CN112891227B

CN112891227B - Biofilm-resistant and remineralization-resistant bifunctional material for blocking treatment of early enamel caries and preparation method thereof

Info

Publication number: CN112891227B
Application number: CN202110111852.8A
Authority: CN
Inventors: 张旭; 刘学; 邓旭亮; 贺健康; 王瑶
Original assignee: STOMATOLOGICAL HOSPITAL TIANJIN MEDICAL UNIVERSITY
Current assignee: STOMATOLOGICAL HOSPITAL TIANJIN MEDICAL UNIVERSITY
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2022-11-08
Anticipated expiration: 2041-01-27
Also published as: CN112891227A

Abstract

The invention provides a biomembrane-remineralization resistant double-function material for blocking and treating early enamel caries and a preparation method thereof, wherein the preparation method comprises the following steps of dissolving carboxymethyl chitosan with certain mass in water (which can be adjusted or not adjusted to be acidic), adding a certain amount of silver ions into the solution to form nano silver particles, and then sequentially dropwise adding calcium ions and phosphate ions into the solution to form nano amorphous calcium phosphate particles to prepare the early enamel caries treatment material; the material of the invention has simple preparation method, and the nano amorphous calcium phosphate can penetrate into the demineralized lesion part of the enamel caries to be effectively mineralized. The acidic solution enables the nano amorphous calcium phosphate to have better permeability, and the nano silver has certain sterilization effect on bacterial biomembranes at carious lesions, thereby achieving the effect of treating dental hard tissue carious teeth with different lesion degrees under special conditions. The material is non-toxic and non-irritant, has good biocompatibility, and has ideal use effect and higher clinical value.

Description

Biofilm-resistant and remineralization-resistant dual-functional material for blocking and treating early enamel caries and preparation method thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a biofilm-resistant remineralization dual-functional material for blocking and treating early dental enamel caries and a preparation method thereof.

Background

Aiming at some special carious people which are temporarily inconvenient or have no condition for invasive treatment, such as infants and disabled people who are uncooperative, old people who are inconvenient to move, even special people such as astronauts and navy soldiers who perform tasks for a long time, or public health incidents with increased occupational exposure risk of stomatologists, the existing technology and material for efficiently and accurately blocking the progress of the carious diseases are still lacked to temporarily replace invasive treatment, and the existing fluoride-based carious disease drug treatment cannot completely and effectively solve the problems. Improper and excessive use of fluoride can also cause dental and bone fluorosis. Moreover, fluoride may accelerate mineralization of the carious surface layer of enamel, which is not conducive to mineral ions entering the lesion for remineralization. The traditional remineralizing agents of mineral ions or polyelectrolyte do not have excellent and rapid bacteriostatic and bactericidal effects, so that the requirements for carrying out caries blockage of various stages cannot be met.

Disclosure of Invention

In view of this, the present invention aims to provide an anti-biofilm-remineralization material for blocking and treating early enamel caries and a preparation method thereof, so as to overcome the defects of the existing mineralized substance, mineralize the lesion part under the surface layer, play a role of inhibiting bacterial plaque biofilm, and achieve the purposes of blocking the caries process and remineralizing demineralized enamel.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a biofilm-resistant remineralizing double-function material for blocking and treating early enamel caries is prepared through dissolving carboxymethyl chitosan in water, adding silver nitrate solution to form nano silver particles, adding Ca and P ions, stabilizing the H ions in the supersaturated solution containing P ions and Ca ions in the supersaturated solution containing Ca ions, and preparing carboxymethyl chitosan-nano amorphous Ca-Ag phosphate solution (CMC/ACP-Ag), which is the neutral biofilm-resistant remineralizing material for treating early enamel caries.

Preferably, an acidic reagent is added into the carboxymethyl chitosan-silver solution before the addition of the calcium ions and the phosphate ions, the pH value is adjusted to 2-3 or 4-6, and after the supersaturated calcium ions and the phosphate ions are stabilized, an acidic carboxymethyl chitosan-nano amorphous calcium-silver phosphate (a-CMC/ACP-Ag) solution, namely an acidic anti-biofilm-remineralization material for treating early enamel caries, is formed.

