CN114903893A - Anti-biofilm-remineralizing material for preventing and blocking secondary caries and preparation method and application thereof - Google Patents
Anti-biofilm-remineralizing material for preventing and blocking secondary caries and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a biofilm-resistant remineralization material for preventing and blocking secondary caries and a preparation method thereof, wherein the biofilm-resistant remineralization material is a phase-transition bovine serum albumin-octenidine solution obtained by dissolving bovine serum albumin, octenidine and tris (2-carbonylethyl) phosphine hydrochloride in hydroxyethyl piperazine ethanethiosulfonic acid buffer solution; the material can quickly form a protein nano film on the surfaces of tooth tissues and filling and repairing materials, the film has stable adhesion, good surface wettability, acid resistance and biofilm resistance, can obviously reduce the adhesion of cariogenic bacteria and resist the damage of metabolic acid production to the tooth tissues and the tooth-prosthesis bonding interface. The nano film can mediate biological mineralization, forms a nondifferential regenerative hydroxyapatite protective layer on the surface of a tooth tissue and a filling and repairing material, has mechanical properties similar to those of natural enamel, can obviously reduce the occurrence of micro-leakage after filling and repairing, and achieves the purpose of preventing and blocking the occurrence of secondary caries.
Description
Technical Field
The invention belongs to the technical field of anti-caries materials, and particularly relates to a material for preventing and blocking secondary caries, which can prevent the formation of cariogenic bacteria biofilm, mediate the surface mineralization of tooth tissues and repair materials, and reduce the micro-leakage after filling and repairing, and a preparation method and application thereof.
Background
Filling and repairing are main methods for preventing pit and furrow caries and treating caries, but the service life of the filling and repairing material is about 5-7 years, and 70% of the repairing body needs to be intervened and treated again. The annual repair failure rate caused by secondary caries is about 12.9 percent, the occurrence of the secondary caries is mainly closely related to biodegradation, stress and micro leakage between tooth tissues and repair materials caused by bacterial biofilms, the research on preventing and blocking the secondary caries mostly focuses on adding an antibacterial agent and/or a mineralizer into the repair materials or adhesives, the clinical transformation is difficult to realize, and the traditional fluoride does not obtain good clinical effect on the aspect of preventing the secondary caries, and has certain potential safety hazard particularly in the high-risk caries crowd. At present, a safe, efficient and stable material for preventing and blocking secondary caries is still lacked.
The international conference of anticarious and remineralizing materials clearly proposes that remineralizing materials need to be combined with antibacterial agents to achieve the aim of anticarious. Bovine serum albumin is prepared into a coating with amyloid characteristics, the coating can be stably adhered to the surface of a base material and is easy to carry out secondary modification, and the coating is expected to be a carrier of an anticarious material. Octenidine is a broad-spectrum antibacterial agent, can improve concentration and generate no drug resistance when being used topically, has simple operation, stable chemical properties, low toxicity to human body and environment, and stable antibacterial activity to gram-positive bacteria, gram-negative bacteria, fungi, etc.
Disclosure of Invention
In view of the above, the present invention aims to provide a biofilm-resistant remineralizing material for preventing and blocking secondary caries and a preparation method thereof, so as to overcome the technical difficulty of the current secondary caries prevention, the material can rapidly form a film on the surfaces of tooth tissues and filling and repairing materials, the film has stable adhesion and good surface wettability, has acid resistance and cariogenic bacteria biofilm resistance, can mediate biomineralization, forms an undifferentiated new hydroxyapatite protective layer on the surfaces of the tooth tissues and the filling and repairing materials, has mechanical properties similar to natural enamel, obviously reduces the occurrence of micro-leakage after filling and repairing, and achieves the purpose of preventing and blocking the occurrence of secondary caries.
In order to achieve the above objects, the anti-biofilm-remineralizing material for preventing and blocking secondary caries according to the present invention is a phase-transition bovine serum albumin-octenidine solution (denoted as PTB-OCT solution) obtained by dissolving bovine serum albumin, octenidine and tris (2-carbonylethyl) phosphine hydrochloride (denoted as TCEP-HCl) in hydroxyethylpiperazine ethanethiosulfonic acid buffer (denoted as HEPES buffer).
