CN114028239A

CN114028239A - Root canal disinfection paste and preparation method thereof

Info

Publication number: CN114028239A
Application number: CN202111382469.2A
Authority: CN
Inventors: 翟远坤; 邵艳艳; 任延方; 滕海英
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-02-11
Anticipated expiration: 2041-11-22
Also published as: CN114028239B

Abstract

The invention belongs to the field of biomedical disinfection materials, and particularly relates to an antibacterial agent combined root canal disinfection paste for root canal treatment and a method for preparing the paste. A novel root canal disinfectant paste for root canal treatment, comprising a solid phase component and a liquid phase component; it is characterized in that the solid phase components are berberine hydrochloride and BaSO₄The liquid phase component is polyethylene glycol solution, Chitosan (CS), acetic acid and NaOH aqueous solution. In the raw material of the root canal sterilizing paste, the solid phase component and the liquid phase component are mixed in a ratio of liquid phase component: the solid phase components (2 ml) were mixed in a ratio of (0.8-1) g. In the solid phase component, the mass percent of BBH is 25-40%, and BaSO₄Is prepared from the following components in percentage by mass60 to 75 percent. The disinfectant paste of the invention takes BBH, CS and PEG as main components, and the components complement each other, thereby expanding antibacterial spectrum and being capable of being used for root canal treatment.

Description

Root canal disinfection paste and preparation method thereof

Technical Field

The invention belongs to the field of biomedical disinfection materials, and particularly relates to an antibacterial agent combined root canal disinfection paste for root canal treatment and a method for preparing the paste.

Background

Root canal therapy is the most fundamental and effective method for treating endodontic diseases and periapical diseases at present, and the key point of successful treatment is to thoroughly eliminate microorganisms and metabolites thereof in a root canal system and then create an environment which is not beneficial to the survival of the microorganisms. Mainly by mechanical preparation, chemical flushing, and sealing in the root canal.

It was shown that primary and secondary infections in root canals were mixed infections of various bacteria and fungi, and that students isolated 32 genera, 47 strains in infected root canals. Wherein the obligate anaerobe is the main dominant bacterium, and the facultative anaerobe is the second. These bacteria are mostly stored in the form of biofilm in root canals, and are more difficult to remove than plankton, and research shows that the resistance of the bacteria to drugs is 1000-fold to 1500-fold that of plankton. These bacteria can also penetrate into the concealed parts of the root canal system, such as dentinal tubules, lateral branches of the root canal, etc., increasing the difficulty of removing microorganisms. In the root canal of secondary infection, enterococcus faecalis is the main dominant bacterium, which can live in adverse environment, and the second is streptococcus digestions, porphyrins, candida, actinomycetes, etc.

At present, the methods of disinfection and sterilization in the root canal mainly used clinically include root canal sealer, ultrasound, photodynamic, etc., but the most widely used method is root canal sealer. Calcium hydroxide is the most commonly used disinfectant in root canals. After years of clinical use, the calcium hydroxide has good biocompatibility and antibacterial property and low cost. However, studies have also found that calcium hydroxide has certain limitations, such as limited antimicrobial spectrum, inability to sterilize some intractable microorganisms (such as enterococcus faecalis) infecting the root canal, reduction of the antimicrobial effect of dentin protein, limitation of the penetration depth and speed to the hidden part of the root canal, weakening of the mechanical strength of dentin, difficulty in removal from the root canal wall, and the like.

Berberine (BBH), also known as Berberine, is a benzylisoquinoline alkaloid, is an active ingredient of many herbs such as coptis chinensis, barberry and phellodendron amurense, is a natural medicine and has a series of pharmacological properties. Berberine has wide antibacterial activity, and has good antibacterial activity for some common and intractable bacteria infected by dental pulp, such as enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Ravorax melanogenesis, Treponema denticola, Porphyromonas undecomposed, Actinomyces, etc.; has the functions of inducing the repair of hard tissues around the root tip and the continuous development of the tooth root; has low toxicity and mutagenicity to human cells. At present, no report exists on preparing a root canal disinfection paste based on berberine. However, in vitro experiments have been conducted to study the inhibition effect of berberine on enterococcus faecalis as a root canal flushing fluid, but the results show that berberine alone has a lower antibacterial effect than the root canal flushing fluids commonly used in clinic, such as sodium hypochlorite and chlorhexidine. This result is similar to another study, in that bacteria still grow when acting alone on floating enterococcus faecalis and need to be combined with other agents to exert inhibitory effect on enterococcus faecalis. Therefore, the present invention has an object to achieve the antibacterial effect due to the conventional endodontic disinfectant paste by using berberine in combination with other antibacterial agents.

