Novel magnetic response root canal sealant for inhibiting bone resorption and preparation method thereof

Abstract

The invention relates to the technical field of root canal sealing agents, and particularly discloses a novel magnetic response root canal sealing agent for inhibiting bone resorption and a preparation method thereof, the novel magnetic response root canal sealing agent comprises a novel magnetic nano material Fe3O4-PAMAM and a root canal sealing agent iRoot SP; wherein the mass fraction of the novel magnetic nano material Fe3O4-PAMAM is 0.25% to 2.00%. According to the invention, the root canal sealant is modified by utilizing the characteristic of directional movement of magnetic nanoparticles in a magnetic field and the performance of PAMAM for inhibiting osteoclast differentiation, so that the improvement of root canal filling permeability and the increase of a bone resorption inhibiting function are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of root canal sealants, and specifically to a novel magnetic-responsive root canal sealant for inhibiting bone resorption and a preparation method thereof. Background Art

[0002] Endodontic periapical diseases are a group of diseases occurring in the dental pulp and periapical tissues. They are inflammatory reactions caused by the invasion of bacteria and their products, which in turn lead to the destruction of periapical tissues. Root canal treatment is the most commonly used and effective treatment method for endodontic periapical diseases. The key point of treatment lies in the control of bacterial infection in the root canal. However, due to the complexity of the root canal anatomical structure, there are complex structures such as accessory canals, apical ramifications, apical forks, root canal isthmuses, and C-shaped root canals, and bacterial biofilms are likely to remain. At the same time, some bacteria such as Enterococcus faecalis can invade the dentinal tubules and grow, reaching hundreds of micrometers deep. Therefore, it is difficult to control the infection caused by Enterococcus faecalis as the main pathogenic bacterium. Even after perfect and standardized root canal treatment, the failure rate of endodontic periapical diseases is still 4% - 15%. Cases where the periapical lesions still do not heal after repeated conventional root canal treatments are called refractory periapical periodontitis. Refractory periapical periodontitis is characterized by obvious periapical bone resorption and has a poor prognosis; in severe cases, it may lead to tooth loss and extensive destruction of the alveolar bone. Therefore, controlling the microbial infection in the root canal and inhibiting periapical bone resorption are beneficial to improving the efficacy of root canal treatment and the prognosis of patients.

[0003] Root canal treatment uses gutta-percha points and root canal sealants to tightly fill the root canal to achieve long-term sealing of the root canal system and prevent the re-infection of the periapical tissues by microorganisms and biofilms in the root canal. Whether the fine areas such as the lateral branches, isthmuses, bifurcations, and blind ends of the root canal system can be effectively filled is the key to tight sealing. This largely depends on the penetration ability and fluidity of the root canal sealant. Improving the existing root canal sealants so that they can penetrate into complex root canal structures and dentinal tubules to achieve tight filling and sealing of the root canal is an important way to improve the success rate of root canal treatment.

[0004] When refractory periapical periodontitis occurs, the uncontrollable microbial infection will directly or indirectly over-activate osteoclasts and promote bone resorption. After the balance between osteogenesis and osteoclastogenesis is disrupted, the periapical bone tissue defect is difficult to repair. Currently, the root canal sealants widely used clinically lack the ability to inhibit bone resorption.

[0005] In summary, the deficiencies of the prior art are as follows: 1. The anatomical structure of the root canal system is complex, and the difficulty of root canal filling is high. Conventional root canal filling materials do not have active fluidity and are difficult to penetrate to the depth of dentinal tubules. The remaining microorganisms and biofilms increase the risk of microleakage, leading to an increased failure rate of root canal treatment; 2. Osteoclasts are active in periapical periodontitis. Conventional root canal filling materials do not have the property of inhibiting osteoclast differentiation; moreover, conventional root canal filling materials cannot inhibit periapical bone resorption.

[0006] Therefore, a novel magnetic-responsive root canal sealer for inhibiting bone resorption and its preparation method are provided; adding materials for inhibiting bone resorption to improve the existing root canal sealer can effectively regulate the osteoblast-osteoclast balance and improve the prognosis of root canal treatment. Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies of the prior art and provide a novel magnetic-responsive root canal sealer for inhibiting bone resorption and its preparation method to solve the problems raised in the above background technology.

[0008] To achieve the above purpose, the present invention provides the following technical solution: A novel magnetic-responsive root canal sealer for inhibiting bone resorption, comprising a novel magnetic nanomaterial Fe3O4-PAMAM and a root canal sealer iRoot SP; wherein, the mass fraction of the novel magnetic nanomaterial Fe3O4-PAMAM is 0.25% - 2.00%.

