CN114533565B - Mineralization material for preventing dentin sensitivity and preventing caries, and preparation method and application thereof - Google Patents
Mineralization material for preventing dentin sensitivity and preventing caries, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a mineralization material for preventing dentin sensitivity and preventing caries, a preparation method and application thereof, and belongs to the technical field of dental materials. The mineralized material for preventing dentin sensitivity and preventing caries is a calcium phosphate nano bionic mineralized material loaded with yolk immunoglobulin, can effectively block exposed dentin tubules, can effectively inhibit growth of cariogenic bacteria streptococcus mutans, and has good safety.
Description
Technical Field
The invention relates to the technical field of dental materials, in particular to a mineralized material for preventing dentin sensitivity and preventing caries, a preparation method and application thereof.
Background
Dentin sensitivity refers to an abnormal soreness symptom which occurs when dentin tubules are directly opened in the oral environment and then subjected to external stimulus such as cold, hot, sour and sweet due to abrasion, acid erosion, caries, wedge-shaped defects and the like. The currently widely accepted pathogenesis of dentinal sensitivity is the "hydrodynamic theory" based on which the opening of dentinal tubules results in the transfer of stimulation from the dentinal fluid within the tubules to the pulp is a key factor in the development of dentinal sensitivity. Thus, for this pathogenesis, the most immediate approach to treating dentinal sensitivity is to occlude the dentinal tubules. Researchers have used various materials or means such as fluoride, oxalate, dentin adhesive and laser treatment for sealing dentinal tubules to treat dentinal sensitivity for decades, and the sealing treatment effect is still not ideal, but although sensitive symptoms can be relieved, the duration depends on the depth of the material sealing dentinal tubules, and problems such as excessive fluorine risk, cytotoxicity, and need of professional doctors to operate exist, so further improvement is still needed.
Because the main component of normal dentin is hydroxyapatite crystal, the mineral compounds with similar composition components are utilized to seal dentinal tubules, which is more in line with the bionics concept. Because of its superior biological properties and better biocompatibility, the calcium phosphate system material can act as a reservoir of calcium ions and phosphate ions in the oral environment, releasing a large amount of calcium and phosphate ions after contacting saliva, and the calcium and phosphate ions rapidly deposit to form insoluble calcifications to seal dentinal tubules and promote remineralization.
Dentin of dentinally sensitive patients is often exposed to the oral cavity, where it mineralizes less than enamel, is more prone to caries and progresses rapidly. The main cariogenic bacteria streptococcus mutans in the oral cavity can be adhered to the tooth surface to generate acidic substances, so that the hard tissues of the tooth body are demineralized and remineralized, and finally caries is caused, thereby influencing the sealing effect. At present, chlorhexidine, fluoride, metal ions, traditional Chinese medicines and the like are doped in dentin desensitizing materials, and although the effect of inhibiting cariogenic bacteria can be provided, the defects of easy generation of drug-resistant strains, cytotoxicity, tooth discoloration, complex extraction process, special taste and the like exist.
Disclosure of Invention
The invention aims to solve the problems, and aims to provide a mineralized material for preventing dentin sensitivity and preventing caries, a preparation method and application thereof.
The invention provides a mineralization material for preventing dentin sensitivity and caries, which has the following characteristics: is a calcium phosphate nano bionic mineralization material loaded with yolk immunoglobulin.
In the mineralization material for preventing dentin sensitivity and caries provided by the invention, the mineralization material can also have the following characteristics: wherein, the particle size of the mineralized material for preventing dentin sensitivity and caries is 60nm-80nm.
The invention provides a preparation method of mineralized material for preventing dentin sensitivity and caries, which has the characteristics that the method comprises the following steps: the mineralized material for preventing and treating dentin sensitivity and caries is prepared by mixing and mineralizing calcium chloride solution, DMEM solution and yolk immunoglobulin powder to obtain a mixture, centrifuging, washing and drying the mixture.
The preparation method of the mineralized material for preventing dentin sensitivity and caries provided by the invention can also have the following characteristics: when the mixture mineralization is carried out, firstly, the DMEM solution and the egg yolk immunoglobulin powder are mixed, and incubated for a first preset time in a shaking table to obtain a first mixed solution, then, the calcium chloride solution is added, and mineralization is carried out for a second preset time in the shaking table to obtain a mixture.
The preparation method of the mineralized material for preventing dentin sensitivity and caries provided by the invention can also have the following characteristics: wherein the mass volume ratio of the yolk immunoglobulin powder to the DMEM solution is 2g:1L.