The anti-biofilm remineralizing material preferably comprises 100mL of HEPES buffer solution in which 0.01-1 g of bovine serum albumin, 0.001-0.8 g of octenidine, and 0.1-3 g of TCEP & HCl are dissolved.
Further, the anti-biofilm remineralizing material is preferably prepared by dissolving 0.2-0.8 g of bovine serum albumin, 0.003-0.4 g of octenidine, and 0.4-1.5 g of TCEP & HCl in 100mL of HEPES buffer solution.
The concentration of the hydroxyethylpiperazine ethanethiosulfonic acid buffer solution is 5-30 mM, and the pH is 7.0-7.4.
The zeta potential of the PTB-OCT solution is 15-20 mV.
The preparation method of the anti-biofilm-remineralizing material for preventing and blocking secondary caries comprises the following steps:
1) fully dissolving bovine serum albumin in a hydroxyethyl piperazine ethanethiosulfonic acid buffer solution to obtain a solution A;
2) completely dissolving octenidine in a hydroxyethyl piperazine ethanethiosulfonic acid buffer solution to obtain a solution B;
3) mixing the solution A and the solution B, and standing for 5-50 minutes to obtain a solution C;
4) dissolving tris (2-carbonyl ethyl) phosphine hydrochloride into a hydroxyethyl piperazine ethanethiosulfonic acid buffer solution to obtain a solution D;
5) and mixing the solution C and the solution D, and standing for 3-90 minutes to obtain a phase-transition bovine serum albumin-octenidine solution, namely an anti-biofilm-remineralization material for preventing and blocking secondary caries.
In the step 3), the solution A and the solution B are preferably mixed and kept stand for 20 to 40 minutes.
In the step 5), the solution C and the solution D are preferably mixed and kept stand for 40-80 minutes.
The invention also provides the application of the anti-biofilm-remineralization material for preventing and blocking secondary caries in the medicines and nursing products for biomimetic remineralization of demineralized dental hard tissues and preventing secondary caries.
The invention relates to an anti-biofilm-remineralization material for preventing and blocking secondary caries, which has the principle of simulating the structural characteristics and self-assembly behavior of enamel mineralization mediated by amelogenin in a human body, realizes in-situ bionic remineralization on the surface of hard tissues of a tooth body, forms a non-differential hydroxyapatite protective coating on the surface of a filling and repairing material and an adhesion interface, and fills the blank that the conventional clinical common caries-preventing product cannot play a role in repairing the filling material and the adhesion interface; meanwhile, the anti-biofilm-remineralization material is loaded with an antibacterial agent, has good antibacterial property on cariogenic bacteria, and can prevent and block the occurrence and the development of caries at the source.
Compared with the prior art, the anti-biofilm-remineralizing material for preventing and blocking secondary caries has the following beneficial effects:
the preparation process of the material is simple, convenient and quick, the condition is mild, the material is green and environment-friendly, the PTB-OCT solution forms a coating (PTB-OCT coating for short) on the surface of the tooth, the adhesion is stable, the surface wettability is good, the adhesion of cariogenic bacteria and metabolites thereof on the surface of hard tissues of the tooth and the surface of the filling and repairing material can be obviously reduced, and the material has good biofilm resistance; meanwhile, the PTB-OCT coating has good acid resistance, can resist the damage of bacteria metabolism acid production to tooth tissues and a tooth-prosthesis bonding interface, and reduces the occurrence of micro leakage; in addition, the PTB-OCT coating can mediate the formation of indiscriminate hydroxyapatite protective coating on the surface of tooth body tissues and filling repair materials, remineralize demineralized tooth body hard tissues, simultaneously play a role in protecting the tooth-prosthesis bonding interface and the surface of the filling prosthesis, and prevent and block the occurrence of secondary caries. The material has good biological safety, low cost and simple operation, and has ideal application effect and potential clinical transformation value.
Drawings
FIG. 1 is a transmission electron micrograph of the PTB solution and PTB-OCT solution prepared in experiment 1.
