CN106798652B

CN106798652B - Acid-resistant, wear-resistant and caries-preventing dentinal desensitizing material and preparation method thereof

Info

Publication number: CN106798652B
Application number: CN201710129402.5A
Authority: CN
Inventors: 黄翠; 喻健; 杨宏业
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2017-03-06
Filing date: 2017-03-06
Publication date: 2019-12-10
Anticipated expiration: 2037-03-06
Also published as: CN106798652A

Abstract

The invention discloses an acid-resistant, wear-resistant and caries-preventing dentin desensitizing material and a preparation method thereof. The EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material is prepared by utilizing the characteristics of high biological activity of epigallocatechin gallate, inhibition of cariogenic bacteria biofilm formation, strong adsorption and acid and wear resistance of mesoporous silicon nanoparticles and mineralization promotion of nano hydroxyapatite. The prepared composite material has the advantages of simple and convenient process, easily controlled parameters, stable material structure, good dispersibility, uniform particle size and high EGCG loading rate and release rate. The EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material prepared by the invention can be used as an acid-resistant, wear-resistant and caries-preventing dentinal desensitizing material, can effectively seal dentinal tubules to treat dentinal hypersensitivity, and can effectively inhibit cariogenic bacteria biofilm from forming to prevent caries.

Description

Acid-resistant, wear-resistant and caries-preventing dentinal desensitizing material and preparation method thereof

Technical Field

the invention relates to the field of biomedical engineering, in particular to an acid-resistant, wear-resistant and caries-preventing dentin desensitizing material and a preparation method thereof.

Background

dentinal sensitivity, one of the most common oral diseases in adults, is primarily derived from exposure of dentinal tubules resulting from loss of enamel and/or recession of the gums, causing transient, sharp pain. According to classical fluid dynamics theory (Brannstrom M. J Am Dent Absoc, 1963;66: 366-. With decades of development, satisfactory results have been obtained for treating dentinal hypersensitivity by occluding the dentinal tubules with oxalate, fluoride, adhesive, laser treatment, and the like. However, in complex oral environments, closed tubules are often difficult to resist variable conditions such as acid attack and mechanical brushing in the daily diet (Wang Z, et al. J Dent, 2010;38(5): 400-.

since the main component in dentin is calcium phosphate, it is more reasonable to use mineral compounds like hydroxyapatite for tubule closure. Nano-hydroxyapatite has the ability to accelerate calcium phosphorus deposition and promote remineralization, and has been used in occlusion tubules to treat dentinal hypersensitivity (Besinis A, et al. Dent Mater, 2014;30(3): 249-. Due to the stable structure, large specific surface area and stable thermochemical performance, in recent years, mesoporous silicon is widely applied to the biomedical field as an ideal carrier (LiX, et al. Int J Nanomedicine, 2016;11: 2471-. In view of this, designing a mesoporous silicon loaded with nano hydroxyapatite has great advantages, on one hand, the mesoporous silica can protect the hydroxyapatite from being dissolved by acid so as to obtain a better remineralization effect, and on the other hand, the mesoporous silica can be used as a powerful weapon for resisting acid erosion and mechanical abrasion through the unique acid-resistant and wear-resistant properties of the mesoporous silica.

Because of the presence of tubules and higher organic content, exposed dentin is generally more susceptible to Caries and progresses rapidly (Takahashi N, et al. Caries Res, 2016;50(4): 422-. Although current desensitizing materials can cut the pathway of cariogenic bacteria into the tubules, the local acidic microenvironment formed by the metabolism of cariogenic bacteria can potentially affect the functional stability and longevity of the desensitizing material (fleming HC, et al Nat Rev Microbiol, 2010;8(9): 623-. Therefore, desensitization materials which inhibit the formation of cariogenic bacteria biomembranes have higher potential.

