1. A method for synthesizing and detecting a modified novel antibacterial resin type pit and trench sealing agent is characterized by comprising the following steps:

step one, preparing a pit and furrow closing agent, and screening dosage relation;

secondly, performing performance detection on the prepared pit and trench sealing agent, grouping according to the mass fraction of DMADDM, and respectively detecting the photocuring depth, cytotoxicity, surface roughness and microhardness of the novel pit and trench sealing agent, wherein each group comprises 5 samples;

step three, detecting the antibacterial performance before and after biological aging;

step four, detecting micro-leakage after biological aging, respectively weighing a certain amount of pit and fissure sealant, and adding DMADDM according to the mass fractions of 0%, 2.5%, 5% and 10% for later use; collecting the third molars in vitro with complete tooth bodies and no carious lesions, randomly dividing into 4 groups, and putting 5 molars in each group.

2. The method for synthesizing and detecting the modified novel antibacterial resin type pit and trench closing agent according to claim 1, wherein in the first step, the specific process for preparing the pit and trench closing agent is as follows: respectively weighing a certain amount of pit and furrow closing agent, adding DMADDM according to the mass fractions of 0%, 2.5%, 5%, 10%, 20% and 30%, and uniformly mixing in a dark place for later use.

3. The method for synthesizing and detecting the modified novel antibacterial resin type pit and fissure sealant according to claim 1, wherein in the second step, the specific process of grouping according to the mass fraction of DMADDM is as follows:

grouping according to the mass fraction of 0%, 2.5%, 5% and 10% DMADDM, and respectively detecting the photocuring depth, cytotoxicity, surface roughness and microhardness of the pit and trench sealing agent, wherein each group comprises 5 samples.

4. The method for synthesizing and detecting the modified novel antibacterial resin type pit and groove sealing agent according to claim 1, wherein in the second step, the photocuring depth detection experiment comprises the following specific steps:

placing the prepared pit and groove sealing agents of different groups into a cylindrical mold with the inner diameter of 4mm and the height of 6mm, and performing photocuring for 20 seconds;

taking out the cured samples, and wiping the uncured liquid film at the top and bottom of each sample by using a thin paper;

recording the height of the sample by a vernier caliper, wherein the height is the curing depth;

according to the light curing depth detection result, the optimal proportion of DMADDM added into the pit and trench sealing agent is screened, and the light curing depth of the resin material is not influenced by the DMADDM with the content of less than 10%.

5. The method for synthesizing and detecting the modified novel antibacterial resin type pit and fissure sealant according to claim 1, wherein in the second step, the cytotoxicity detection is carried out by the following specific steps:

placing the prepared pit and trench sealing agents of different groups into a cylindrical mold with the inner diameter of 13mm and the height of 1mm, carrying out photocuring for 20 seconds, and then placing the prepared sample into deionized water to soak for 24 hours;

sterilizing the sample at low temperature by using ethylene oxide, soaking the sample in a cell culture medium for 24 hours to obtain a leaching solution, filtering the leaching solution, and diluting the leaching solution with a culture solution in different proportions for later use;

adding the leaching solution into a 96-well plate which is paved with human oral keratinocytes, culturing for 24 hours in a cell culture box, adding a CCK-8 reagent, reacting for 1 hour, and measuring the reading of the reagent under the wavelength of 595nm by using an enzyme-labeling instrument to obtain the cytotoxicity.

6. The method for synthesizing and detecting the modified novel antibacterial resin type pit and groove closing agent according to claim 5, wherein the different proportions of the dilution of the culture solution are as follows: 32-fold, 64-fold and 128-fold dilution.

7. The method for synthesizing and detecting the modified novel antibacterial resin type pit and groove sealing agent according to claim 1, wherein in the second step, the surface roughness detection comprises the following specific steps:

sample preparation: placing the prepared pit and trench sealing agents of different groups into a cylindrical mold with the inner diameter of 13mm and the height of 1mm, and performing photocuring for 20 seconds;

and (3) detecting the samples under an atomic force microscope, and observing 3 areas of each sample to obtain a surface topography image and an average roughness Ra value.