Preferably, the mass ratio of the carboxymethyl chitosan to the water solvent is (1); ca in the supersaturated solution containing hydrogen phosphate ions and the supersaturated solution containing calcium ions: the molar ratio of P is 1; the concentration of silver ions added into the CMC or a-CMC solution is 5-10ppm.

Preferably, the mass fraction of the acidic reagent in the carboxymethyl chitosan solution is 5% -10%; the acidic reagent is one or more of citric acid, lactic acid, gluconic acid, hydrochloric acid or acetic acid.

Preferably, the CMC/ACP-Ag nano-composite has an average particle size of 20-300nm.

Preferably, the average particle size of the a-CMC/ACP-Ag nano composite is 20-300nm.

The present invention also provides a method for preparing the biofilm-remineralization resistant dual-functional material for blocking and treating early enamel caries as described above, comprising the steps of,

1) Sequentially adding carboxymethyl chitosan and potassium dihydrogen phosphate into deionized water under the condition of fully stirring to obtain a solution (1); the temperature is 20-30 ℃;

2) Under the condition of full stirring, adding a certain mass of calcium nitrate tetrahydrate into deionized water to prepare a solution (2);

3) Slowly dripping the solution (2) into the solution (1) under the condition of fully stirring, and then adding dipotassium hydrogen phosphate into the obtained mixed solution to obtain the CMC/ACP nano composite;

4) Adding a silver nitrate solution into the mixed solution obtained in the step 3) under the condition of fully stirring to obtain a CMC/ACP-Ag solution, namely a neutral anti-biofilm-remineralizing material for treating early enamel caries;

in the steps 1) to 4), the stirring speed is 1000r/min to 2000r/min; preferably, the concentration of silver ions added to the CMC/ACP solution is from 5 to 10ppm.

1) Sequentially adding carboxymethyl chitosan and an acidic reagent into deionized water under the condition of fully stirring to obtain a solution (1); the temperature is 20-30 ℃;

2) Under the condition of fully stirring, adding a certain mass of calcium nitrate tetrahydrate into deionized water to prepare a solution (2);

3) Slowly dripping the solution (2) into the solution (1) under the condition of fully stirring, and then adding dipotassium hydrogen phosphate into the obtained mixed solution to obtain the a-CMC/ACP nano composite;

4) Adding a silver nitrate solution into the mixed solution obtained in the step 3) under the condition of fully stirring to obtain an a-CMC/ACP-Ag solution, namely the acidic anti-biofilm-remineralizing material for treating early enamel caries;

in the steps 1) to 4), the stirring speed is 1000r/min to 2000r/min;

preferably, the mass ratio of the mixed solution containing the CMC/ACP complex obtained in the step 3) to the acidic reagent added in the step 1) is 30-50; preferably, 40-45; the concentration of silver ions added into the a-CMC/ACP solution is 5-10ppm.

Preferably, the mass ratio of the carboxymethyl chitosan to the calcium nitrate tetrahydrate to the dipotassium phosphate is 100-200:28-94:9-42.

The invention also provides the application of the anti-biofilm-remineralization difunctional material or the anti-biofilm-remineralization difunctional material prepared by the preparation method in medicines or nursing products for biomimetic remineralization of demineralized hard tissues and caries blockage.

Compared with the prior art, the anti-biofilm-remineralization bifunctional material has the following advantages:

the material of the invention has simple preparation method, and the nano amorphous calcium phosphate can penetrate into the demineralized lesion part of the enamel caries to effectively mineralize. The acid solution can acid-erode the surface layer of the early enamel caries, so that the permeability of the nano amorphous calcium phosphate is better, the silver ions have certain sterilization effect on bacterial biomembranes at the caries lesion, and the effect of treating dental hard tissue caries with different lesion degrees under special conditions can be achieved. The material is non-toxic and non-irritant, has good biocompatibility, and has ideal use effect and higher clinical value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation of the invention. In the drawings:

fig. 1 is a scanning electron microscope image of artificial enamel caries prepared in experiment one.