Dentinal desensitizing anticariogenic materials of the present stage are mainly used to inhibit the formation of cariogenic bacteria biofilm by doping the desensitizing agent with antibacterial ingredients such as triclosan, chlorhexidine, stannous fluoride, etc., but the consequent problems of drug resistance, tooth staining, state instability and cytotoxicity limit their application to some extent (Raut SA, et al. Environ Toxicol Chem, 2010;29(6): 1287-1291; Miller S, et al. Int Dent J, 1994;44(1 Suppl 1): 83-98; Shen Y, et al. Sci Rep, 2016;6: 27537.). In recent years, researches show that epigallocatechin gallate (EGCG) extracted from green tea has high biological activity and biocompatibility, can be used as an MMP inhibitor to resist collagen degradation, and can also be used for effectively inhibiting biofilm formation of streptococcus mutans (DuX, et al, J Dent, 2012;40(6):485 and 492), and the characteristics can be used as a safe, stable and efficient anticarious drug for inhibiting biofilm formation.

Therefore, according to the characteristics and advantages of the nano-hydroxyapatite, the mesoporous silicon and the EGCG, the invention designs and synthesizes the EGCG-loaded nano-hydroxyapatite/mesoporous silicon multifunctional composite material, namely the acid-resistant, wear-resistant and caries-preventing dentinal desensitization material, which can treat dentinal sensitivity by sealing dentinal tubules on one hand and prevent caries by inhibiting the formation of cariogenic bacteria biofilm on the other hand. However, no studies have been reported so far.

disclosure of Invention

The invention aims to provide an acid-resistant, wear-resistant and caries-preventing dentinal desensitizing material and a preparation method thereof.

The technical scheme provided by the invention is as follows: an acid-resistant, wear-resistant and caries-preventing dentin desensitizing material is an EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material.

the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material has the advantages that the particle size of the nano hydroxyapatite is 40-80 nm, the particle size of the mesoporous silicon is 400-600 nm, the nano hydroxyapatite is uniformly loaded on the surface of the mesoporous silicon, and the EGCG loading rate and the EGCG release rate are high.

The preparation method of the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material comprises the following steps:

(1) Respectively dissolving calcium nitrate tetrahydrate and diammonium hydrogen phosphate in double distilled water at room temperature, performing ultrasonic dispersion, and stirring on a magnetic stirrer to respectively obtain a solution I and a solution II;

(2) Adding dry mesoporous silicon powder into double distilled water, performing ultrasonic dispersion to obtain dispersion liquid III, then placing on a magnetic stirrer, dropwise adding the solution I into the dispersion liquid III, adjusting and keeping the pH value between 10 and 10.5, and stirring for 12 hours to obtain mixed liquid IV;

(3) Dropwise adding the solution II into the mixed solution IV, adjusting and keeping the pH value between 10 and 10.5, and stirring for 30 hours to obtain a mixed solution V;

(4) After aging for 24 hours, centrifuging the mixed solution V, cleaning with double distilled water and absolute ethyl alcohol, filtering, drying and calcining to obtain the nano hydroxyapatite/mesoporous silicon composite material;

(5) under the dark, dissolving EGCG powder in absolute ethyl alcohol, performing ultrasonic dispersion, placing on a magnetic stirrer for stirring, then adding a dried nano hydroxyapatite/mesoporous silicon composite material, stirring at room temperature, and then placing on an oscillating table for oscillating for 72 hours;

(6) centrifuging, washing with absolute ethyl alcohol, filtering, and drying in vacuum to obtain the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material.

The technical scheme of the specific parameters of the combined embodiment is as follows:

(1) Respectively dissolving 0.944-3.776 g of calcium nitrate tetrahydrate and 0.3168-1.2672 g of diammonium phosphate in 60 mL of double distilled water at room temperature, performing ultrasonic treatment for 5 minutes at the power of 400W, and stirring for 15 minutes on a magnetic stirrer at the stirring speed of 200-400 rpm to respectively obtain a solution I and a solution II;