8. The method for synthesizing and detecting the modified novel antibacterial resin type pit and groove sealing agent according to claim 1, wherein in the second step, the microhardness detection comprises the following specific processes:

sample preparation: placing the prepared pit and groove sealing agents of different groups into a cylindrical mold with the inner diameter of 6mm and the height of 1mm, and performing photocuring for 20 seconds;

placing the sample in deionized water, soaking for 24 hours, and taking out for later use;

vickers hardness was recorded using a microhardness tester and 3 zones were observed per sample, each zone being pressed 20g for 10 seconds.

9. The method for synthesizing and detecting the modified novel antibacterial resin type pit and groove sealing agent as claimed in claim 1, wherein in the third step, the specific process of the antibacterial performance detection before and after biological aging is as follows:

respectively weighing a certain amount of pit and trench sealing agent, adding DMADDM according to the mass fractions of 0%, 2.5%, 5% and 10%, wherein each group of 6 samples is divided into a pre-aging group and a post-aging group;

the treatment is carried out according to 2 ways respectively: immediate MTT analysis; soaking the sample in a culture saliva biofilm medium for 30 days for biological aging, replacing the culture solution every day, and then performing MTT analysis;

the metabolic activity of the biomembrane reflected by the MTT analysis result is an important index for reflecting the antibacterial performance of the material.

10. The method for synthesizing and detecting the modified novel antibacterial resin type pit and groove closing agent according to claim 9, wherein the specific process of detecting the metabolic activity of the surface biological membrane before and after biological aging is as follows:

preparing a nest and furrow closing agent resin sheet by taking a 48-hole plate cover as a mould, taking out prepared resins with different DMADDM mass fractions, soaking the resins in deionized water for 24 hours, and then sterilizing the resins at low temperature by using ethylene oxide for later use;

dividing the sample into a pre-aging group and an aging group, and performing biological aging on the aging group;

placing the resin piece of the pit and fissure sealant in a 24-well plate, culturing the salivary fluid biofilm for 24 hours, and transferring the sample to a new well plate;

adding 1mL of fresh MTT solution with the concentration of 0.5mg/mL into each hole, incubating for 1 hour in a dark place, and transferring the sample into a new hole plate;

adding 1mL of dimethyl sulfoxide solution into each hole, uniformly shaking the mixture in a shaking table for 20 minutes to completely decolorize the biological membrane, and uniformly blowing the biological membrane;

200. mu.L of the solution was transferred to a 96-well plate for each well, and the OD value at a wavelength of 540 was measured using a microplate reader and recorded as the metabolic activity of the biofilm.

Background

At present, caries is the oral disease with the highest incidence rate, and the incidence rate of Chinese children caries is the first of children diseases. The Chinese children caries rate is up to 40-70%, wherein the pit and furrow caries rate is over 80%. The unique anatomical physiological characteristics of the primary molars and the occlusal grooves of young permanent teeth of children make food residues and bacterial plaques difficult to clean, so that the caries develops very quickly. Since the onset of clinical application of the pit and fissure sealing technique in 1960, the pit and fissure sealing technique has been recognized as an effective method for preventing pit and fissure caries in deciduous molars and young permanent molars.

The anticaries effect of pit and fissure sealants is directly related to the retention rate of the sealant, which is affected by the properties of the sealant material. The current pit and groove sealant materials applied to pit and groove sealing are mainly divided into two categories of light-cured composite resin and glass ions. The photocuring composite resin is most widely applied in clinic due to good aesthetic property and superior physical property. The clinical challenges currently faced by photocurable composite resins are: more bacterial plaque biofilm may be accumulated on the surface, polymerization shrinkage exists, micro leakage can be formed between the sealing agent and the tooth surface, conditions are provided for invasion of microorganisms, caries is caused, and materials are easy to age and fall off easily. Therefore, in order to prevent secondary caries after pit and furrow closure, the novel pit and furrow closure agent should have good and long-lasting antibacterial and anti-aging properties.