Chitosan (CS) is a polysaccharide consisting of glucosamine and N-acetyl glucosamine, which is deacetylated from chitin found in the shells of crustaceans. CS has antibacterial activity against a variety of gram-positive and gram-negative bacteria, and its antibacterial effect depends on its molecular weight and degree of deacetylation. CS has good biocompatibility and is a good drug carrier, and can slowly and stably release the active ingredients of the drug. The addition of the gel of CS improves the injectability and consistency of the composite. Research shows that CS has a killing effect on enterococcus faecalis in a planktonic state, but has a reduced antibacterial activity on enterococcus faecium in a biofilm state, and the bacteria still survive for 72 hours after the action. In addition, since CS needs to be dissolved by acid, the acidity thereof can cause degradation of dentin, which is a disadvantage that we need to overcome when CS is used in the root canal. One study finds that the antibacterial effect of CS on enterococcus faecalis is not better than that of calcium hydroxide which is a clinically common root canal disinfection paste, but CS does not have the defect that calcium hydroxide reduces the biomechanical property of dentin. Therefore, the invention takes CS as an auxiliary component of the berberine root canal sterilization paste, hopefully utilizes the antibacterial action of the berberine to enhance the antibacterial activity of the berberine and overcome the disadvantages caused by the acidity of a CS solvent, and also hopefully avoids the defect that the traditional calcium hydroxide root canal sterilization paste causes the reduction of the biomechanical property of dentin.

PEG is an artificially synthesized high molecular polymer, is one of the most common carriers in root canal medicaments, and can increase the solubility and the antibacterial activity of berberine hydrochloride. It has very low toxicity, high solubility and very low immunogenicity and antigenicity. BaSO₄Is a common anti-radiation agent in root canal therapy materials, has no toxicity, and is often prepared into barium meal serving as a contrast agent of the digestive tract, BaSO₄Not only can enhance the X-ray opacity, but also can increase the viscosity of the paste.

Disclosure of Invention

Aiming at the defects of the existing root canal disinfection agent, the invention provides a novel root canal disinfection material which can resist or kill dominant bacteria enterococcus faecalis in the root canal with continuous infection and secondary infection and has good biocompatibility.

In order to realize the tasks, the invention is realized by the following technical scheme: a novel root canal disinfectant paste for root canal treatment, comprising a solid phase component and a liquid phase component; it is characterized in that the solid phase components are berberine hydrochloride and BaSO₄The liquid phase component is polyethylene glycol solution, Chitosan (CS), acetic acid and NaOH aqueous solution.

In the raw material of the root canal sterilizing paste, the solid phase component and the liquid phase component are mixed in a ratio of liquid phase component: the solid phase components (2 ml) were mixed in a ratio of (0.8-1) g.

In the solid phase component, the mass percent of BBH is 25-40%, and BaSO₄The mass percentage of the component (A) is 60-75%;

in the liquid phase component, the concentration of acetic acid is 0.1mol/L, the mass percentage concentration of CS is 1 percent, PEG is a solution with the relative molecular weight of 400, and the ratio of PEG400 to CS solution is as follows: CS was mixed at a volume ratio of 9:1, so that the final concentration of CS was 0.1%.

A preferred root canal sterilizing paste is prepared by mixing a 1% CS solution in the liquid phase component with 1mol/L acetic acid in a ratio of 10 mg: 1ml of the mixture.

A preferred endodontic paste is one which is adjusted to a neutral or slightly alkaline pH of 7-8 using a 1% NaOH solution.

The preparation method of the novel root canal sterilization paste is characterized by comprising the following steps:

(ii) adding CS in the liquid phase fraction at a ratio of 10 mg: dissolving 1ml of the mixture in 1mol/L acetic acid solution to obtain a CS solution with the mass concentration of 1%, wherein the mass concentration of PEG and the CS solution is as follows: mixing the CS in a volume ratio of 9: 1; then mixing the solid-phase component and the liquid-phase component in a liquid-phase component: mixing solid phase components 10ml (0.4-0.5) g uniformly, stirring with magnetic stirrer 800r/min for 1h, and concocting to obtain uniform paste; the pH of the paste was then adjusted to 7-8 with 1% NaOH solution. The paste was aspirated into a 1ml syringe and stored at 4 ℃.