[0009] Furthermore, the mass fraction of the novel magnetic nanomaterial Fe3O4-PAMAM is 1%.

[0010] A preparation method of a novel magnetic-responsive root canal sealer for inhibiting bone resorption as described above, the specific steps are as follows: Step 1: Synthesis of Fe3O4-PAMAM nanoparticles: Step 101: Preparation of Fe3O4 nanoparticles: Fe3O4 nanoparticles are synthesized by a solvothermal method. Add 2.43 g of FeCl3·6H2O, 0.45 g of Na3CT, and 3.60 g of NaAc to 60 mL of ethylene glycol, and stir with a magnetic stirrer for 1 hour until all are dissolved to obtain a mixed solution; Step 102: Pour the above mixed solution into a stainless steel autoclave and heat at 200 °C for 12 hours; Step 103: After the reaction is completed, cool to room temperature, collect the obtained product using a magnet, wash successively with ethanol and deionized water, and store the prepared Fe3O4 nanoparticles in deionized water for subsequent use; Step 2: Synthesis of Fe3O4-PAMAM nanoparticles: Step 201: Synthesize Fe3O4-PAMAM nanoparticles by condensation reaction. Specifically, dissolve 25 mg of Fe3O4 nanoparticles, 144 mg of EDCI, and 87 mg of NHS in 15 mL of a mixed solution composed of PBS and DMF and activate for 1 hour; Step 202: Then add 100 - 500 mg of PAMAM and shake for 12 hours; Step 203: After the reaction, collect the product using a magnet to obtain Fe3O4-PAMAM nanoparticles, wash them with ethanol and deionized water, and store them in deionized water for subsequent use; Step 3: Add the synthesized Fe3O4-PAMAM nanoparticles to the commercial root canal sealer iRoot SP at a mass fraction of 0.25% - 2.00%, and use a spatula to mix until evenly mixed to obtain the root canal sealer.

[0011] As a preferred technical solution of the present invention, the amount of PAMAM added in Step 202 is 300 mg.

[0012] Compared with the prior art, the beneficial effects of the present invention are: The present invention synthesizes a new type of magnetic nanoparticle Fe3O4-PAMAM and introduces it into the root canal sealer to prepare a new type of magnetic-responsive root canal sealer with bone resorption inhibition. Under the action of a magnetic field, the permeability of root canal filling is improved, overcoming the problem that the existing root canal sealer cannot inhibit bone resorption.

[0013] The present invention has the characteristics of magnetic response. Since the magnetic nanoparticles will move directionally in the magnetic field, after being used to modify the root canal sealer, it does not affect the solubility of the root canal sealer. At the same time, it can drive the root canal sealer, enhance its ability to penetrate into complex root canal structures and dentinal tubules, improve the permeability of the root canal sealer, and prevent reinfection caused by bacteria entering.

[0014] Fe3O4-PAMAM can inhibit osteoclast differentiation, so that the prepared new type of root canal sealer can inhibit the absorption of periapical bone tissue, which is beneficial to improving the prognosis of refractory periapical periodontitis. Description of the Drawings

[0015] Figure 1 It is the flowchart of the method of the present invention; Figure 2 It is the grafting density diagram of PAMAM after mixing different dosages of PAMAM and Fe3O4 of the present invention; Figure 3 It is the solubility result diagram of the root canal sealer containing different mass fractions of Fe3O4-PAMAM nanoparticles of the present invention; Figure 4 This is the biocompatibility result diagram of the root canal sealer containing Fe3O4-PAMAM nanoparticles with different mass fractions of the present invention; Figure 5 This is the schematic diagram of the root canal filling process of the modified root canal sealer of the present invention under the action of a magnetic field; Figure 6 This is the permeability result diagram of the root canal filling of the root canal sealer containing Fe3O4-PAMAM nanoparticles with different mass fractions of the present invention; Figure 7 This is the result diagram of the effect of Fe3O4-PAMAM nanoparticles on osteoclast differentiation in vitro; Figure 8 This is the diagram of the effect of the modified root canal sealer containing 1% Fe3O4-PAMAM nanoparticles of the present invention on bone resorption in a rat model of refractory apical periodontitis. Detailed implementation manners

[0016] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0017] The present invention utilizes the characteristics of the directional movement of magnetic nanoparticles in a magnetic field and the performance of PAMAM in inhibiting osteoclast differentiation to synthesize a new type of magnetic nanomaterial Fe3O4-PAMAM, which is used to prepare a new type of magnetic-responsive root canal sealer for inhibiting bone resorption. The mass fraction of the new type of magnetic nanomaterial Fe3O4-PAMAM is 0.25% - 2.00%, achieving an improvement in the permeability of root canal filling and an increase in the function of inhibiting bone resorption.