The clinical common pit and fissure sealant is a fourth generation product represented by 3M-Clinpro and has the advantages of releasing fluoride ions, good fluidity and the like. The 5-year retention rate is 82%, and the 10-year retention rate is only 57%. Thus, its sustained release of fluoride ion to promote remineralization and antibacterial effects has been controversial. Quaternary Ammonium Monomers (QAMs) are broad-spectrum antimicrobial agents and are widely used in the fields of industry, medical treatment, medicine and the like. The QAMs can form a polymer network with the methacrylate component of the adhesive, thereby fixing into the penetrating resin and exerting long-lasting antibacterial action. There is some controversy over the antibacterial mechanisms of QAMs, and it is widely believed that QAMs can bind to the bacterial cell membrane, causing the bacteria to burst and lyse. Dimethylamino Dodecyl Methacrylate (DMADDM) is a novel quaternary ammonium salt monomer, and has been added to thermosetting resins, adhesives, and the like for systematic study. Research shows that the dental material added with DMADDM has certain inhibition effect on oral biomembranes at different stages, and the mechanical property of the material is not obviously influenced. Meanwhile, in vitro experiments show that the material added with DMADDM has a regulating effect on the strain balance of three-strain biomembranes of streptococcus mutans (streptococcus mutans), streptococcus sanguis (streptococcus sanguinis) and streptococcus gordonii (streptococcus gordonii); in addition, related researches show that the material added with DMADDM can delay the biological aging process and achieve the anti-aging effect.

Through the above analysis, the problems and defects of the prior art are as follows: the resin pit and groove sealant has polymerization shrinkage, so that micro leakage and local biofilm accumulation are caused; the existing pit and fissure sealant has insufficient long-acting caries preventing performance.

The difficulty in solving the above problems and defects is: on the premise of not influencing the polymerization shrinkage and mechanical properties of the existing pit and fissure sealant, the problem of insufficient antibacterial property is solved, so that good long-acting anticarious property is realized.

The significance of solving the problems and the defects is as follows: in order to further improve the antibacterial performance of the photocuring pit and furrow closing agent and improve the caries prevention capability of the closing agent, DMADDM is added into the resin pit and furrow closing agent Clinpro, and the excellent antibacterial performance is utilized. Meanwhile, the modified pit and furrow closing agent is subjected to biological aging by using the oral biomembrane, so that the modified pit and furrow closing agent has long-acting and repeatable antibacterial capability while keeping good material performance, thereby effectively preventing pit and furrow caries.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for synthesizing and detecting a modified novel antibacterial resin type pit and fissure sealant.

The invention is realized in this way, a method for synthesizing and detecting a modified novel antibacterial resin type pit and groove sealing agent, the method for synthesizing and detecting the modified novel antibacterial resin type pit and groove sealing agent comprises:

step one, preparing a pit and furrow closing agent, and screening dosage relation;

secondly, performing performance detection on the prepared pit and trench sealing agent, grouping according to the mass fraction of DMADDM, and respectively performing light curing depth, cytotoxicity, surface roughness and microhardness on the pit and trench sealing agent, wherein each group comprises 5 samples;

step three, detecting the antibacterial performance before and after biological aging;

and step four, detecting the micro-leakage after biological aging, respectively weighing a certain amount of pit and fissure sealant, and adding DMADDM according to the mass fractions of 0%, 2.5%, 5% and 10% for later use. Collecting the third molars in vitro with complete tooth bodies and no carious lesions, randomly dividing into 4 groups, and putting 5 molars in each group.

Further, in the first step, the specific process for preparing the pit and fissure sealant comprises the following steps: respectively weighing a certain amount of pit and furrow closing agent, adding DMADDM according to the mass fractions of 0%, 2.5%, 5%, 10%, 20% and 30%, and uniformly mixing in a dark place for later use.

Further, in the second step, the specific process of grouping according to the mass fraction of the DMADDM is as follows:

grouping according to the mass fraction of 0%, 2.5%, 5% and 10% DMADDM, and respectively carrying out light curing depth, cytotoxicity, surface roughness and microhardness on the pit and trench sealing agent, wherein each group comprises 5 samples.