Compared with the prior art, the invention has the beneficial effects.

The disinfectant paste of the invention takes BBH, CS and PEG as main components, and the components complement each other, thereby expanding antibacterial spectrum and being capable of being used for root canal treatment. Moreover, the paste has neutral pH, does not contain metal ions capable of chelating with hard tissues of the tooth body, and has milder property. The paste has good biocompatibility and no toxicity to dental pulp stem cells and periodontal ligament stem cells.

The berberine hydrochloride root canal sterilizing paste for disinfecting the root canals has the following advantages: first, berberine in the solid phase component is a natural component, has good antibacterial effect, can promote the repair of hard tissues around the root tips, has good biocompatibility and the like. And in addition, the chitosan can also be used as a drug carrier to increase the stability of the drug in the root canal, so that the chitosan is safe for the root canal disinfection and is expected to improve the root canal disinfection effect. Thirdly, liquidPEG in the phase component is an artificially synthesized high molecular polymer, is one of the most common carriers in root canal medicaments, and can increase the solubility and antibacterial activity of berberine hydrochloride. It has very low toxicity, high solubility and very low immunogenicity and antigenicity. PEG itself has an antibacterial effect against a variety of bacteria. Finally, BaSO in the solid phase component₄The paste X can be given linearity. In conclusion, the berberine hydrochloride root canal disinfection paste of the invention has the following potential when being used as a diagnosis and sealing material for disinfecting a root canal: can resist or kill various microorganisms in infected root canals, has good infiltration effect on hidden parts of dentin, has no negative influence on the biomechanical property of dentin, has good biocompatibility, can induce the formation of hard tissues at the root tip, promotes the inflammation around the root tip to subside, and does not generate complications such as postoperative pain and swelling during the use.

The berberine hydrochloride root canal sterilization paste is currently in an initial exploration stage, the optimized proportion of the material is found only through in vitro tests, and the performances of convenient use in the root canal, better antibacterial effect and good biocompatibility are verified compared with the traditional interdiagnostic seal medicine for root canal sterilization. Animal experiments are needed to verify that the berberine hydrochloride root canal disinfection paste has good biocompatibility, permeability in root canals and antibacterial effect, and has no negative influence on the biomechanical property of dentin, the capability of inducing the repair and regeneration of hard tissues around the root tips and the like because of the traditional interdiagnostic sealing medicine for root canal disinfection. And clinical tests are needed to verify the biological safety, curative effect, related complications and the like of the medicine after being applied to a human body. The berberine hydrochloride root canal disinfection paste is used for the root canal diagnosis and sealing medicine, is suitable for the patients with pulposis, periapical disease, periapical shade of which the periodontal pocket is communicated with periapical lesion, completely presents horn-shaped apical tip to the underdeveloped tooth root and periapical tissue damage, and has better effect than the prior other types of diagnosis and sealing medicine for root canal disinfection.

Drawings

FIG. 1 is a diagram of a finished product of the berberine hydrochloride root canal sterilizing paste of the present invention.

FIG. 2 is a X-ray contrast graph of the berberine hydrochloride root canal sterilizing paste of the present invention (left) and calcium hydroxide paste (right) respectively filled in the isolated root canal of a human.

FIG. 3 is a graph (left) showing the results of the pH test of the berberine hydrochloride root canal sterilizing paste of the present invention, and a pH reading table (right).

FIG. 4 is a graph showing the results of the bacteriostatic experiments of PEG solutions of BBH of different concentrations in example 3, in which 4-1 (A)₁-A₆Results of bacteriostatic experiments), 4-2 (A)₇-A₉Results of bacteriostatic experiments).

FIG. 5 is a graph showing the results of the bacteriostatic test of comparative example 1, wherein 5-1 (bacteriostatic effect of PEG solution of BBH and sterile aqueous solution of BBH), and 5-2 (bacteriostatic effect of solvent PEG solution and sterile water).

FIG. 6 is a graph showing the antibacterial effect of BBH root canal disinfectant paste and calcium hydroxide root canal disinfectant paste by filter paper diffusion method.