FIG. 2 shows the appearance, transmission electron microscope image and particle size measurement results of the CMC/ACP-Ag solution in the first example. The solution is colorless and clear in appearance (a), and the CMC/ACP-Ag complex is in a nano-scale particle state under the condition of pH =7.0 as shown by a transmission electron microscope (b, c, d); the particle size detection result shows that the average diameter of the CMC/ACP-Ag composite particles is about 20-300nm (e).

FIG. 3 shows the appearance, transmission electron microscope image and particle size measurement results of the a-CMC/ACP-Ag solution in the second example. The solution is colorless and clear in appearance (a), and the transmission electron microscope result shows that the a-CMC/ACP-Ag complex is in a nano-scale particle state under the condition of pH =2.0 (b, c, d); the particle size detection result shows that the average diameter of the a-CMC/ACP-Ag composite particles is about 20-300nm (e).

Fig. 4 is a scanning electron microscope image of enamel caries model after remineralization treatment in experiment two.

FIG. 5 is a confocal laser microscopy image of enamel remineralization treated in experiment two, wherein a is the untreated group of artificial caries and b is the treated group.

Fig. 6 is a confocal laser microscope image of the enamel bacteriostatic effect after remineralization agent treatment in experiment three.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to examples.

Example one

An anti-biofilm-remineralizing material for use in the prevention and treatment of early enamel caries is prepared by the steps of,

synthesis of neutral carboxymethyl chitosan-amorphous calcium phosphate-silver compound

Under the full stirring of a magnetic stirrer at normal temperature, adding 200mg of carboxymethyl chitosan into 30ml of deionized water, after complete dissolution, adding 1mL of 0.05mmol/L silver nitrate solution, reacting for 30min at normal temperature and normal pressure, adding 27.84mg of monopotassium phosphate, and reacting for 15min to obtain a solution (1); under the conditions of normal temperature and full stirring of a magnetic stirrer, 56.64mg of calcium nitrate tetrahydrate is added into 10ml of deionized water to form a solution (2);

and (3) dropwise adding the solution (2) into the solution (1) at normal temperature under the full stirring of a magnetic stirrer, wherein the average particle size of the formed CMC/ACP-Ag nano-composite is 20-300nm, namely the neutral anti-biofilm-remineralizing material for treating early enamel caries.

Example two

synthesis of acidic carboxymethyl chitosan-amorphous calcium phosphate-silver compound

Under the full stirring of a magnetic stirrer at normal temperature, adding 200mg of carboxymethyl chitosan into 30mL of deionized water, after complete dissolution, adding 1mL of 0.05mmol/L silver nitrate solution, reacting for 30min at normal temperature and normal pressure, adding 2g of citric acid (or 2mL of lactic acid, 2mL of gluconic acid and 3mL of acetic acid), and reacting for 15min to obtain a solution (1); under the conditions of normal temperature and full stirring of a magnetic stirrer, 56.64mg of calcium nitrate tetrahydrate is added into 10ml of deionized water to form a solution (2);

under the condition of normal temperature and full stirring of a magnetic stirrer, the solution (2) is dropwise added into the solution (1), and 27.84mg of dipotassium hydrogen phosphate is added to form a-CMC/ACP-Ag nano composite with the average particle size of 20-300nm, namely the acidic anti-biofilm-remineralization material for treating early enamel caries.

Experiment one

Collecting 20 complete non-carious-damage and non-obvious-abrasion fresh in-vitro teeth which are pulled out due to the inhibition of the growth of the wisdom teeth, washing the teeth clean by deionized water, preparing a 5mm by 1mm enamel sample and a 4mm by 1.5mm dentin sample by a slow tooth grinder under the cooling of running water, and finely polishing by SiC abrasive paper. Enamel specimens 35% phosphoric acid surface acid etching artificial enamel caries models were prepared and 5 were randomly selected for SEM characterization as a blank control. The characterization result is shown in fig. 1, and the fish scale glaze column is etched by acid to form gully and porous shapes.