Further, in the second step, the specific process of the light curing depth detection experiment is as follows:

placing the prepared pit and groove sealing agents of different groups into a cylindrical mold with the inner diameter of 4mm and the height of 6mm, and performing photocuring for 20 seconds;

taking out the cured samples, and wiping the uncured liquid film at the top and bottom of each sample by using a thin paper;

recording the height of the sample by a vernier caliper, wherein the height is the curing depth;

according to the light curing depth detection result, the optimal proportion of DMADDM added into the pit and trench sealing agent is screened, and the light curing depth of the resin material is not influenced by the DMADDM with the content of less than 10%.

Further, in the second step, the specific process of cytotoxicity detection is as follows:

placing the prepared pit and trench sealing agents of different groups into a cylindrical mold with the inner diameter of 13mm and the height of 1mm, carrying out photocuring for 20 seconds, and then placing the prepared sample into deionized water to soak for 24 hours;

sterilizing the sample at low temperature by using ethylene oxide, soaking the sample in a cell culture medium for 24 hours to obtain a leaching solution, filtering the leaching solution, and diluting the leaching solution with a culture solution in different proportions for later use;

adding the leaching solution into a 96-well plate which is paved with human oral keratinocytes, culturing for 24 hours in a cell culture box, adding a CCK-8 reagent, reacting for 1 hour, and measuring the reading of the reagent under the wavelength of 595nm by using an enzyme-labeling instrument to obtain the cytotoxicity.

Further, the different proportions of the culture solution are specifically as follows: 32-fold, 64-fold and 128-fold dilution.

Further, in the second step, the surface roughness detection specifically comprises the following steps:

sample preparation: placing the prepared pit and trench sealing agents of different groups into a cylindrical mold with the inner diameter of 13mm and the height of 1mm, and performing photocuring for 20 seconds;

and (3) detecting the samples under an atomic force microscope, and observing 3 areas of each sample to obtain a surface topography image and an average roughness Ra value.

Further, in the second step, the specific process of microhardness detection is as follows:

sample preparation: placing the prepared pit and groove sealing agents of different groups into a cylindrical mold with the inner diameter of 6mm and the height of 1mm, and performing photocuring for 20 seconds;

placing the sample in deionized water, soaking for 24 hours, and taking out for later use;

vickers hardness was recorded using a microhardness tester and 3 zones were observed per sample, each zone being pressed 20g for 10 seconds.

Further, in the third step, the specific process of detecting the antibacterial performance before and after biological aging is as follows:

respectively weighing a certain amount of pit and trench sealing agent, adding DMADDM according to the mass fractions of 0%, 2.5%, 5% and 10%, wherein each group of 6 samples is divided into a pre-aging group and a post-aging group;

the treatment is carried out according to 2 ways respectively: immediate MTT analysis; soaking the sample in a culture saliva biofilm medium for 30 days for biological aging, replacing the culture solution every day, and then performing MTT analysis;

the metabolic activity of the biomembrane reflected by the MTT analysis result is an important index for reflecting the antibacterial performance of the material.

Further, the specific process of the surface biological membrane metabolic activity detection before and after biological aging is as follows:

preparing a nest and furrow closing agent resin sheet by taking a 48-hole plate cover as a mould, taking out prepared resins with different DMADDM mass fractions, soaking the resins in deionized water for 24 hours, and then sterilizing the resins at low temperature by using ethylene oxide for later use;

dividing the sample into a pre-aging group and an aging group, and performing biological aging on the aging group;

placing the resin piece of the pit and fissure sealant in a 24-well plate, culturing the salivary fluid biofilm for 24 hours, and transferring the sample to a new well plate;

adding 1mL of fresh MTT solution with the concentration of 0.5mg/mL into each hole, incubating for 1 hour in a dark place, and transferring the sample into a new hole plate;

adding 1mL of dimethyl sulfoxide solution into each hole, uniformly shaking the mixture in a shaking table for 20 minutes to completely decolorize the biological membrane, and uniformly blowing the biological membrane;

200. mu.L of the solution was transferred to a 96-well plate for each well, and the OD value at a wavelength of 540 was measured using a microplate reader and recorded as the metabolic activity of the biofilm.