[0018] Magnetic nanoparticles have characteristics such as nanoscale particle size, superparamagnetism, good biocompatibility, chemical stability, and magnetic responsiveness. They can move directionally under a magnetic field, and their surfaces can be modified differently to further increase biological activity. Adding magnetic nanoparticles into the root canal sealer, the particles can move directionally under the action of a magnetic field, driving the root canal sealer and enhancing its ability to penetrate into complex root canal structures and dentinal tubules. Polyamidoamine dendrimer (PAMAM) has the potential to inhibit osteoclast differentiation. Grafting PAMAM onto magnetic nanoparticles to synthesize the new type of magnetic nanoparticle Fe3O4-PAMAM can utilize its directional movement characteristics to improve the permeability of the root canal sealer and reduce the destruction of periapical bone resorption tissue by inhibiting osteoclast formation, thereby preventing and treating refractory apical periodontitis.

[0019] The present invention synthesizes a new type of magnetic nanomaterial Fe3O4-PAMAM; 1.1. Synthesis of Fe3O4-PAMAM nanoparticles: 1.1.1 Preparation of Fe3O4 nanoparticles Fe3O4 nanoparticles were synthesized by the solvothermal method. 2.43 g of FeCl3·6H2O, 0.45 g of Na3CT, and 3.60 g of NaAc were added to 60 mL of ethylene glycol and stirred magnetically for 1 hour until completely dissolved. The above mixed solution was poured into a stainless steel autoclave and heated at 200 °C for 12 hours. After the reaction, it was cooled to room temperature, and the obtained product was collected using a magnet and washed successively with ethanol and deionized water. The prepared Fe3O4 nanoparticles were stored in deionized water for subsequent use.

[0020] 1.1.2 Synthesis of Fe3O4-PAMAM nanoparticles Fe3O4-PAMAM nanoparticles were synthesized by a condensation reaction. 25 mg of Fe3O4 nanoparticles, 144 mg of EDCI, and 87 mg of NHS were dissolved in 15 mL of a mixed solution (2:1) composed of PBS and DMF and activated for 1 hour. Then, 100 mg, 300 mg, and 500 mg of PAMAM were added respectively and shaken for 12 hours. After the reaction, the product was collected using a magnet, washed with ethanol and deionized water, and stored in deionized water for subsequent use.

[0021] 1.2 Ratio optimization 1.2.1 Zeta potential detection At room temperature, 10 mg of nanoparticles were uniformly dispersed in deionized water, and the surface Zeta potential of the particles was detected. Each group was detected three times and the average value was taken.

[0022] 1.2.2 Thermogravimetric analysis (TGA) 25 mg of powdered nanoparticles were placed in an alumina crucible. Under nitrogen protection, it was heated from room temperature to 900 °C at a rate of 15 °C / minute to obtain the weight loss change of the nanoparticles during the heating process.

[0023] As Figure 2 shown, according to the experimental results, when the input amount is 300 mg, the grafting density of PAMAM reaches the peak value. Therefore, an input amount of 300 mg is selected for subsequent work.

[0024] The present invention uses a novel magnetic nanomaterial Fe3O4-PAMAM to prepare a novel magnetic-responsive root canal sealer for inhibiting bone resorption; 2.1 Preparation of the modified root canal sealer; The synthesized Fe3O4-PAMAM nanoparticles were added to the commercial root canal sealer iRoot SP at different mass fractions of 0.25%, 0.50%, 1.00%, and 2.00%. A mixing spatula was used to mix them evenly and set aside for later use.

[0025] 2.2. Performance testing to optimize the formulation; 2.2.1. Solubility testing; First, the mixed root canal sealer was loaded into a cylindrical silicone mold (inner diameter 5 mm, height 2.5 mm), and the surface was carefully leveled with a glass plate to ensure uniformity. After curing, it was demolded and sterilized with ethylene oxide. Each sample (n = 6) in all groups was weighed three times, and the average value was calculated as the initial mass (M1). Then, all samples were placed in 10 mL of sterile double-distilled water and incubated at 37 °C for 14 days. After removal, the surface moisture was carefully removed and weighed three times again, and the average value was calculated as the final mass (M2). The percentage of mass loss was determined by the following formula: S = (M1 - M2) / M1 × 100%. The results are as Figure 3 shown. It can be seen that the solubility of the root canal sealers in each group met the requirement of less than 3% specified in the international standard ISO6876:2012 for root canal filling paste materials, meeting the requirements.