Experiment two

The surface of artificial caries model enamel prepared by 35% phosphoric acid surface acid etching is divided into two parts by taking a midline as a boundary, one side of the surface is covered by nail polish, and the other side of the surface is treated by materials. The two mineralized materials in the step (2) of the first and second examples are applied to the surface of artificial carious enamel, 3min later, 3% (w/v) NaClO solution is applied, a small brush is applied to the surface of the enamel and then naturally dried, the application and drying are repeated for at least 3 times, and then the enamel is soaked in artificial saliva for 24h and the operation is repeated for three days. As a control group, an artificial caries model was treated with 0.2% (w/v) NaF solution in the same manner (without the step of coating 3% (w/v) NaClO solution). Before detection, the surface of enamel is repeatedly washed with deionized water for three times, and after drying and dehydration, the surface of enamel is sprayed with gold for detection by a scanning electron microscope. The results show that the CMC/ACP-Ag groups (a, b) a-CMC/ACP-Ag groups (c, d) and the 0.2% (w/v) NaF groups (e, f) have remineralization effects, while the a-CMC/ACP-Ag groups have large and more compact crystal formation amount, and the artificial saliva groups (g, h) generate a small amount of scattered crystals, so the effect is not ideal. In addition, the enamel samples treated by the materials are treated for 1h in a way of keeping away from light by adopting rhodamine B dye solution, and after the enamel surface is repeatedly washed for three times by deionized water, the detection is carried out by a laser confocal microscope. Because tiny gaps are left on the surface of enamel after acid etching, dye liquor is easy to permeate and remain, and the enamel is red under a mirror; after being treated by the remineralizing agent, the gap area is reduced or disappeared due to remineralization, and the coloring agent is not easy to remain, so that the coloring agent is not developed. As shown in FIG. 4, the CMC/ACP-Ag solution, the a-CMC/ACP-Ag solution and the 0.2% (w/v) NaF solution all have remineralization effects, the CMC/ACP-Ag group and the a-CMC/ACP-Ag group are more obvious, and the artificial saliva group is not obviously remineralized.

Experiment three

Thawing frozen Streptococcus mutans (S.m), inoculating in BHI liquid culture medium, culturing at 37 deg.C under anaerobic environment (10)％H ₂ 、80％N ₂ 、10％CO ₂ ) And (3) standing and culturing for 24h for resuscitation, selecting the bacterial liquid by using a sterile inoculating loop, streaking on a BHI solid culture medium, culturing for 24h at 37 ℃ in a microaerobic environment, and observing the colony morphology to confirm that the bacterial is pure culture. Selecting a single colony, inoculating the single colony in a BHI liquid culture medium, and performing static culture at 37 ℃ in an anaerobic environment. The growth state of the bacteria was monitored in the logarithmic growth phase using spectrophotometry with an OD600nm =1.0 (about 1X 10) ⁸ CFU/mL). The bacterial culture was then centrifuged at 5000rpm for 5 minutes, the pellet was washed 3 times with Phosphate Buffered Saline (PBS), diluted with fresh BHI and resuspended at 1X 10 ⁶ CFU/mL。

The treated dental films were placed right side up in a 48-well plate. 400 μ L of the adjusted bacterial solution was injected into each well and incubated in an anaerobic chamber at 37 ℃ for 24h. After incubation was complete, the media and bacteria were removed from the dental film surface and the dental film was transferred to a new 24-well plate. The non-adherent bacteria on the surface were gently washed off with deionized water and stained with Baclight Live/Dead solution at the recommended concentration for 15min. And washing away the redundant dye liquor by using deionized water, and observing by using a confocal laser scanning microscope. For each sample, 5 randomly selected areas at 200 x magnification were used to acquire fluorescence images. The experimental results are shown in FIG. 5, the CMC/ACP-Ag group and the a-CMC/ACP-Ag group have the best antibacterial effect, the 0.2% (w/v) NaF group has the second effect, and the artificial saliva group has no obvious antibacterial effect.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. An anti-biofilm-remineralizing bifunctional material for blocking treatment of early enamel caries, characterized in that: after carboxymethyl chitosan is dissolved in water, adding a silver nitrate solution into the solution to form nano silver particles; then adding calcium ions and phosphate ions into the solution to stabilize hydrogen phosphate ions in the supersaturated phosphate ion-containing solution and calcium ions in the supersaturated calcium ion-containing solution to form a carboxymethyl chitosan-nano amorphous calcium phosphate-silver solution, namely a neutral anti-biofilm-remineralization material for treating early enamel caries;

the mass ratio of the carboxymethyl chitosan to the aqueous solvent is (1); adding the silver ion into the CMC solution with the concentration of 5-10ppm; ca in the supersaturated solution containing hydrogen phosphate ions and the supersaturated solution containing calcium ions: the molar ratio of P is 1;

the average grain diameter of the carboxymethyl chitosan-nano amorphous calcium phosphate-silver is 20-300nm.