By combining all the technical schemes, the invention has the advantages and positive effects that: the invention screens the dosage relation of DMADDM and Clinpro according to the photocuring depth of the modified resin pit and furrow closing agent material, and screens out the optimal dosage range through experiments. On the basis, the influence of DMADDM on the material performance and the anti-caries performance of the pit and fissure sealant is examined by taking a plurality of performances such as cytotoxicity, hardness, surface performance, antibacterial performance and the like as indexes. Meanwhile, a saliva biomembrane is cultured in vitro, the modified resin sealing agent is subjected to biological aging, the antibacterial capability after aging is inspected, and the result proves that the resin pit and furrow sealing agent modified by the DMADDM monomer has long-acting and lasting antibacterial capability while maintaining good mechanical property and safety, so that the long-acting anti-caries capability of the resin pit and furrow sealing agent on pit and furrow caries is improved.

Meanwhile, the invention utilizes the capability of DMADDM capable of covalently combining resin monomers and the antibacterial capability thereof to carry out antibacterial modification on the resin pit and furrow closing agent. The modified pit and fissure sealant realizes effective antibacterial and anticarious abilities on the basis of not influencing the original material performance and safety. Meanwhile, the modified resin sealant material can also effectively resist the aging effect of oral biofilms, has long-acting and sustainable antibacterial effect while keeping the excellent mechanical property, and makes up the deficiency of the long-acting anti-caries property of the existing pit and fissure sealant. The quaternary ammonium salt modified pit and furrow sealing agent developed by the invention has a long-acting antibacterial function, can effectively improve the anticarious success rate, and is expected to become a novel clinically ideal pit and furrow sealing material.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

FIG. 1 is a flow chart of a method for synthesizing and detecting a modified novel antibacterial resin type pit and fissure sealant provided by an embodiment of the invention.

FIG. 2 is a graphical representation of the depth of photocuring of antimicrobial pit and trench sealants modified with DMADDM at various mass fractions as provided in the examples of the present invention.

FIG. 3 is a schematic representation of the cytotoxicity of antibacterial pit and fissure sealants modified with DMADDM at different mass fractions, as provided in the examples of the present invention.

FIG. 4 is a graphical representation of the surface roughness of antimicrobial pit and trench sealants modified with varying mass fractions of DMADDM as provided by the examples of the present invention.

FIG. 5 is a graphical representation of microhardness of antimicrobial socket closure agents modified with varying mass fractions of DMADDM as provided in the examples of the present invention.

FIG. 6 is a graphical representation of the metabolic activity of surface biofilms before and after biological aging of modified antimicrobial pit and fissure sealants containing DMADDM at different mass fractions, as provided in the examples of the present invention.

Figure 7 is a graphical representation of the microleakage scores of biological aged antimicrobial pit sealers modified with varying mass fractions of DMADDM as provided in the examples of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a method for synthesizing and detecting a modified novel antibacterial resin type pit and fissure sealant, which is described in detail below by combining the accompanying drawings.

As shown in fig. 1, the method for synthesizing and detecting a modified novel antibacterial resin type pit and fissure sealant provided by the embodiment of the present invention includes:

s101: preparing pit and furrow closing agent and screening the dosage relation.

S102: and (3) carrying out performance detection on the prepared pit and trench sealing agent, grouping according to the mass fraction of DMADDM, and respectively carrying out light curing depth, cytotoxicity, surface roughness and microhardness on the pit and trench sealing agent, wherein each group comprises 5 samples.

S103: and (4) detecting the antibacterial performance before and after biological aging.

S104: and (3) detecting micro-leakage after biological aging, respectively weighing a certain amount of pit and fissure sealant, and adding DMADDM according to the mass fractions of 0%, 2.5%, 5% and 10% for later use. Collecting the third molars in vitro with complete tooth bodies and no carious lesions, randomly dividing into 4 groups, and putting 5 molars in each group.