(2) adding 0.24-0.96 g of dry mesoporous silicon powder into 20-40 mL of double distilled water, performing ultrasonic dispersion for 5 minutes under the power of 400W to obtain dispersion liquid III, then placing the dispersion liquid III on a magnetic stirrer, slowly dropwise adding the solution I into the dispersion liquid III at the stirring speed of 200-400 rpm, adjusting and keeping the pH value between 10-10.5, and stirring for 12 hours to obtain a mixed solution IV;

(3) Slowly dripping the solution II into the mixed solution IV at the stirring speed of 200-400 rpm, adjusting and keeping the pH value between 10 and 10.5, and stirring for 30 hours to obtain a mixed solution V;

(4) After 24 hours of aging, centrifuging the mixed solution V for 15 minutes at 6000-8000 rpm, respectively cleaning 3 times by using double distilled water and absolute ethyl alcohol, filtering, drying at 80 ℃ overnight, and calcining at 550 ℃ for 6 hours to obtain the nano hydroxyapatite/mesoporous silicon composite material;

(5) Dissolving 20 mg of EGCG powder in 10 mL of absolute ethyl alcohol in the dark, performing ultrasound for 5 minutes under the power of 400W, stirring for 15 minutes on a magnetic stirrer at the stirring speed of 300 rpm, then adding 100 mg of dried nano hydroxyapatite/mesoporous silicon composite material, stirring for 2 hours at room temperature, and placing on a shaking table at the shaking speed of 180 rpm for shaking for 72 hours;

(6) centrifuging at 8000 rpm for 15 min, washing with anhydrous ethanol for 3 times, filtering, and vacuum drying to obtain EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material.

The invention has the outstanding characteristics and beneficial effects that:

The preparation process of the acid-resistant, wear-resistant and caries-preventing dentin desensitizing material is simple and convenient, and the process parameters are easy to control.

The acid-resistant, wear-resistant and caries-preventing dentin desensitizing material has stable structure, good dispersibility and uniform particle size.

and thirdly, the EGCG load rate and the release rate of the acid-resistant, wear-resistant and caries-preventing dentin desensitizing material are higher.

The acid-resistant, wear-resistant and caries-preventing dentin desensitizing material can be applied to sealing dentinal tubules, treating dentin sensitivity and inhibiting the formation of cariogenic bacteria biofilm to prevent caries.

Drawings

FIG. 1 shows the acid-resistant, wear-resistant and caries-resistant dentinal desensitizing material (i.e., EGCG-loaded nano-hydroxyl) obtained in example 2 of the present invention

Apatite/mesoporous silicon composite) in the following steps: wherein the finger represents nano hydroxyapatite, the sphere represents mesoporous silicon, and the scale is 100 nm. As can be seen from the figure, the particle diameter of the nano-hydroxyapatite is 40-80 nm, the particle diameter of the mesoporous silicon is 400-600 nm, the dispersibility of the nano-hydroxyapatite and the mesoporous silicon is good, and the nano-hydroxyapatite is uniformly loaded on the surface of the mesoporous silicon.

Fig. 2 is a thermogravimetric analysis curve diagram of the acid-resistant, wear-resistant and caries-preventing dentinal desensitizing material (i.e., the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) obtained in example 2 of the present invention. As can be seen from the graph, the EGCG loading rate was 11.29%.

fig. 3 is a graph showing the EGCG release curve of the acid-resistant, wear-resistant and caries-preventing dentinal desensitizing material (i.e., the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) obtained in example 2 of the present invention. As can be seen, the release rate of EGCG in 96 hours is 54.49% + -3.79%.

Fig. 4 is a scanning electron microscope image of the dentinal tubule sealing effect of the acid-resistant, wear-resistant and caries-preventing dentinal desensitizing material (i.e., EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) obtained in example 2 of the present invention. Wherein, fig. 4A shows that all tubules on the surface of the untreated dentin sheet are in an open state, and fig. 4B shows that all tubules on the surface of the dentin sheet treated by the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material are tightly sealed; fig. 4C shows that all tubules on the surface of the untreated dentin sheet are still open and the mouths of the tubules are enlarged after being soaked in strong acid, and fig. 4D shows that most tubules on the surface of the dentin sheet treated by the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material are still closed after being soaked in strong acid; fig. 4E shows that all tubules on the surface of the untreated dentin sheet are still open after mechanical brushing, and fig. 4F shows that all tubules on the surface of the dentin sheet treated by the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material are still tightly sealed after mechanical brushing.