2. Anti-biofilm-remineralizing bifunctional material for use in the block treatment of early enamel caries according to claim 1, characterized in that: before adding calcium ions and phosphate ions, adding an acidic reagent into a carboxymethyl chitosan solution, adjusting the pH to 2-3 or 4-6, and stabilizing supersaturated calcium ions and phosphate ions to form an acidic carboxymethyl chitosan-nano amorphous calcium phosphate-silver solution, namely an acidic anti-biofilm-remineralization material for treating early enamel caries; the average grain diameter of the acidic carboxymethyl chitosan-nano amorphous calcium phosphate-silver is 20-300nm.

3. The biofilm-resistant remineralizing bifunctional material for blocking treatment of early enamel caries according to claim 2, characterized in that: the mass fraction of the acidic reagent in the carboxymethyl chitosan solution is 5-10%; the acidic reagent is one or more of citric acid, lactic acid, gluconic acid, hydrochloric acid or acetic acid.

4. A method for preparing the anti-biofilm-remineralizing bifunctional material for blocking treatment of early enamel caries according to claim 1, characterized by: comprises the following steps of (a) carrying out,

1) Under the condition of fully stirring, adding carboxymethyl chitosan into deionized water, and adding a silver nitrate solution after dissolving to obtain a solution (1); the temperature is 20-30 ℃;

2) Under the condition of fully stirring, adding calcium nitrate tetrahydrate into deionized water to prepare a solution (2);

3) Slowly dripping the solution (2) into the solution (1) under the condition of fully stirring, and then adding dipotassium hydrogen phosphate into the obtained mixed solution to obtain a CMC/ACP-Ag solution, namely a neutral anti-biofilm-remineralization material for treating early enamel caries;

in the steps 1) to 3), the stirring speed is 1000r/min to 2000r/min.

5. The method of manufacturing according to claim 4, characterized in that: in the steps 1) to 3), the concentration of silver ions added into the CMC solution is 5-10ppm.

6. A process for the preparation of a biofilm-remineralising resistant bifunctional material for use in the block treatment of early enamel caries according to claim 2, characterized in that: comprises the following steps of (a) carrying out,

1) Under the condition of fully stirring, sequentially adding carboxymethyl chitosan, an acidic reagent and a silver nitrate solution into deionized water to obtain a solution (1); the temperature is 20-30 ℃;

3) Slowly dripping the solution (2) into the solution (1) under the condition of fully stirring, and then adding dipotassium hydrogen phosphate into the obtained mixed solution to obtain an a-CMC/ACP-Ag solution, namely an acidic biofilm-resistant remineralizing material for treating early enamel caries;

in the steps 1) to 3), the stirring speed is 1000r/min to 2000r/min;

the mass ratio of the mixed solution containing the CMC/ACP compound obtained in the step 3) to the acidic reagent added in the step 1) is 30-50; the concentration of silver ions added into the a-CMC solution is 5-10ppm.

7. The method of claim 6, wherein: the mass ratio of the mixed solution containing the CMC/ACP compound obtained in the step 3) to the acidic reagent added in the step 1) is 40-45.

8. The production method according to claim 4 or 6, characterized in that: the mass ratio of the carboxymethyl chitosan to the calcium nitrate tetrahydrate to the dipotassium phosphate is 100-200:9-94:7-42.

9. Use of the biofilm-resistant remineralizing bifunctional material according to any one of claims 1 to 3, or prepared according to the preparation method of any one of claims 4 to 8, in the preparation of drugs or care products for biomimetic remineralization of demineralized dental hard tissues and caries blockage.