In S101 provided by the embodiment of the present invention, a specific process for preparing a pit and fissure sealant is as follows: respectively weighing a certain amount of pit and furrow closing agent, adding DMADDM according to the mass fractions of 0%, 2.5%, 5%, 10%, 20% and 30%, and uniformly mixing in a dark place for later use.

In S102 provided in the embodiment of the present invention, a specific process of grouping according to the mass fraction of DMADDM is as follows:

grouping according to the mass fraction of 0%, 2.5%, 5% and 10% DMADDM, and respectively carrying out light curing depth, cytotoxicity, surface roughness and microhardness on the pit and trench sealing agent, wherein each group comprises 5 samples.

In S102 provided by the embodiment of the present invention, the specific process of the photocuring depth detection experiment is as follows:

placing the prepared pit and groove sealing agents of different groups into a cylindrical mold with the inner diameter of 4mm and the height of 6mm, and performing photocuring for 20 seconds;

taking out the cured samples, and wiping the uncured liquid film at the top and bottom of each sample by using a thin paper;

recording the height of the sample by a vernier caliper, wherein the height is the curing depth;

according to the light curing depth detection result, the optimal proportion of DMADDM added into the pit and trench sealing agent is screened, and the light curing depth of the resin material is not influenced by the DMADDM with the content of less than 10%.

In S102 provided by the embodiment of the present invention, the specific process of detecting cytotoxicity is as follows:

placing the prepared pit and trench sealing agents of different groups into a cylindrical mold with the inner diameter of 13mm and the height of 1mm, carrying out photocuring for 20 seconds, and then placing the prepared sample into deionized water to soak for 24 hours;

after the sample is sterilized by ethylene oxide at low temperature, the sample is put into a cell culture medium to be soaked for 24 hours, and after leaching liquor is obtained, the leaching liquor is filtered and diluted for standby use (32 times, 64 times and 128 times) according to different proportions with a culture solution.

Adding the leaching solution into 96-well plate filled with Human Oral Keratinocyte (HOK), culturing in cell incubator for 24 hr, adding CCK-8 reagent, reacting for 1 hr, and measuring reading with microplate reader at 595nm wavelength to obtain cytotoxicity.

In S102 provided by the embodiment of the present invention, the specific process of detecting the surface roughness is as follows:

sample preparation: the prepared pit and trench sealants of different groups are placed into a cylindrical mold with the inner diameter of 13mm and the height of 1mm, and are subjected to photocuring for 20 seconds.

And (3) detecting the samples under an atomic force microscope, and observing 3 areas of each sample to obtain a surface topography image and an average roughness Ra value.

In S102 provided by the embodiment of the present invention, the microhardness detection specifically includes:

sample preparation: placing the prepared pit and groove sealing agents of different groups into a cylindrical mold with the inner diameter of 6mm and the height of 1mm, and performing photocuring for 20 seconds;

placing the sample in deionized water, soaking for 24 hours, and taking out for later use;

vickers hardness was recorded using a microhardness tester and 3 zones were observed per sample, each zone being pressed 20g for 10 seconds.

In S103 provided by the embodiment of the present invention, the specific process of detecting the antibacterial performance before and after biological aging is as follows:

respectively weighing a certain amount of pit and trench sealing agent, adding DMADDM according to the mass fractions of 0%, 2.5%, 5% and 10%, dividing each group of 6 samples into a pre-aging group and a post-aging group, and respectively treating according to 2 modes: (1) immediate MTT analysis; (2) soaking the sample in a culture saliva biofilm medium for 30 days for biological aging, replacing the culture solution every day, and then performing MTT analysis; the metabolic activity of the biomembrane reflected by the MTT analysis result is an important index for reflecting the antibacterial performance of the material.

Wherein the metabolic activity of the surface biomembrane before and after biological aging is detected

Preparing a nest and furrow closing agent resin sheet by taking a 48-hole plate cover as a mould, taking out prepared resins with different DMADDM mass fractions, soaking the resins in deionized water for 24 hours, and then sterilizing the resins at low temperature by using ethylene oxide for later use;

the samples were divided into pre-aging and post-aging groups, which were subjected to biological aging.