Fig. 5 is a scanning electron microscope image of the dentinal desensitizing material (i.e., EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) for acid-resistance, wear-resistance and caries prevention obtained in example 2 of the present invention, which inhibits the formation of cariogenic bacteria streptococcus mutans biofilm. Wherein, fig. 5A shows that a large amount of streptococcus mutans biofilm is formed on the surface of an untreated dentin sheet, and fig. 5B shows that the streptococcus mutans biofilm formed on the surface of a dentin sheet treated by the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material is remarkably reduced.

Detailed Description

The following examples are intended only to further illustrate the present invention, and those skilled in the art can embody the present invention in other various ways in light of the disclosure of the present specification.

example 1

(1) Weighing 0.944 g of calcium nitrate tetrahydrate and 0.3168g of diammonium phosphate at room temperature, respectively adding the calcium nitrate tetrahydrate and the 0.3168g of diammonium phosphate into a beaker filled with 60 mL of double distilled water for dissolving, performing ultrasonic treatment for 5 minutes under the power of 400W, and stirring the mixture on a magnetic stirrer at the stirring speed of 200 rpm for 15 minutes to respectively obtain a solution I and a solution II;

(2) Weighing 0.24 g of dry mesoporous silicon powder, adding the dry mesoporous silicon powder into a beaker filled with 20 mL of double distilled water, performing ultrasonic dispersion for 5 minutes under the power of 400W to obtain a solution III, then placing the solution III on a magnetic stirrer, slowly dropwise adding the solution I into the solution III at the stirring speed of 200 rpm, adjusting and keeping the pH value between 10 and 10.5, and stirring for 12 hours to obtain a solution IV;

(3) Slowly dripping the solution II into the solution IV at a stirring speed of 200 rpm, adjusting and keeping the pH value between 10 and 10.5, and stirring for 30 hours to obtain solution V;

(4) After aging for 24 hours, centrifuging the solution V at 6000 rpm for 15 minutes, respectively washing with double distilled water and absolute ethyl alcohol for 3 times, filtering, drying at 80 ℃ overnight, and calcining at 550 ℃ for 6 hours to obtain the nano hydroxyapatite/mesoporous silicon composite material which is colorless powder.

(5) weighing 20 mg of EGCG powder, adding the EGCG powder into a beaker filled with 10 mL of absolute ethyl alcohol to dissolve the EGCG powder in the dark, performing ultrasonic treatment for 5 minutes under the power of 400W, and stirring the EGCG powder for 15 minutes on a magnetic stirrer at the stirring speed of 300 rpm to obtain solution VI;

(6) weighing 100 mg of dry nano hydroxyapatite/mesoporous silicon composite material, adding the dry nano hydroxyapatite/mesoporous silicon composite material into the solution VI, stirring the mixture for 2 hours at room temperature, and placing the mixture on an oscillating table at an oscillating speed of 180 rpm to oscillate for 72 hours;

(7) Centrifuging at 8000 rpm for 15 minutes, cleaning with absolute ethanol for 3 times, filtering, and vacuum drying to obtain EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material, which is an acid-resistant, wear-resistant, and caries-preventing dentin desensitizing material, and is colorless powder.

(8) the morphology of the composite material was observed by using a transmission electron microscope (JEM-2100, JEOL), the loading rate of EGCG in the composite material was measured by using a thermogravimetric analyzer (STA449F3, NETZSCH), and the release rate of EGCG in the composite material was calculated by using an ultraviolet-visible spectrophotometer (UV-2401 PC, Shimadzu Co.).