The preparation method of the mineralized material for preventing dentin sensitivity and caries provided by the invention can also have the following characteristics: wherein the concentration of the calcium chloride solution is 1mol/L,
the volume ratio of the calcium chloride solution to the first mixed solution is 1:50.
The preparation method of the mineralized material for preventing dentin sensitivity and caries provided by the invention can also have the following characteristics: wherein the temperature of the shaking table is 37 ℃, the rotating speed is 170rpm-200rpm, the first preset time is 11h-13h, and the second preset time is 20min-40min.
The preparation method of the mineralized material for preventing dentin sensitivity and caries provided by the invention can also have the following characteristics: wherein, the conditions of centrifugation are: the rotation speed is 9000rpm, the time is 5min, and the drying is freeze drying.
The invention provides application of mineralization material for preventing dentin sensitivity and caries in closed dentinal tubules.
The invention provides an application of mineralized material for preventing dentin sensitivity and preventing caries.
Effects and effects of the invention
According to the mineralized material for preventing dentin sensitivity and caries, the preparation method and the application thereof, because the mineralized material is a calcium phosphate nano bionic mineralized material loaded with egg yolk immunoglobulin (IgY), the nano-structured calcium phosphate particles have the advantage of higher specific surface area, have small size and relatively stable, can better permeate into a demineralized area, and achieve the function of deep mineralization sealing; igY is used as an immune anticarious substance, is derived from eggs laid by immune hens, has the function of specifically inhibiting streptococcus mutans, can block interaction between bacteria and tooth surfaces, and specifically comprises the steps of blocking adhesion of receptors participating in dental surface colonization or aggregation (glucan binding sites) or blocking activation of glucosyltransferase, has the advantages of no pathogenicity, no sensitization, safety and effectiveness in reaching food non-toxicity standards and the like, has relatively stable biological activity at pH of 4.0-11, ensures that antibodies at local parts of the oral cavity are maintained at a higher level, and is suitable for application in oral environment. Therefore, the mineralized material can effectively inhibit the growth of cariogenic bacteria streptococcus mutans while effectively closing the exposed dentinal tubules, and has good safety.
In addition, in the preparation method, sodium dihydrogen phosphate in the DMEM solution is used as a phosphorus source, and the self-assembly and degradation process is regulated by simply regulating the environment supersaturation degree and the calcium chloride as a calcium source, so that nano calcium phosphate particles can be formed by mineralization, and the slow release of the medicament can be realized by loading the medicament (IgY).
In addition, the main component of the mineralized material is calcium phosphate similar to teeth, and has good biocompatibility; the mineralization material can induce the self-mineralization in dentin tubules through simple coating, thereby achieving the effects of sealing the dentin tubules and treating dentin sensitivity; the mineralized material can also be specifically antibacterial so as to prevent caries.
Drawings
FIG. 1 is a transmission electron microscope image of an ethanol suspension of the material prepared in the examples and comparative examples of the present invention, wherein FIG. 1a is a transmission electron microscope image of a calcium phosphate material prepared in the comparative examples, and FIG. 1b is a transmission electron microscope image of a IgY-loaded calcium phosphate nano biomimetic mineralization material prepared in the examples;
FIG. 2 is a Fourier infrared scan of materials prepared in examples and comparative examples of the present invention;
FIG. 3 is a scanning electron microscope image of test example 1 according to the present invention, wherein FIG. 3a and FIG. 3b are a surface and a cross-section scanning electron microscope image of a dentin sheet treated with deionized water after being placed in artificial saliva for 1 day, FIG. 3c and FIG. 3d are a surface and a cross-section scanning electron microscope image of a dentin sheet treated with IgY-loaded calcium phosphate nano biomimetic mineralization material after being placed in artificial saliva for 1 day, FIG. 3e and FIG. 3f are a surface and a cross-section scanning electron microscope image of a dentin sheet treated with deionized water after being placed in artificial saliva for 7 days, FIG. 3g and FIG. 3h are a surface and a cross-section scanning electron microscope image of a dentin sheet treated with IgY-loaded calcium phosphate nano biomimetic mineralization material after being placed in artificial saliva for 1 day after being placed in deionized water after being soaked in strong acid, and FIG. 3j is a surface scanning electron microscope image after being placed in strong acid after being placed in IgY-loaded calcium phosphate nano biomimetic mineralization material after being placed in artificial saliva for 1 day;
FIG. 4 is a graph of bacterial colonies after co-cultivation of the material suspensions prepared in examples and comparative examples with Streptococcus mutans suspensions.