The pieces of pit-gap blocker resin were placed in 24-well plates and the specimens were transferred to new plates 24 hours after incubation of salivary biofilms.

1mL of fresh MTT solution at a concentration of 0.5mg/mL was added to each well, and after 1 hour of incubation in the dark, the samples were transferred to new well plates.

Adding 1mL of dimethyl sulfoxide (DMSO) solution into each hole, uniformly shaking the mixture in a shaking table for 20 minutes to completely decolor the biological membrane, and uniformly blowing the biological membrane.

200. mu.L of the solution was transferred to a 96-well plate for each well, and the OD value at a wavelength of 540 was measured using a microplate reader and recorded as the metabolic activity of the biofilm.

The technical solution of the present invention is further described below with reference to experiments.

1 materials, instruments

Resin pit and fissure sealants clinopro ((3M ESPE, st. paul, MN, USA), DMADDM (dimethylamino dodecyl methacrylate), in vitro human molars, HOK cells, SHI medium, MTT (methylthiazolyltetrazole), microhardness tester, microplate reader, atomic force microscope, cell incubator, bacterial incubator, electronic die, and the like.

2 pit and furrow closing agent preparation and dosage relation screening, performance detection, antibacterial performance detection before and after biological aging and micro-leakage detection after biological aging

2.1 preparation of pit and furrow closing agent and dosage relationship screening

Respectively weighing a certain amount of pit and furrow closing agent, adding DMADDM according to the mass fractions of 0%, 2.5%, 5%, 10%, 20% and 30%, and uniformly mixing in a dark place for later use.

Photocuring depth detection experiment

The prepared pit and groove closing agents of different groups are placed into a cylindrical mold with the inner diameter of 4mm and the height of 6mm, and are subjected to photocuring for 20 seconds.

The cured samples were removed and the uncured liquid film on the top and bottom of each sample was wiped off with a tissue paper.

The vernier caliper records the specimen height, which is the cure depth.

According to the light curing depth detection result, the optimal proportion of DMADDM added into the pit and trench sealing agent is screened, and the light curing depth of the resin material is not influenced by the DMADDM with the content of less than 10%.

2.2 Performance testing

Grouping according to the mass fraction of 0%, 2.5%, 5% and 10% DMADDM, and respectively carrying out light curing depth, cytotoxicity, surface roughness and microhardness on the pit and trench sealing agent, wherein each group comprises 5 samples.

2.2.1 cytotoxicity assays

Placing the prepared pit and trench sealing agents of different groups into a cylindrical mold with the inner diameter of 13mm and the height of 1mm, carrying out photocuring for 20 seconds, and then placing the prepared sample into deionized water to soak for 24 hours.

After the sample is sterilized by ethylene oxide at low temperature, the sample is put into a cell culture medium to be soaked for 24 hours, and after leaching liquor is obtained, the leaching liquor is filtered and diluted for standby use (32 times, 64 times and 128 times) according to different proportions with a culture solution.

Adding the leaching solution into 96-well plate filled with Human Oral Keratinocyte (HOK), culturing in cell incubator for 24 hr, adding CCK-8 reagent, reacting for 1 hr, and measuring reading with microplate reader at 595nm wavelength to obtain cytotoxicity.

2.2.2 surface roughness

Sample preparation: the prepared pit and trench sealants of different groups are placed into a cylindrical mold with the inner diameter of 13mm and the height of 1mm, and are subjected to photocuring for 20 seconds.

And (3) detecting the samples under an atomic force microscope, and observing 3 areas of each sample to obtain a surface topography image and an average roughness Ra value.

2.2.3 microhardness test

Sample preparation: the prepared pit and groove closing agents of different groups are placed into a cylindrical mold with the inner diameter of 6mm and the height of 1mm, and are subjected to photocuring for 20 seconds.

And soaking the sample in deionized water for 24 hours, and taking out for later use.