FIG. 7 is a dendogram comparing the zone diameter analysis of BBH root canal sterilizing paste with calcium hydroxide root canal sterilizing paste.

FIG. 8 is a diagram showing the preparation method of the eluent (left), and the eluent of the berberine hydrochloride root canal sterilizing paste (upper right) and the calcium hydroxide paste (lower right).

FIG. 9 is a graph showing the results of cytotoxicity of berberine hydrochloride root canal sterilizing paste and calcium hydroxide leaching solution to PDLSCs (9a) and DPSCs (9 b).

Detailed Description

In order to make the technical purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is further described with reference to specific examples, but the implementation is intended to explain the present invention and should not be construed as a limitation of the present invention, and those who do not specify specific techniques or conditions in the examples follow the techniques or conditions described in the literature in the field or follow the product specification.

The experimental method comprises the following steps:

example 1.

0.2g of BBH powder and 0.6g of BaSO were weighed₄The powders were mixed to a solid phase powder. 10mg of CS powder was weighed and dissolved in 1ml of 1mol/L acetic acid solution to obtain 1% (w/v) CS. Then 0.2mL of 1% CS was mixed with 1.8mL of PEG400 to obtain a liquid phaseAnd (3) components. Then, mixing the following components in a liquid phase: mixing the solid phase components at a ratio of 2ml to 0.8g, stirring with a magnetic stirrer at a speed of 800r/min for 1h, and blending to obtain uniform paste; then, the pH of the paste was adjusted to 7 with a 1% NaOH solution to obtain a berberine hydrochloride root canal sterilizing paste having a BBH effective concentration of 0.1 g/mL. The paste was aspirated into a 1ml syringe and stored at 4 ℃.

Example 2.

0.4g of BBH powder and 0.6g of BaSO were weighed₄The powders were mixed to a solid phase powder. 10mg of CS powder was weighed and dissolved in 1ml of 1mol/L acetic acid solution to obtain 1% (w/v) CS. Then, 0.2mL of 1% CS was mixed with 1.8mL of PEG400 to obtain a liquid phase component. Then, mixing the following components in a liquid phase: mixing the solid phase components at a ratio of 2ml to 1g, stirring with a magnetic stirrer at a speed of 800r/min for 1h, and blending to obtain uniform paste; then, the pH of the paste was adjusted to 7 with a 1% NaOH solution to obtain a berberine hydrochloride root canal sterilizing paste having a BBH effective concentration of 0.2 g/mL. The paste was aspirated into a 1ml syringe (FIG. 1) and stored at 4 ℃.

Example 3.

The invention adopts PEG400 solutions of BBH with different concentrations to perform bacteriostatic activity experiments on enterococcus faecalis.

1, 2, 4, 8, 16, 32, 64, 128, 256mg of BBH powder was weighed and dissolved using PEG400 as a solvent to prepare BBH solutions of 1, 2, 4, 8, 16, 32, 64, 128, 256 mg/mL.

The solutions of each group were subjected to the antibacterial activity test (FIGS. 4-1 and 4-2), and the results are shown in Table 1. . 1. No bacteriostatic zone appears in BBH solutions of 2, 4 and 8mg/mL, and the BBH solutions have no bacteriostatic activity. 16. The average inhibition zone of 32 and 64mg/mL BBH solutions is less than 10mm, and enterococcus faecalis is not considered to be sensitive to the BBH solutions with the concentrations. The average zone of inhibition for 128mg/mL BBH solution was between 10-15mm, and the sensitivity of enterococcus faecalis to BBH solution of this concentration was considered as an intermediary. The mean zone of inhibition of BBH solution at 256mg/mL >15mm, and enterococcus faecalis is considered highly sensitive to BBH solutions of this concentration. And compared with other groups, the 128 and 256mg/mL groups have obvious difference in bacteriostatic effect, and the bacteriostatic effect between the two groups is also obvious (P is less than 0.05).

TABLE 1 results of the bacteriostatic test for BBH solutions of different drug contents in each group of this example

"+" indicates statistical differences compared to the other groups.

Comparative example 1.