[0026] 2.2.2. Biocompatibility testing; The cured and sterilized root canal sealers were separately placed in 10 mL of DMEM medium, supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin, and cultured in an incubator at 37 °C for 24 hours. The supernatant was collected and filtered through a 0.22 μm filter. Mouse fibroblast L929 cells were seeded in a 96-well plate at a density of 5000 cells per well and cultured under conditions of 37 °C and 5% CO2 for 24 hours. After incubation for 24 hours, the medium was replaced with 100 μL of the root canal sealer leachate, with fresh DMEM as the control (n = 5). After further incubation for 24 hours, the biocompatibility was evaluated by the CCK-8 assay. The leachate was removed, and the wells were rinsed three times with PBS. Then, 100 μL of medium and 10 μL of CCK-8 reagent were added to each well, and the mixture was incubated at 37 °C and 5% CO2 in the dark for 1 hour. An experimental group containing only medium and CCK-8 solution was set as the blank group to avoid the influence of the color of the test solution and the culture medium itself. The supernatant was aspirated and transferred to a new 96-well plate, and the absorbance (OD) at a wavelength of 450 nm was measured. The results are as Figure 4 shown. It can be seen that compared with the 0% control group, the cell viability in the 0.25%, 0.5%, 1%, and 2% groups was not damaged, indicating that this material has good biosafety. In summary, all modified sealers have good biocompatibility.

[0027] 2.2.3. Permeability detection; Single-root extracted teeth without caries, with completely developed apical foramina, root canal curvatures less than 15°, and no visible cracks on the root surface were selected. Each tooth was sectioned at the cementoenamel junction to remove the crown. The initial root canal patency was established using a #10 K file. The working length was determined by setting the clinical endpoint to 0.5 mm from the anatomic apex, and root canal preparation was performed. The final preparation size was set to #40 / 06. After shaping, the root canals were irrigated with 2 mL of 5.25% NaClO and 1 mL of 17% EDTA solution. Subsequently, each root canal was irrigated with 1 mL of normal saline. The root canal sealer was mixed with 0.1% rhodamine B, and after drying the root canals with paper points, root canal filling was performed using the single-cone technique. As Figure 5 shown, after root canal filling, a circular magnet (10 mm in diameter, 5 mm in thickness) was placed at each of the apical, buccal, and lingual positions 5 mm from the sample, and the action time was 5 minutes. Subsequently, the pulp chamber was sealed with flowable resin. All teeth were stored at 37 °C and 100% relative humidity for 7 days to ensure complete fixation.

[0028] Horizontal sections were obtained at three predetermined levels along the root (2 mm, 6 mm, and 10 mm from the apex) using a DTQ-5 low-speed precision cutting machine. Then the sections were polished to a thickness of 1 ± 0.1 mm using a polishing machine. Each sample (n = 5) was examined using a confocal laser scanning microscope with an absorption wavelength of 543 nm. All images were taken at a magnification of 4× to obtain a complete view of the sample. The captured images were analyzed to calculate the penetrability. The perimeter of the root canal wall was measured using a measuring tool and denoted as A. The perimeter of the wall area of the dentinal tubules into which the root canal sealer penetrated at any distance was measured using the same method and denoted as B. The percentage of root canal sealer penetration was calculated according to the formula: D = A / B × 100%.

[0029] The experimental results are as Figure 6 shown. As the mass fraction of particle addition increased, the permeability increased and reached a stable and relatively high permeability at 1%. Therefore, the screening range of Fe3O4-PAMAM nanoparticle addition was 0.25% - 2%, and the optimal addition mass fraction was 1%.