FIG. 2 shows zeta potential values of bovine serum albumin solution, OCT solution, PTB solution and PTB-OCT solution in experiment 2.
Fig. 3 is the acid resistance characterization result of the PTB-OCT coating formed in experiment 3.
FIG. 4 is a scanning electron micrograph of the coating formed by PTB-OCT solution, OCT solution and PTB solution, respectively, inducing remineralization of the enamel surface in experiment 4.
FIG. 5 is a confocal laser microscopy image of anti-Streptococcus mutans biofilm in PTB-OCT, and PTB solutions for experiment 5.
FIG. 6 is a sectional microscope image of microleakage staining after resin fill cooling and heating cycles in experiment 6.
Detailed Description
The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.
Example 1
1) At normal temperature, 66mg of bovine serum albumin is fully dissolved in 10mL of HEPES buffer solution to obtain solution A;
2) fully dissolving 1mg octenidine in 10mL HEPES buffer solution to obtain solution B;
3) mixing the solution A and the solution B, and standing for 30 minutes to obtain a solution C;
4) dissolving 143mg of TCEP & HCl in 10mL of HEPES buffer solution to obtain a solution D;
5) and mixing the solution C and the solution D, and standing for 1 hour to obtain a PTB-OCT solution, namely an anti-biofilm-remineralizing material for preventing and blocking secondary caries.
Comparative example 1
In example 1, the solution B was replaced with an equal volume of ultrapure water, and the other steps were the same as in example 1, to obtain a phase-converted bovine serum albumin solution.
Comparative example 2
In example 1, the solution a was replaced with an equal volume of ultrapure water, and the other steps were the same as in example 1, to obtain an octenidine solution.
Example 2
1) At normal temperature, 66mg of bovine serum albumin is fully dissolved in 10mL of HEPES buffer solution to obtain solution A;
2) fully dissolving 10mg octenidine in 10mL HEPES buffer solution to obtain solution B;
3) mixing the solution A and the solution B, and standing for 30 minutes to obtain a solution C;
4) 71mg of TCEP & HCl was dissolved in 10mL of HEPES buffer solution to obtain solution D;
5) and mixing the solution C and the solution D, and standing for 1 hour to obtain a PTB-OCT solution, namely an anti-biofilm-remineralizing material for preventing and blocking secondary caries.
Example 3
1) Fully dissolving 100mg of bovine serum albumin in 10mL of HEPES buffer solution at normal temperature to obtain solution A;
2) fully dissolving 60mg octenidine in 10mL HEPES buffer solution to obtain solution B;
3) mixing the solution A and the solution B, and standing for 30 minutes to obtain a solution C;
4) dissolving 143mg of TCEP & HCl in 10mL of HEPES buffer solution to obtain a solution D;
5) and mixing the solution C and the solution D, and standing for 1 hour to obtain a PTB-OCT solution, namely an anti-biofilm-remineralizing material for preventing and blocking secondary caries.
Example 4
1) Fully dissolving 20mg of bovine serum albumin in 10mL of HEPES buffer solution at normal temperature to obtain solution A;
2) fully dissolving 10mg octenidine in 10mL HEPES buffer solution to obtain solution B;
3) mixing the solution A and the solution B, and standing for 30 minutes to obtain a solution C;
4) 286mg of TCEP & HCl is dissolved in 10mL of HEPES buffer solution to obtain a solution D;
5) and mixing the solution C and the solution D, and standing for 1 hour to obtain a PTB-OCT solution, namely an anti-biofilm-remineralizing material for preventing and blocking secondary caries.
Example 5
1) Fully dissolving 200mg of bovine serum albumin in 10mL of HEPES buffer solution at normal temperature to obtain solution A;
2) fully dissolving 100mg of octenidine in 10mL of HEPES buffer solution to obtain a solution B;
3) mixing the solution A and the solution B, and standing for 30 minutes to obtain a solution C;
4) 71mg of TCEP & HCl was dissolved in 10mL of HEPES buffer solution to obtain solution D;
5) and mixing the solution C and the solution D, and standing for 1 hour to obtain a PTB-OCT solution, namely an anti-biofilm-remineralizing material for preventing and blocking secondary caries.