Two parts of 64mg BBH powder are weighed and dissolved by PEG400 and sterile aqueous solution respectively to prepare BBH solution with the concentration of 64mg/mL, and the PEG solution, the aqueous solution of BBH, the PEG400 solution and the sterile aqueous solution of BBH are subjected to antibacterial experiments respectively, and the results are shown in Table 2. PEG400 showed a certain antibacterial effect when used as a solvent for BBH, and did not show any antibacterial effect when used as a solvent for BBH in a sterile aqueous solution (FIG. 5-1). In the absence of BBH added to both solvents, the antimicrobial activity assay alone did not show any antimicrobial effect (fig. 5-2). It was demonstrated that BBH can exert antibacterial activity when solubilized with PEG400, and that the antibacterial activity is produced by BBH.

TABLE 2 results of the bacteriostatic activity test of each experimental group of this comparative example

Grouping	Diameter of bacteriostatic circle (mm)
		64mg/mL BBH in PEG solution	7.4
64mg/mL BBH in water	6
		PEG400	6
Sterile water	6

Comparative example 2.

Using the root canal disinfectant paste calcium hydroxide paste which is the most commonly used clinically as a control group, the antibacterial activity against enterococcus faecalis was compared for different concentrations of the BBH root canal disinfectant paste (prepared as in examples 1 and 2, with BBH concentrations of 0.1g/mL and 0.2g/mL, respectively). The antibacterial effect is measured by a filter paper diffusion method, the diameter of the inhibition zone is measured after 7 days, the measurement is repeated three times, and an average value is obtained. As shown in Table 3, the calcium hydroxide paste most frequently used in clinic has a poor antibacterial effect against enterococcus faecalis, while the BBH root canal disinfectant pastes of examples 1 and 2 of the present invention have significant antibacterial effects, and the difference is statistically significant compared with the calcium hydroxide group (P < 0.05).

TABLE 3 results of bacteriostatic experiments for this comparative example

Group of	Diameter of antibacterial ring (mm, X + -S)
		0.1g/mL BBH root canal disinfection paste	11.34±0.28
0.2g/mL BBH root canal disinfection paste	14.65±0.14
		Calcium hydroxide paste	6.54±0.22

Firstly, in-vitro antibacterial experiment:

examination of root canal Disinfection pastes (BBH) prepared in examples 1 and 2 by Filter paper diffusion method₁And BBH₂Group) has inhibitory activity against enterococcus faecalis in vitro, and Calcium Hydroxide (CH) paste, which is the most commonly used root canal disinfectant paste in clinic, is used as a control group (denoted as CH group), and sterile water is used as a blank control group.

Enterococcus faecalis ATCC29212(enterococcus faecalis, E.faecalis) was placed in a blood dish for activation culture for 24 hours at an incubation temperature of 37 ℃. The next day, a single colony on a blood-taking plate with an inoculating loop is placed in a TSB culture medium, and is cultured for 24 hours at the constant temperature of 37 ℃ and 180r/min in a shaking incubator, which is the first generation. Inoculating the bacterial liquid into a TSB culture medium at a ratio of 1:100 the next day, and culturing for 24h at a constant temperature of 37 ℃ and 180r/min in a shaking culture box, wherein the second generation is the bacterial liquid. The operation is repeated the next day and is transmitted to the third generation, and the obtained liquid of the enterococcus faecalis is used for the next experiment.

Respectively taking 100uL of bacterial suspension, inoculating the bacterial suspension to an MRS agar plate culture medium, uniformly coating, standing for about 15min, and drying until no macroscopic liquid exists on the surface of the culture medium. Placing a filter paper piece with the diameter of 6mm on the surface of the agar by using a sterile forceps, and lightly pressing by using the forceps to ensure that the filter paper piece is completely contacted with the surface of the agar. The centre-to-centre spacing of the individual sheets should be greater than 24mm and the centre of the sheets should be greater than 15mm from the edge of the plate. Each set of pastes was injected one drop each onto the surface of the paper and spread evenly. The plate was incubated in a 37 ℃ incubator for 7 days. The medicine contained in the paper sheet can continuously diffuse to the area around the paper sheet to form a decreasing concentration gradient, the growth of the bacteria to be detected is inhibited within the bacteriostatic concentration range around the paper sheet, so that a transparent bacteriostatic circle is generated, after 7 days, the diameter of the bacteriostatic circle is measured by a vernier caliper cross method, and the average value is repeatedly taken for 3 times