Vickers hardness was recorded using a microhardness tester and 3 zones were observed per sample, each zone being pressed 20g for 10 seconds.

2.3 antimicrobial Performance testing before and after biological aging

Respectively weighing a certain amount of pit and trench sealing agent, adding DMADDM according to the mass fractions of 0%, 2.5%, 5% and 10%, dividing each group of 6 samples into a pre-aging group and a post-aging group, and respectively treating according to 2 modes: (1) immediate MTT analysis; (2) soaking the sample in a culture saliva biofilm medium for 30 days for biological aging, replacing the culture solution every day, and then performing MTT analysis; the metabolic activity of the biomembrane reflected by the MTT analysis result is an important index for reflecting the antibacterial performance of the material.

Wherein the metabolic activity of the surface biomembrane before and after biological aging is detected

Preparing a nest and furrow closing agent resin sheet by taking a 48-hole plate cover as a mould, taking out prepared resins with different DMADDM mass fractions, soaking the resins in deionized water for 24 hours, and then sterilizing the resins at low temperature by using ethylene oxide for later use;

the samples were divided into pre-aging and post-aging groups, which were subjected to biological aging.

The pieces of pit-gap blocker resin were placed in 24-well plates and the specimens were transferred to new plates 24 hours after incubation of salivary biofilms.

1mL of fresh MTT solution at a concentration of 0.5mg/mL was added to each well, and after 1 hour of incubation in the dark, the samples were transferred to new well plates.

Adding 1mL of dimethyl sulfoxide (DMSO) solution into each hole, uniformly shaking the mixture in a shaking table for 20 minutes to completely decolor the biological membrane, and uniformly blowing the biological membrane.

200. mu.L of the solution was transferred to a 96-well plate for each well, and the OD value at a wavelength of 540 was measured using a microplate reader and recorded as the metabolic activity of the biofilm.

(4) Detection of microleakage after biological aging

Respectively weighing a certain amount of pit and furrow closing agent, and adding DMADDM according to the mass fractions of 0%, 2.5%, 5% and 10% for later use. Collecting the third molars in vitro with complete tooth bodies and no carious lesions, randomly dividing into 4 groups, and putting 5 molars in each group. Figure 7 is a graphical representation of the microleakage scores of biological aged antimicrobial pit sealers modified with varying mass fractions of DMADDM as provided in the examples of the present invention.

A. Microleakage detection

1. Sample preparation: the molar surfaces were cleaned, acid etched with 35% phosphoric acid for 30 seconds, rinsed with water for 15 seconds, and dried for 15 seconds. Coating pit and groove closing agents containing DMADDM with different mass fractions in the molar pit and photocuring for 20 seconds.

2. Molar samples were cryogenically sterilized using ethylene oxide.

3. Biological aging: the molar samples were placed in sterile centrifuge tubes. Saliva and SHI medium 1: after 50 dilutions, 5mL of saliva culture medium mixture was added to the centrifuge tube containing the molar sample to culture the salivary biofilm. The samples were soaked in cultured salivary biofilm medium for 30 days for bioaging, and the culture medium was changed daily.

4. Dyeing: and taking out the molar sample after biological aging, washing with water and drying. Evenly coating nail polish on the surfaces of teeth which are 1mm away from the sealant, soaking the dried molar sample into 0.5% fuchsin solution, placing the sample in a thermostat at 37 ℃ for 24 hours, taking out the sample, and washing the sample with water for 30 seconds.

5. The molar samples were cut longitudinally from the center of the occlusal plane along the long axis of the tooth using a slow speed cutter.

6. The sections of the teeth were examined under a stereomicroscope for dye penetration, and each section was recorded as a sample, with 10 samples per group. The microleakage was evaluated using the following scale: 0 min-no dye penetration; 1 minute-1/3 for the dye to penetrate to the depth of the pit and groove; 2 min-the dye penetrates to 2/3 of the depth of the pit and groove; and 3 min, namely the dye penetrates into the bottom of the pit and groove. Each group of scores was recorded.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.