[0030] 2.3. Evaluation of the performance of inhibiting bone resorption; 2.3.1. Detection of the effect of Fe3O4-PAMAM nanoparticles on osteoclast differentiation in vitro; Mouse macrophages Raw264.7 were seeded in 48-well plates at a density of 10,000 cells per well. 500 μL of α-MEM medium and 100 ng / mL RANKL were added to each well. Fe3O4-PAMAM nanoparticles at 0, 50, 100, and 200 μg / mL were added to each group (n = 3), and the cells were cultured at 37 °C in a 5% CO2 incubator. The medium was changed on the 3rd day. After obvious osteoclast formation was observed on the 5th day, osteoclast formation was evaluated by TRAP staining. Three random fields were selected from each well for recording, and the osteoclast formation area and number were calculated. The results are as Figure 7 shown. The particles could directly inhibit osteoclast formation, and the inhibitory effect gradually increased with the increase in concentration. A relatively stable inhibitory effect on osteoclast differentiation could be achieved at 100 μg / mL, suggesting that this material has the ability to inhibit bone resorption.

[0031] 2.3.2 Detection of the effect of the modified root canal sealer on bone resorption in rats with refractory periapical periodontitis; Fifteen Wistar rats (male, 8 weeks old) were randomly divided into a blank control group, an unmodified root canal sealer group, and a 1% Fe3O4-PAMAM nanoparticle-modified root canal sealer group (n = 5). The mandibular first molars were pulp-opened and fully exposed to the oral environment for 2 weeks to induce chronic periapical periodontitis. Then the root canals were prepared to #20, rinsed and disinfected with 2.5% sodium hypochlorite, and temporarily sealed with formocresol + glass ionomer for 1 week. Then the pulp-opening cavity was opened again, the medicine was cleaned and sealed, the root canal system was inoculated with Enterococcus faecalis, and the cavity was sealed with glass particles for 4 weeks to complete the establishment of the refractory periapical periodontitis model. Finally, the cavity was opened again, the root canal system was cleaned, prepared to #25, and root canal filling was performed using the single-cone method. After filling, a circular magnet with a diameter of 10 mm and a thickness of 5 mm was placed on each buccal and lingual side of each root canal for 5 minutes. The cavity was sealed with resin to complete root canal treatment; the results are as Figure 8 shown. Compared with the control group, in the in vivo model, the modified root canal sealer had a good effect on inhibiting bone resorption, suggesting its potential to improve the prognosis of periapical inflammation.

[0032] The above embodiments only represent the implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims

1. A novel magnetic-responsive root canal sealer for inhibiting bone resorption, characterized in that, It includes the novel magnetic nanomaterial Fe3O4-PAMAM and the root canal sealer iRoot SP; among them, the mass fraction of the novel magnetic nanomaterial Fe3O4-PAMAM is 0.25% - 2.00%.

2. The novel magnetic response root canal sealer for inhibiting bone resorption according to claim 1, wherein: The mass fraction of the novel magnetic nanomaterial Fe3O4-PAMAM is 1%.

3. A preparation method of the novel magnetic response root canal sealer for inhibiting bone resorption according to any one of claims 1-2, characterized in that: Step 1: Synthesis of Fe3O4 nanoparticles: Step 101: Preparation of Fe3O4 nanoparticles: Solvothermal method is used to synthesize Fe3O4 nanoparticles. Add 2.43 g of FeCl3·6H2O, 0.45 g of Na3CT, and 3.60 g of NaAc into 60 mL of ethylene glycol, and stir with a magnetic stirrer for 1 hour until all are dissolved to obtain a mixed solution. Step 102: Pour the above mixed solution into a stainless-steel autoclave and heat at 200 °C for 12 hours. Step 103: After the reaction, cool to room temperature, collect the obtained product with a magnet, wash it successively with ethanol and deionized water, and store the prepared Fe3O4 nanoparticles in deionized water for subsequent use. Step 2: Synthesis of Fe3O4-PAMAM nanoparticles: Step 201: Condensation reaction is used to synthesize Fe3O4-PAMAM nanoparticles. Specifically, dissolve 25 mg of Fe3O4 nanoparticles, 144 mg of EDCI, and 87 mg of NHS in 15 mL of a mixed solution composed of PBS and DMF and activate for 1 hour. Step 202: Then add 100 - 500 mg of PAMAM and shake for 12 hours. Step 203: After the reaction, collect the product with a magnet to obtain Fe3O4-PAMAM nanoparticles, wash them with ethanol and deionized water, and store them in deionized water for subsequent use. Step 3: Add the synthesized Fe3O4-PAMAM nanoparticles into the commercial root canal sealer iRoot SP according to a mass fraction of 0.25% - 2.00%, and use a spatula to mix until evenly mixed to obtain the root canal sealer.

4. The preparation method according to claim 3, characterized in that: The added amount of PAMAM in Step 202 is 300 mg.

Images