The experimental results are as follows: the inhibition zones of 0.1g/mL, 0.2g/mL berberine root canal disinfectant paste and calcium hydroxide paste control groups are respectively 11.34 + -0.28 mm, 14.65 + -0.14 mm and 6.54 + -0.22 mm (figure 6). The root canal sterilization paste containing berberine hydrochloride of 0.2g/mL has the best antibacterial effect, and the root canal sterilization paste containing berberine hydrochloride of 0.1g/mL has the second best antibacterial effect, and the diameters of the antibacterial zones are obviously different from those of the calcium hydroxide paste group and the sterile water group, and are also obviously different from each other (figure 7). The calcium hydroxide paste group had a weak bacteriostatic effect, and the sterile water group had no bacteriostatic effect at all. Therefore, we selected 0.2g/mL berberine hydrochloride root canal sterilizing paste for subsequent experiments.

Secondly, testing physical and chemical properties of the materials:

and (3) testing the pH value: the pH value of the berberine hydrochloride root canal sterilization paste is detected by using a pH test paper (on the left in figure 3), and is read to be between 7 and 8 when being compared with a pH ratio card (on the right in figure 3).

Injectability testing: the berberine hydrochloride root canal sterilization paste is filled into a 1ml syringe, and is injected by using a needle head with the same type as calcium hydroxide, the paste can be smoothly ejected by using the similar force, and the ejected state is continuous and occasionally drops.

X-ray opacity: 6 extracted third molars were collected in the alveolar surgery, prepared for opening the marrow and root canals, and then a berberine hydrochloride root canal disinfectant paste (fig. 2 left) and a strong calcium oxide paste (fig. 2 right) were injected into the root canals, respectively, and apical discs were photographed to compare their X-ray resistances.

Third, material safety detection

Primary culture of human periodontal ligament stem cells (hPDLSCs) and human dental pulp stem cells (hdscs):

taking complete and healthy premolars (excluding standard: deciduous teeth, premolars with caries or defect, age > 25 years, systematic medical history, periodontal disease and poor oral hygiene) which need to be removed due to orthodontics in the sai oral hospital, scraping periodontal membrane tissue and dental pulp tissue of tooth apex 1/3 with informed consent, and uniformly inoculating the tissues in a 25cm2 culture dish for primary culture. When the cells are fused to 70-80%, 0.25% trypsin is used for digestion and passage.

Preparation of a drug eluent: taking 24 individual tubes of premolar teeth in vitro, scraping out the periphery of the rootPeriodontal ligament and dental calculus, opening marrow in the coronal part, uncovering the apical part of the pulp chamber, trimming the coronal part of each tooth, ensuring that the length of each tooth is 15mm, extracting the marrow, determining the working length, preparing the conventional root canal of each tooth to a Protaper rotary nickel titanium file F2 by a coronal-down technology, flushing each file by using 1% NaClO solution, and fully flushing by using 5mL of physiological saline solution after preparation. After the teeth were sterilized, 12 were used as experimental groups and 12 were used as control groups. The root canal of the experimental group was filled with berberine hydrochloride paste of 0.2g/mL, the root canal of the control group was filled with calcium hydroxide paste, and the root canal of the control group was sealed with vitreous ion cement. The teeth were placed upright in a 5ml EP tube containing 1ml DMEM medium, the root tips were immersed in the DMEM medium (FIG. 8, left side), and stored in a 37 ℃ constant temperature and humidity incubator. The experimental groups (marked as B) were taken at 1 day, 7 days, 14 days and 21 days respectively₁、B₇、B₁₄、B₂₁Group, upper right of fig. 8) and control group (each designated as CH)₁、CH₇、CH₁₄、CH₂₁Panel, bottom right of fig. 8) were subjected to cytotoxicity assay.

In vitro cytotoxicity test: the experimental and control groups were evaluated for cytotoxicity using hPDSCs and hDPSCs at the 3 rd generation logarithmic growth phase. The 96-well plate was previously supplemented with 0.1mL of DMEM medium (Shanghai-derived leaf Biotech Co., Ltd.) to be 2X 10⁴One cell/well is inoculated in a 96-well plate, after 1d of culture, 100uL of eluent prepared by berberine root canal sterilization paste and calcium hydroxide paste is added, 1 blank control group (without any medicine) is arranged, and 6 multiple wells are arranged in each group. 2 time points (24h and 48h) were set. To avoid cell contamination when absorbance was measured, 1 96 well cell culture plate was used at each time point. A total of 2 plates. And (3) sucking out the original culture medium of each well before measuring the absorbance at each time point, replacing the fresh culture medium, adding 10 mu L of CCK-8 reagent into each well, placing the wells into an incubator for continuous culture for 1h, and immediately measuring the OD value of each well by using an enzyme-linked immunosorbent assay detector at the wavelength of 450 nm. The final results were calculated as the average of 6 well plates and the relative cell proliferation was calculated: relative cell growth rate (RGR)%, OD value of experimental group/control group/OD value of blank control group × 100%, as shown in Table4。