Example 6
1) Fully dissolving 100mg of bovine serum albumin in 10mL of HEPES buffer solution at normal temperature to obtain solution A;
2) fully dissolving 60mg octenidine in 10mL HEPES buffer solution to obtain solution B;
3) mixing the solution A and the solution B, and standing for 30 minutes to obtain a solution C;
4) 430mg of TCEP & HCl was dissolved in 10mL of HEPES buffer to obtain solution D;
5) and mixing the solution C and the solution D, and standing for 1 hour to obtain a PTB-OCT solution, namely an anti-biofilm-remineralization material for preventing and blocking secondary caries.
In order to prove the beneficial effects of the invention, the inventor carries out a large number of laboratory research experiments, and the specific experiments are as follows:
experiment 1: in comparative example 1, the solution C and the solution D were mixed, and samples were taken after standing for 5 minutes and 30 minutes, respectively, the samples were dropped on a copper mesh, and transmission electron microscopy was performed after negative staining with phosphotungstic acid, and the results showed that nanoparticles having a diameter of about 10 to 100nm were formed after standing for 5 minutes, and were varied in shape and size and dispersed in distribution (FIG. 1 a); after 30 minutes of standing, the oligomer was partially aggregated and connected in a chain form (FIG. 1 b). In example 1, the solution C and the solution D are mixed, and the mixture is respectively sampled after standing for 3 minutes and 20 minutes, the sampled sample is dropped on a copper mesh, and transmission electron microscope detection is carried out after phosphotungstic acid negative staining, and the result shows that nanoparticles with the diameter of about 20-100 nm are formed after standing for 3 minutes, have different sizes and are scattered and distributed (figure 1C); after standing for 20 minutes, the oligomers partially aggregated and connected into chains and sheets (FIG. 1 d). Indicating that the nanoparticles increased slightly in size with the addition of OCT and accelerated aggregation of oligomers and thus accelerated film formation with the addition of OCT.
Experiment 2: the zeta potential of each solution was measured using a particle size potentiometer, taking 1mL each of a bovine serum albumin solution (solution A), an octenidine solution (OCT solution) obtained in comparative example 2, a phase-transition bovine serum albumin solution (PTB solution) obtained in comparative example 1, and a PTB-OCT solution obtained in example 1, and the results are shown in FIG. 2. As can be seen, the PTB-OCT solution of example 1 is positively charged with a zeta potential of 17.9. + -. 2.1 mV.
Experiment 3: the silicon wafer was placed in the PTB-OCT solution obtained in example 1, left to stand for 1 hour, treated with a hydrochloric acid solution having a pH of 2, an aqueous sodium hydroxide solution having a pH of 11, an aqueous lactic acid solution having a pH of 4, and a clinical 35% phosphoric acid gel for 30 seconds, rinsed thoroughly with deionized water, and dried. All silicon wafers are soaked in 0.1mg/mL thioflavin T (ThT) solution for dyeing for 1 minute, washed by deionized water, and observed by a laser confocal microscope for peeling of the PTB-OCT coating formed on the surface of the silicon wafer, and the result is shown in figure 3. As can be seen, hydrochloric acid and sodium hydroxide destroy most of the PTB-OCT coating, while lactic acid and phosphoric acid cause only a small amount of peeling of the PTB-OCT coating.
