliva for culture to obtain a substrate with a saliva-obtaining film formed on the surface;
(2) Co-culturing the activated bacterial liquid, the substrate with the saliva-obtained film formed on the surface and a brain heart infusion culture medium containing sucrose, and forming a dental plaque biomembrane on the surface of the substrate;
(3) Collecting the substrate with the dental plaque biomembrane formed on the surface, cleaning and removing redundant plankton and culture medium, and then fixing the substrate with the dental plaque biomembrane formed on the surface by using a fixing agent to obtain the isolated dental plaque biomembrane model for evaluating the efficacy of oral products.
9. The method according to claim 8, wherein the volume ratio of the activated bacterial liquid to the sucrose-containing brain heart infusion medium is 1: (8-10).
10. The method according to claim 8, wherein the concentration of the bacterial strain-increasing liquid is 10 6 ～10 9 CFU/mL, preferably 10 7 ～10 8 CFU/mL。
11. The method according to claim 8, wherein the co-cultivation is performed for a period of 20 to 80 hours.
12. The method of claim 8, wherein the oral care product is a dental rinse and the co-cultivation time required for the plaque biofilm model is 40 to 80 hours or the oral care product is an electric toothbrush and the co-cultivation time required for the plaque biofilm model is 20 to 30 hours.
13. The method of claim 8, wherein the washing to remove excess plankton and media is performed with PBS buffer or water; the fixing agent is selected from paraformaldehyde solution; the fixing process includes: and (3) contacting the substrate with the dental plaque biomembrane formed on the surface with 1-8% of paraformaldehyde solution, standing for 5-10 min, and cleaning the obtained substrate to obtain the dental plaque biomembrane model.
14. The method according to claim 8, wherein the sucrose content in the brain heart infusion medium containing sucrose is 1 to 5 mass%.
15. The method according to claim 8, wherein the culturing is performed under anaerobic conditions at 35 to 40 ℃.
16. The method of claim 6, wherein the treatment is preceded by a staining treatment of the plaque biofilm model prior to the treatment of the isolated plaque biofilm model for evaluating efficacy of the oral product.
17. The method of claim 6, wherein the change in plaque biofilm comprises at least one of:
morphological changes of the plaque biofilm;
the absorbance value of the plaque biofilm varies;
The surface color difference value of the dental plaque biomembrane changes.
18. The method of claim 6 wherein the oral care product to be tested is a dental rinse, step (a) comprising:
fixing the dental plaque biomembrane model, and adjusting the vertical distance between the nozzle of the dental irrigator and the opposite surface of the dental plaque biomembrane to be 0.1-1 cm;
placing a blocking piece between the nozzle of the dental flusher and the dental plaque biomembrane, starting the dental flusher to flush the blocking piece for 3-7 s, taking away the blocking piece, enabling the dental flusher to flush the dental plaque biomembrane for 1-5 s, and airing the dental plaque biomembrane; or alternatively
The oral care product to be tested is an electric toothbrush, and step (a) comprises:
fixing the dental plaque biomembrane model, simulating a human oral cavity wetting environment, enabling an electric toothbrush to horizontally slide and brush the dental plaque biomembrane for 5-8 s, simulating a human mouth rinsing action after brushing, cleaning the dental plaque biomembrane with water for 1 or 2 times, and airing the dental plaque biomembrane.
19. A method of evaluating the plaque cleaning efficacy of an oral product comprising:
(1) Activating oral Streptococcus mutans strain, selecting 1-ring activated strain, adding into a test tube containing 10mL brain heart infusion medium, sealing the test tube with sterile cotton plug, anaerobic culturing at 37deg.C for 24 hr to obtain strain multiplication solution, and diluting the strain multiplication solution to 10 with PBS buffer solution 7 ～10 8 CFU/mL, obtaining bacterial suspension;
(2) Soaking the hydroxyapatite sheet in 1mL of artificial saliva, culturing at 37 ℃ for 24 hours to form an artificial acquired membrane, discarding liquid, and flushing redundant artificial saliva on the surface of the hydroxyapatite sheet with sterile PBS;
(3) Co-culturing the bacterial suspension with a brain heart infusion culture medium containing 1 mass% of sucrose and a hydroxyapatite sheet formed with an artificially obtained film, wherein the volume of the bacterial suspension is 9 times that of the bacterial suspension, the co-culture time required by a dental plaque biomembrane model for evaluating the efficacy of a tooth irrigator is 48-72 hours, and the co-culture time required by a dental plaque biomembrane model for evaluating an electric toothbrush is 24 hours;
(4) Transferring the hydroxyapatite sheet with the dental plaque biomembrane to a new 24-hole plate after the co-culture is finished, adding deionized water to the wall, cleaning to remove redundant plankton and culture medium, fixing for 5-10min by using 4% paraformaldehyde, cleaning redundant formaldehyde, cleaning crystal violet staining solution for 1min, and cleaning to remove unbound staining solution after the staining is finished; determining one or more of plaque biofilm morphology, absorbance value and surface color difference value of the obtained plaque biofilm model;
(5) Aiming at the evaluation product being a dental irrigator, fixing the dental plaque biomembrane finally obtained in the step (4), and adjusting the vertical distance between a nozzle of the dental irrigator and the opposite surface of the dental plaque biomembrane to be not more than 1cm; placing an acrylic plate between the nozzle of the dental flusher and the dental plaque biomembrane, starting the dental flusher to flush the acrylic plate for 5s, taking away the acrylic plate, enabling the dental flusher to flush the dental plaque biomembrane for 2s, and airing the dental plaque biomembrane; determining one or more of the three types of the obtained dental plaque biofilm morphology, absorbance value and surface color difference value, and comparing with the corresponding parameters determined in the step (4) so as to evaluate the dental plaque cleaning efficacy of the dental irrigator; and/or
Fixing the dental plaque biomembrane model finally obtained in the step (4) aiming at the evaluation product, simulating the wet environment of the oral cavity of a human body, enabling the electric toothbrush to horizontally slide and brush the dental plaque biomembrane for 5-8 s, simulating the mouth rinsing action of the human body after the brushing is finished, cleaning the dental plaque biomembrane model for 1 or 2 times by using water, and airing the dental plaque biomembrane; determining one or more of the three of the resulting plaque biofilm morphology, absorbance values, surface color difference values, and comparing with the corresponding parameters determined in step (4) to evaluate the plaque cleaning efficacy of the electric toothbrush.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117292837A (en) * | 2023-09-13 | 2023-12-26 | 广州星际悦动股份有限公司 | Dental plaque generation prediction method, device, equipment and storage medium |
| CN117990728A (en) * | 2024-04-07 | 2024-05-07 | 北京大学口腔医学院 | In vitro efficacy evaluation device and method for protein denaturation blocking type tooth desensitizer |
| CN117264818B (en) * | 2023-09-15 | 2024-05-17 | 中山大学附属口腔医院 | Oral bacteria biological film culture medium and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117292837A (en) * | 2023-09-13 | 2023-12-26 | 广州星际悦动股份有限公司 | Dental plaque generation prediction method, device, equipment and storage medium |
| CN117264818B (en) * | 2023-09-15 | 2024-05-17 | 中山大学附属口腔医院 | Oral bacteria biological film culture medium and application thereof |
| CN117990728A (en) * | 2024-04-07 | 2024-05-07 | 北京大学口腔医学院 | In vitro efficacy evaluation device and method for protein denaturation blocking type tooth desensitizer |
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