Table 4: relative cell proliferation rate (%, X + -S) of hDPSCs and hPDSCs under the action of each eluent group

The experimental results are as follows: the eluent of berberine hydrochloride root canal sterilization paste at 1, 7, 14 and 21 days has no obvious toxicity to PDLSCs (figure 9a) and DPSCs (figure 9b) at 24h and 48 h. The calcium hydroxide paste eluate has no significant toxicity to PDLSCs and DPSCs at 24h, but the calcium hydroxide paste eluate after 7, 14 and 21 days has toxicity to PDLSCs and DPSCs at 48h, but only the 21-day group has significant toxicity compared with the control group, and the 21-day group has significantly reduced relative cell proliferation rate compared with other groups.

Therefore, the calcium hydroxide is not suitable for being used for too long time in the root canal, otherwise toxicity is generated on periapical tissues, the berberine hydrochloride root canal disinfection paste is mild in property, and cytotoxicity does not occur after the calcium hydroxide is used for 3 weeks, so that the berberine has the potential of being applied to infected root canal disinfection.

The specific method of use of the root canal sterilizing paste of the present invention is as follows:

when the paste is used for disinfecting the root canal, the paste is injected into the root canal by a syringe, then the coronal sealing is carried out by a temporary filling material (such as zinc oxide resin cement, glass ion cement and the like), the coronal sealing material and the paste in the root canal are removed after 1-2 weeks of sealing, and then the subsequent root canal filling is carried out.

The above embodiments are described in more detail and specifically, but the invention is not limited thereto. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A for root canal therapyA novel root canal disinfectant paste comprising a solid phase component and a liquid phase component; it is characterized in that the solid phase components are berberine hydrochloride and BaSO₄The liquid phase component is polyethylene glycol solution, Chitosan (CS), acetic acid and NaOH aqueous solution.

2. The novel endodontic paste for endodontic treatment according to claim 1, wherein the solid phase component and the liquid phase component are mixed in a liquid phase: the solid phase components (2 ml) were mixed in a ratio of (0.8-1) g.

3. The novel root canal disinfectant paste for endodontic treatment according to claim 1, wherein in said solid phase component, the mass percentage of BBH is 25% to 40%, BaSO₄The mass percentage of the component (A) is 60-75%.

4. The novel root canal disinfectant paste for root canal treatment according to claim 1, wherein in said liquid phase component, the concentration of acetic acid is 0.1mol/L, the concentration of CS is 1% by mass, PEG is a solution having a relative molecular weight of 400, and PEG400 and CS solutions are mixed in a ratio of PEG 400: CS was mixed at a volume ratio of 9:1, so that the final concentration of CS was 0.1%.

5. The novel endodontic paste for endodontic treatment according to claim 4, wherein the ratio of 1% CS solution to 1mol/L acetic acid in the liquid phase composition is 10 mg: 1ml of the mixture.

6. The novel endodontic paste for endodontic treatment according to claim 1, which is preferably a endodontic paste adjusted to have a PH of 7 to 8, which is neutral or weakly alkaline using a 1% NaOH solution.

7. The method for preparing a novel endodontic paste for endodontic treatment according to any one of claims 1 to 6, wherein the CS in the liquid phase component is added in a ratio of 10 mg: dissolving 1ml of the mixture in 1mol/L acetic acid solution to obtain a CS solution with the mass concentration of 1%, wherein the mass concentration of PEG and the CS solution is as follows: mixing the CS in a volume ratio of 9: 1; then mixing the solid-phase component and the liquid-phase component in a liquid-phase component: mixing solid phase components 10ml (0.4-0.5) g uniformly, stirring with magnetic stirrer 800r/min for 1h, and concocting to obtain uniform paste; then adjusting the pH value of the paste to 7-8 by using 1% NaOH solution; the paste was aspirated into a 1ml syringe and stored at 4 ℃.