Example 2

(1) Weighing 1.888g of calcium nitrate tetrahydrate and 0.6336g of diammonium phosphate, respectively adding the weighed materials into a beaker filled with 60 mL of double distilled water for dissolving, performing ultrasonic treatment for 5 minutes under the power of 400W, and stirring the materials on a magnetic stirrer for 15 minutes at the stirring speed of 200 plus 400 rpm to respectively obtain a solution I and a solution II;

(2) weighing 0.48 g of dry mesoporous silicon powder, adding the dry mesoporous silicon powder into a beaker filled with 20 mL of double distilled water, performing ultrasonic dispersion for 5 minutes under the power of 400W to obtain a solution III, then placing the solution III on a magnetic stirrer, slowly dropwise adding the solution I into the solution III at the stirring speed of 300 rpm, adjusting and keeping the pH value between 10 and 10.5, and stirring for 12 hours to obtain a solution IV;

(3) Slowly dripping the solution II into the solution IV at a stirring speed of 300 rpm, adjusting and keeping the pH value between 10 and 10.5, and stirring for 30 hours to obtain solution V;

(4) After aging for 24 hours, centrifuging the solution V for 15 minutes at 8000 rpm, respectively cleaning with double distilled water and absolute ethyl alcohol for 3 times, filtering, drying at 80 ℃ overnight, and calcining at 550 ℃ for 6 hours to obtain the nano hydroxyapatite/mesoporous silicon composite material which is colorless powder.

example 3

(1) Weighing 3.776g of calcium nitrate tetrahydrate and 1.2672g of diammonium phosphate, respectively adding the weighed materials into a beaker filled with 60 mL of double distilled water at room temperature, dissolving the materials in the beaker, performing ultrasonic treatment for 5 minutes under the power of 400W, and stirring the materials for 15 minutes on a magnetic stirrer at the stirring speed of 400 rpm to respectively obtain a solution I and a solution II;

(2) weighing 0.96 g of dry mesoporous silicon powder, adding the dry mesoporous silicon powder into a beaker filled with 40 mL of double distilled water, performing ultrasonic dispersion for 5 minutes under the power of 400W to obtain a solution III, then placing the solution III on a magnetic stirrer, slowly dropwise adding the solution I into the solution III at the stirring speed of 400 rpm, adjusting and keeping the pH value between 10 and 10.5, and stirring for 12 hours to obtain a solution IV;

(3) Slowly dripping the solution II into the solution IV at a stirring speed of 400 rpm, adjusting and keeping the pH value between 10 and 10.5, and stirring for 30 hours to obtain solution V;

Performance testing

(1) Test of closed dentinal tubules and acid and abrasion resistance

Selecting complete non-carious third molars, cooling with water, removing enamel perpendicular to long axis of tooth body, and making thick product

Dentin sheets with a thickness of 1 mm. After gradient polishing, the dentin tubules were left open in a 1% (w/v) citric acid solution for 20 seconds to simulate a dentin sensitivity model and rinsed thoroughly with double distilled water. The dentin surface was treated by dipping slurries (prepared from 10 mg of the material in powder-to-liquid ratio: 100 μ L double distilled water) of the acid-resistant, wear-resistant, and caries-preventing dentinal desensitizing material (i.e., EGCG-loaded nano hydroxyapatite/mesoporous silicon composite) prepared in example 123 respectively in polishing cups for 30 seconds, twice in total. Subsequently, the dentin sheet was subjected to two treatments, respectively: one was immersed in a 6% (w/v) citric acid solution (pH 1.5) for 1 minute for the test of acid resistance, and the other was mechanically brushed on the dentin sheet surface using a soft-bristle toothbrush for 3 minutes (150 times/minute) for the test of abrasion resistance. Each treated dentin section sample was fully rinsed with double distilled water, dried, and sprayed with gold, and then the dentin tubule blocking condition was observed using a scanning electron microscope (Sigma, Zeiss).