Detailed Description
In order to make the technical means, creation characteristics, achievement purposes and effects of the present invention easy to understand, the following examples are specifically described with reference to the accompanying drawings for the mineralization material for preventing dentin sensitivity and anticaries, the preparation method and the application thereof.
< example >
The embodiment provides a mineralization material for preventing dentin sensitivity and preventing caries and a preparation method thereof.
The preparation method of the mineralized material for preventing dentin sensitivity and caries comprises the following steps:
step 1, preparing calcium chloride (CaCl) 2 ) A solution. 1.11g of calcium chloride powder was dissolved in 10mL of distilled water at room temperature, and 1mol/L CaCl was prepared by vortexing uniformly 2 A solution.
Step 2, according to the mass volume ratio of 2g:1L of powdered IgY is added into fresh DMEM medium (DMEM solution), and after uniform vortex, the mixture is placed in a shaking table with the parameters of 37 ℃ and 180rpm and incubated for 12 hours, so as to obtain a first mixed solution. The first mixed liquid is a suspension.
Step 3, adding 20 mu L of CaCl into each milliliter of the first mixed solution 2 Ratio of solution (1 mol/L), caCl was added 2 The solution (1 mol/L) was added to the first mixed solution and the mixture was further mineralized for 30min in a shaker at a parameter of 37℃and 180 rpm.
Step 4, rapidly subpackaging the mixture (4) into EP pipes with the specification of 2mL, centrifuging in a 9000rpm centrifuge for 5min, removing the supernatant after centrifuging, washing with distilled water for three times, and freeze-drying a part of the supernatant to obtain calcium phosphate nano biomimetic mineralized material powder loaded with IgY, namely mineralized material for preventing dentin sensitivity and caries; the other part of ethanol is used for suspending ultrasound, a Transmission Electron Microscope (TEM) is used for observing the shape, structure and size of the material, and a special absorption peak is observed through Fourier infrared scanning.
Comparative example
Comparative examples provide a calcium phosphate material and a method of making the same.
The preparation method of the calcium phosphate material comprises the following steps:
step 1, preparing calcium chloride (CaCl) 2 ) A solution. 1.11g of calcium chloride powder was dissolved in 10mL of distilled water at room temperature, and 1mol/L CaCl was prepared by vortexing uniformly 2 A solution.
Step 2, adding 20. Mu.L of CaCl per ml of DMEM medium 2 Ratio of solution (1 mol/L), caCl was added 2 The solution (1 mol/L) was added to fresh DMEM medium and mineralized for 30min in a shaker at 37℃and 180 rpm.
Step 3, after mineralization, rapidly subpackaging the product into an EP tube with the specification of 2mL, centrifuging in a 9000rpm centrifuge for 5min, removing the supernatant after centrifuging, cleaning with distilled water for three times, and freeze-drying a part of the supernatant to obtain calcium phosphate material powder; the other part of ethanol is used for suspending ultrasound, a Transmission Electron Microscope (TEM) is used for observing the shape, structure and size of the material, and a special absorption peak is observed through Fourier infrared scanning.
Fig. 1 is a transmission electron microscope image of an ethanol suspension of the material prepared in the examples and the comparative examples of the present invention, wherein fig. 1a is a transmission electron microscope image of the calcium phosphate material prepared in the comparative examples, and fig. 1b is a transmission electron microscope image of the IgY-loaded calcium phosphate nano biomimetic mineralization material prepared in the examples.
As shown in fig. 1, the transmission electron microscope result of fig. 1a shows that the calcium phosphate material is uniform spherical particles and has a smooth surface; the transmission electron microscope result in fig. 1b shows that compared with the calcium phosphate material in fig. 1a, the diameter of the calcium phosphate nano biomimetic mineralization material loaded with IgY is obviously reduced, about 60nm-80nm, the surface of the spherical particles is rough, which suggests that the size of the formed calcium phosphate product is smaller possibly due to the loading of IgY, and the calcium phosphate nano biomimetic mineralization material also presents a non-smooth surface structure.
Fig. 2 is a fourier infrared scan of the materials prepared in the examples and comparative examples of the present invention.
As shown in FIG. 2, the Fourier infrared scanning finds that the calcium phosphate nano-composite biomimetic mineralization of the loaded IgY is 2965cm -1 、2998cm -1 And 1536cm -1 Absorption peaks were observed, which are in agreement with the standard infrared absorption peaks of IgY alone, further suggesting successful loading of IgY protein molecules into calcium phosphate products.