Experiment 4: collecting complete non-carious and non-obvious mill removed due to treatmentConsuming 20 impacted teeth, washing with 3% sodium hypochlorite and normal saline, and removing soft tissues and dirt. Permanent tooth mesial, distal, buccal and lingual enamel slabs were cut using a low speed dental mill under running water cooling to prepare about 4mm x 2mm (length x width x thickness) enamel specimens. All samples were treated with 35% phosphoric acid etching agent for 30 seconds, rinsed thoroughly with deionized water, and then placed in the PTB-OCT solution obtained in example 1 and allowed to stand for 1 hour. 50mM CaCl is sequentially dipped by a special dental medicine coating rod 2 Aqueous solution and 30mM KH 2 PO 4 The aqueous solution was applied for 10 seconds, thoroughly dried and repeated three times. SEM examination was performed after gradient dehydration and drying, and the characterization results are shown in FIG. 4. It can be seen from the figure that the normal permanent enamel appearance, the surface is a layer of compact enamel without enamel columns (figure 4a), after being treated by 35% phosphoric acid etching agent, the surface presents fish scales, the enamel without enamel columns disappears, the enamel columns and enamel column interstitials can be seen (figure 4b), after being induced and mineralized by the PTB-OCT coating formed on the surface, the fish scales structure on the enamel surface is completely covered by crystals, the new crystals are in a sheet shape and similar in size, and are aggregated and fused with each other to form a thicker mineral layer (figures 4c, d). The PTB-OCT solution can form a PTB-OCT coating on the surface of the enamel, and can induce the formation of a new hydroxyapatite layer after being simply and quickly coated with the calcium-phosphorus solution, promote the in-situ remineralization of demineralized enamel and repair the dental lesion. Whereas the acid-etched enamel treated with PTB solution in comparative example 1 (fig. 4e) and OCT solution in comparative example 2 (fig. 4f) both appeared fish-scaly and failed to repair the demineralized enamel.
Experiment 5: the enamel pieces were placed in the PTB-OCT solution obtained in example 1, the PTB solution obtained in comparative example 1, and the OCT solution obtained in comparative example 2, and left to stand for 1 hour. Centrifuging Streptococcus mutans bacterial liquid at 5000rpm for 5 minutes, resuspending, and adjusting bacterial liquid OD with spectrophotometer 600 To 0.8 (about 1X 10) 8 CFU/mL). 1mL of the resuspended suspension was added to a well plate of enamel plate and incubated at 37 ℃ for 24 hours and 48 hours under anaerobic conditions. After the culture is finished, washing with sterile deionized water. Mixing the acridine orange solution and the hexylidine bromide solution according to the volume ratio of 1:1 to form a working solution, transferring the tooth enamel sheet to a new pore plate, adding 1mL of sterile PBS and 20 mu L of the working solution into each pore, and keeping the mixture at room temperature in the darkAfter standing for 5 minutes, the surface of the enamel piece is lightly washed with sterile PBS to remove excessive staining agent, and the adhesion condition of bacteria and the distribution condition of live and dead bacteria on the surface of the enamel piece are observed by using a laser confocal microscope, and the characterization result is shown in FIG. 5. As can be seen, after 24 hours and 48 hours of bacterial culture, Streptococcus mutans biofilm was formed on the surface of the enamel slab (see FIGS. 5a and b), bacterial adhesion was significantly reduced but mostly viable on the enamel surface after PTB solution treatment (see FIGS. 5c and d), and dead bacteria was found on the enamel surface after OCT solution treatment (see FIGS. 5e and f), and the PTB-OCT coating exhibited good anti-fouling and anti-bacterial properties and significantly reduced bacterial adhesion and mostly dead bacteria was found (see FIGS. 5g and h).
Experiment 6: preparing V-shaped holes with the length of 3mm, the height of 2mm and the depth of 2mm on the cheek and the tongue side in vitro, washing and drying the holes, coating 3M adhesive, curing by light for 20 seconds, filling the holes with 3M Z350 paste resin, and curing by light for 20 seconds. After filling, a PTB-OCT coating is constructed on the tooth surface and the resin surface by randomly selecting one side of the in-vitro tooth from the filled tooth surface and the resin surface by using the PTB-OCT solution in the embodiment 1, and the coating is repeated for 3 times by coating calcium chloride solution and dipotassium hydrogen phosphate solution for 10 seconds respectively. The separated tooth is placed in deionized water and is kept at the constant temperature of 37 ℃ for 24 hours. It was then placed in a water bath at 55 ℃ and 4 ℃ for 30 seconds each, with 1 minute as a cycle, and repeated 500 times. Uniformly coating 2 layers of waterproof nail polish on the surface of the tooth body beyond 1mm from the edge of the resin, and sealing the root part with wax after the waterproof nail polish is completely dried. The excised teeth were completely placed in 1% methylene blue solution for 24 hours, and the excess dye solution was rinsed off with running water. The tooth root was removed along the 1mm below the enamel cementum interface using a low speed dental mill, the sample was cut into dental sections of approximately 1mm thickness along the long axis of the tooth, the dye penetration was observed using a stereomicroscope, and the characterization results are shown in fig. 6. It can be seen that there was a varying degree of penetration of the methylene blue dye into the tooth-resin interface (fig. 6a), and that the PTB-OCT coating was constructed to significantly reduce the incidence of microleakage (fig. 6 b).