from the results of fig. 4, it can be seen that all tubules on the surface of the untreated dentin sheet are in an open state (fig. 4A), and all tubules on the surface of the dentin sheet treated by the dentin desensitizing material (i.e. the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) which is acid-resistant, wear-resistant and anti-caries are tightly sealed (fig. 4B); after being soaked by strong acid, all tubules on the surface of the untreated dentin sheet are still in an open state, the tube openings are expanded (figure 4C), and most of tubules on the surface of the dentin sheet treated by an acid-resistant, wear-resistant and caries-proof dentin desensitizing material (namely EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) are still sealed (figure 4D); after mechanical brushing, all tubules on the surface of the untreated dentin sheet are still in an open state (fig. 4E), and all tubules on the surface of the dentin sheet treated by the acid-resistant, wear-resistant and caries-proof dentin desensitizing material (namely the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) are still tightly sealed (fig. 4F). The results directly prove that the acid-resistant, wear-resistant and caries-preventing dentinal desensitizing material (namely the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) can effectively seal dentinal tubules, is acid-resistant and wear-resistant, and can be applied to treatment of dentinal hypersensitivity. This result is the effect obtained in example 2, which is also obtained in examples 1 and 3.

(2) Test for caries prevention Performance by inhibiting formation of cariogenic bacteria biofilm

Preparing dentin sheets and establishing a sensitive model according to the method in the step (1), sterilizing the front and back surfaces of the dentin sheets by ultraviolet light for 1 hour respectively, and then respectively dipping slurry (prepared by powder-liquid ratio of 10 mg material: 100 mu L double distilled water) of the acid-resistant, wear-resistant and caries-preventing dentin desensitizing material (namely EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) prepared in the step (123) into a polishing cup to treat the dentin surface

polishing the surface for 30 seconds twice, placing the dentin sheet samples in 24-well plates, adding 1 mL of Streptococcus mutans inoculation medium per well (Streptococcus mutans is cultured in brain heart infusion broth for anaerobic overnight at 37 ℃ C., adjusted to a concentration of 10 ⁷ CFU/mL, the inoculation medium is obtained by diluting with brain heart infusion broth containing 1% (w/v) sucrose), culturing at 37 ℃ for 24 hours under anaerobic conditions for formation of Streptococcus mutans biofilm, then washing with PBS three times to elute non-adherent bacteria, fixing each dentin sheet sample with 2.5% glutaraldehyde at 4 ℃ for 4 hours, dehydrating with an ethanol gradient (30%, 50%, 70%, 80%, 90% and 100%), drying, spraying gold, and observing the morphology and adhesion of the Streptococcus mutans biofilm on the surface using a scanning electron microscope (Sigma, Zeiss).

from the results of fig. 5, it can be seen that a large amount of streptococcus mutans biofilm is formed on the surface of the untreated dentin sheet (fig. 5A), and the streptococcus mutans biofilm formed on the surface of the dentin sheet treated by the acid-resistant, wear-resistant and caries-resistant dentin desensitizing material (i.e. the EGCG-loaded nano-hydroxyapatite/mesoporous silicon composite material) is significantly reduced (fig. 5B). The above results directly prove the acid and the resistance

The dentin desensitizing material (namely the EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material) for grinding and preventing the caries can effectively inhibit the formation of cariogenic bacteria biomembrane and can be applied to the prevention of the dentin caries. This result is the effect obtained in example 2, which is also obtained in examples 1 and 3.

Claims

1. an acid-resistant, wear-resistant and caries-preventing dentinal desensitization material is characterized by being an EGCG-loaded nano hydroxyapatite/mesoporous silicon composite material, the particle size of the nano hydroxyapatite is 40-80 nm, the particle size of the mesoporous silicon is 400-600 nm, the nano hydroxyapatite is uniformly loaded on the surface of the mesoporous silicon, the EGCG loading rate is 11.29%, and the EGCG release rate within 96 hours is 54.49% +/-3.79%.

2. A preparation method of an acid-resistant, wear-resistant and caries-preventing dentin desensitization material, which is characterized in that,