Test example 1 ]
Test example 1 was used to study the closed dentinal tubule properties of IgY-loaded calcium phosphate nanobiomimetic mineralization materials prepared in the examples.
The test method is as follows:
firstly, collecting healthy and complete permanent teeth which are prevented from being removed freshly, cutting the healthy and complete permanent teeth perpendicular to a long axis of a tooth body below a glazed dentin boundary by using a slow cutting machine and a slow saw under the cooling of running water, removing exposed middle dentin of crown enamel, and continuously cutting to prepare a dentin sheet sample with the thickness of 1 mm+/-0.1 mm. The gradient SiC sand paper polishes the dentin sheet for 60s under the flushing of running water to smooth the surface, ultrasonic (power 400W) cleaning is carried out for 15min after polishing, the dentin sheet is soaked in 17% EDTA to etch 5min open dentin tubules so as to establish a sensitive dentin model, and then deionized water is used for thoroughly flushing.
Then, dipping the slurry of the calcium phosphate nano biomimetic mineralized material loaded with IgY prepared in the example by using a medical small brush (prepared by 10mg of material according to powder-liquid ratio: 200 mu L of liquid) to treat the surface of a dentin sheet for 2min, then placing the dentin sheet in artificial saliva at 37 ℃ for 1 day and 7 days (once per day), randomly taking out the dentin sheet, fully flushing with deionized water, drying, spraying gold, and observing the surface and section sealing condition of the dentin tubule by using a scanning electron microscope; the plaque after 1 day of treatment was then selected for acid resistance testing by immersing in 6% (w/v)) citric acid solution (ph=1.5) for 1 minute, and after the same removal, the plaque was rinsed with deionized water, dried, and sprayed with gold, and then observed for dentinal tubule occlusion using a scanning electron microscope. Dentin sheets treated with deionized water were used as control groups.
The test results were as follows:
fig. 3 is a scanning electron microscope image of test example 1 according to the present invention, wherein fig. 3a and 3b are a surface and a cross-section scanning electron microscope image of a dentin sheet treated with deionized water after being placed in artificial saliva for 1 day, fig. 3c and 3d are a surface and a cross-section scanning electron microscope image of a dentin sheet treated with IgY-loaded calcium phosphate nano biomimetic mineralization material after being placed in artificial saliva for 1 day, fig. 3e and 3f are a surface and a cross-section scanning electron microscope image of a dentin sheet treated with deionized water after being placed in artificial saliva for 7 days, fig. 3g and 3h are a surface and a cross-section scanning electron microscope image of a dentin sheet treated with IgY-loaded calcium phosphate nano biomimetic mineralization material after being placed in artificial saliva for 7 days, fig. 3i is a surface scanning electron microscope image of a dentin sheet treated with strong acid after being placed in artificial saliva for 1 day, and fig. 3j is a surface scanning electron microscope image of a dentin sheet treated with IgY-loaded calcium phosphate nano biomimetic mineralization material after being placed in strong acid.
As shown in FIG. 3, the surface and cross-section tubules of the deionized water treated dentin sheet were open after EDTA treatment (FIGS. 3a,3b,3e,3 f). The tubules on the surface of dentin sheet treated with IgY-loaded calcium phosphate nano biomimetic mineralizing material were mostly closed, only a few tubules were exposed (fig. 3 c), and the cross-sectional view revealed that a layer of dense mineralizer was present on the surface covering the tubules (fig. 3 d). All tubules on the surface of the dentin sheet treated for 7 days were completely closed (fig. 3 g), and dense mineralized structures were present in the deep portions of the dentin tubules (fig. 3 h). After the strong acid soaking treatment, all tubules on the surface of the dentin sheet treated by deionized water are still in an open state (figure 3 i), and most of the tubules on the surface of the dentin sheet using the calcium phosphate nano biomimetic mineralized material loaded with IgY are partially detached, but most of the tubules are still closed (figure 3 j).
The results directly prove that the calcium phosphate nano bionic mineralization material loaded with the IgY can effectively seal dentin tubules, has better acid resistance, and can self-mineralize the sealed dentin tubules by utilizing mineralized substances in the dentin tubules, thereby achieving the purpose of treating dentin hypersensitivity.
Test example 2 ]
Test example 2 was used to investigate the anticariogenic properties of IgY-loaded calcium phosphate nanobiomimetic mineralization material prepared in the examples to inhibit the growth of cariogenic bacteria.