Claims (8)
1. Anti-biofilm-remineralizing material for preventing and blocking secondary caries, characterized in that: the anti-biofilm-remineralization material is a phase-transition bovine serum albumin-octenidine solution obtained by dissolving bovine serum albumin, octenidine and tris (2-carbonylethyl) phosphine hydrochloride in a hydroxyethyl piperazine ethanethiosulfonic acid buffer solution.
2. Anti-biofilm-remineralizing material for the prevention and the blockade of secondary caries according to claim 1, characterized in that: 0.01-1 g of bovine serum albumin, 0.001-0.8 g of octenidine, and 0.1-3 g of tris (2-carbonylethyl) phosphine hydrochloride are dissolved in 100mL of hydroxyethylpiperazine ethanethiosulfonic acid buffer solution.
3. Anti-biofilm-remineralizing material for the prevention and the blockade of secondary caries according to claim 1, characterized in that: 0.2 to 0.8g of bovine serum albumin, 0.003 to 0.4g of octenidine, and 0.4 to 1.5g of tris (2-carbonylethyl) phosphine hydrochloride are dissolved in 100mL of hydroxyethylpiperazine ethanethiosulfonic acid buffer solution.
4. Anti-biofilm-remineralizing material for the prevention and the blockade of secondary caries according to claim 1, characterized in that: the concentration of the hydroxyethyl piperazine ethanethiosulfonic acid buffer solution is 5-30 mM, and the pH value is 7.0-7.4.
5. Anti-biofilm-remineralizing material for the prevention and the blockade of secondary caries according to claim 1, characterized in that: the zeta potential of the phase-transition bovine serum albumin-octenidine solution is 15-20 mV.
6. A method for preparing an anti-biofilm-remineralizing material for preventing and blocking secondary caries according to any one of claims 1 to 5, characterized by comprising the steps of:
1) fully dissolving bovine serum albumin in a hydroxyethyl piperazine ethanethiosulfonic acid buffer solution to obtain a solution A;
2) completely dissolving octenidine in a hydroxyethyl piperazine ethanethiosulfonic acid buffer solution to obtain a solution B;
3) mixing the solution A and the solution B, and standing for 5-50 minutes to obtain a solution C;
4) dissolving tris (2-carbonyl ethyl) phosphine hydrochloride into a hydroxyethyl piperazine ethanethiosulfonic acid buffer solution to obtain a solution D;
5) and mixing the solution C and the solution D, and standing for 3-90 minutes to obtain a phase-transition bovine serum albumin-octenidine solution, namely an anti-biofilm-remineralization material for preventing and blocking secondary caries.
7. The method for the preparation of anti-biofilm-remineralizing material for the prevention and the blockade of secondary caries according to claim 6, characterized in that: in the step 3), mixing the solution A and the solution B, and standing for 20-40 minutes; and in the step 5), mixing the solution C with the solution D, and standing for 40-80 minutes.
8. Use of the anti-biofilm-remineralizing material for preventing and blocking secondary caries according to any one of claims 1 to 5 in pharmaceutical and care products for the biomimetic remineralization of demineralized dental hard tissues and for the prevention of secondary caries.
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| US20140051036A1 (en) * | 2012-08-15 | 2014-02-20 | Helmholtz-Zentrum Fuer Infektionsforschung Gmbh | Tooth filling materials and dental varnish for inhibiting the formation of a biofilm of streptococcus mutans and the production thereof |
| CN111000747A (en) * | 2018-12-31 | 2020-04-14 | 美釉(西安)生物技术有限公司 | Mineralized material for preventing enamel demineralization and application thereof |
| 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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