The test method is as follows:
the materials prepared in examples and comparative examples were prepared as suspensions having a concentration of 10mg/mL, respectively, and subjected to sufficient ultrasound. Culturing Streptococcus mutans in brain-heart extract broth overnight, and adjusting concentration to 10 5 Bacterial suspension of CFU/mL is inoculated with material suspension and bacterial suspension of the same volume in a 96-well plate, after anaerobic culture for 24 hours, 5 mu L of bacterial liquid is diluted and coated on the plate, and after continuous culture in an incubator for 48 hours, the colony number on the plate is observed. The image was recorded and the average value of the total number of colonies was taken and the data graph was drawn.
FIG. 4 is a graph of bacterial colonies after co-cultivation of the material suspensions prepared in examples and comparative examples with Streptococcus mutans suspensions.
As shown in FIG. 4, the number of bacterial colonies on the plates of the calcium phosphate nano biomimetic mineralization material treated group loaded with IgY is significantly reduced, and the difference is statistically different (P < 0.05) compared with the control group.
The results directly prove that the calcium phosphate nano bionic mineralization material loaded with IgY can effectively inhibit the growth of cariogenic bacteria and can be applied to the prevention of caries.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Claims (8)
1. A mineralizing material for preventing dentin sensitivity and caries, which is characterized in that: the preparation method of the calcium phosphate nano biomimetic mineralization material for loading yolk immunoglobulin comprises the following steps:
mixing and mineralizing calcium chloride solution, DMEM solution and yolk immunoglobulin powder to obtain a mixture, centrifuging, washing, drying to obtain mineralized material for preventing dentin sensitivity and caries,
when the mixture mineralization is carried out, firstly, the DMEM solution and the egg yolk immunoglobulin powder are mixed, and incubated for a first preset time in a shaking table to obtain a first mixed solution, then, the calcium chloride solution is added, and mineralization is carried out for a second preset time in the shaking table to obtain the mixture.
2. The mineralization material for preventing dentin sensitivity and caries according to claim 1, wherein:
wherein, the particle size of the mineralized material for preventing dentin sensitivity and caries is 60nm-80nm.
3. The mineralization material for preventing dentin sensitivity and caries according to claim 1, wherein:
wherein the mass-to-volume ratio of the yolk immunoglobulin powder to the DMEM solution is 2g:1L.
4. The mineralization material for preventing dentin sensitivity and caries according to claim 1, wherein:
wherein the concentration of the calcium chloride solution is 1mol/L,
the volume ratio of the calcium chloride solution to the first mixed solution is 1:50.
5. The mineralization material for preventing dentin sensitivity and caries according to claim 1, wherein:
wherein the temperature of the shaker at the time of incubating the first predetermined time in the shaker is 37 o C，The rotation speed is 170rpm-200rpm, the first preset time is 11h-13h,
mineralizing said second predetermined time in said cradle at a temperature of 37 o C, the rotating speed is 170rpm-200rpm, and the second preset time is 20min-40min.
6. The mineralization material for preventing dentin sensitivity and caries according to claim 1, wherein:
wherein, the centrifugation conditions are: the rotation speed is 9000rpm, the time is 5min,
the drying is freeze drying.
7. Use of mineralizing material for preventing and treating dentin sensitivity and caries in preparing medicine for sealing dentinal tubules.
8. Use of mineralized material for preventing and treating dentin sensitivity and caries in preparing medicine for preventing caries is provided.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101579293A (en) * | 2009-06-16 | 2009-11-18 | 赵凯 | IgY antibody clearing agent for preventing dental decayed tooth |
| CN112716812A (en) * | 2021-01-11 | 2021-04-30 | 天津医科大学口腔医院 | Biofilm-resistant remineralizing material for root canal irrigation and blocking treatment of dental caries and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20040126384A1 (en) * | 2000-10-10 | 2004-07-01 | Yang Rong Jian | Combination of IgY against dental caries |
| WO2015023773A2 (en) * | 2013-08-14 | 2015-02-19 | University Of Tennessee Research Foundation | Tooth remineralization compositions and methods |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101579293A (en) * | 2009-06-16 | 2009-11-18 | 赵凯 | IgY antibody clearing agent for preventing dental decayed tooth |
| CN112716812A (en) * | 2021-01-11 | 2021-04-30 | 天津医科大学口腔医院 | Biofilm-resistant remineralizing material for root canal irrigation and blocking treatment of dental caries and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李艳萍 ; 刘兴容 ; 徐皑 ; 李伟 ; .酪蛋白磷酸肽钙磷液对发酵乳杆菌产酸影响的实验研究.牙体牙髓牙周病学杂志.2006,(第11期),第627-629页. * |
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