Preparation and application of selenium-containing bioglass cap pith material

Abstract

The invention discloses selenium-containing bioglass, preparation thereof and application of the selenium-containing bioglass in a pulp covering material for repairing dental pulp injury, or application of the selenium-containing bioglass in expression promoters of RUNX transcription factor 2, dentin saliva phosphoprotein and dentin matrix acid phosphoprotein 1. The preparation method comprises the following steps: preparing Se-Ca-Si-P quaternary system sol by adopting a sol-gel method, sealing and aging at room temperature until the sol is gel-shaped, and drying; and grinding the dried product, calcining, carrying out secondary grinding on the calcined powder, and screening to obtain the selenium-containing bioglass. The material not only can rapidly induce bone-like apatite deposition, has excellent antioxidant and antibacterial activity, but also can remarkably promote proliferation and migration of dental pulp stem cells, effectively up-regulate expression of a dental differentiation gene RUNX transcription factor 2, dentin saliva phosphoprotein and dentin matrix acid phosphoprotein 1, and can promote the growth of dental pulp stem cells. The material can be used as a pulp capping material in the aspects of dental pulp injury repair, dentin regeneration and the like.

Description

Technical Field

[0001] This invention relates to a method for preparing selenium-containing bioglass (Se-Ca-Si-P) and its application, and relates to the field of oral biomedical materials technology. Background Technology

[0002] The dental pulp is the core of tooth function and is sensitive to external stimuli. Deep caries and trauma can easily damage the pulp, leading to pain, infection, and even tooth loss. However, existing clinically used pulp capping materials such as calcium hydroxide (Ca(OH)2) and mineral trioxide aggregates (MTA) have significant drawbacks, such as high alkalinity and low bioactivity, which affect their pulp repair efficacy. Furthermore, the pathological process of pulp injury is usually accompanied by bacterial invasion. Bacterial proliferation leads to excessive production of reactive oxygen species (ROS), which in turn triggers oxidative stress in cells, causing damage and death of pulp cells, further exacerbating pulp inflammation and tissue destruction.

[0003] Bioglass (BG) is a multi-metal oxide composite material with a SiO2-CaO network structure as its main matrix. Due to its excellent osteogenic activity and ability to promote the adhesion and proliferation of human dental pulp stem cells (HDPSCs), it is considered a potential material for promoting tooth repair and regeneration (Dental Materials 38 (2022) 725-747). However, traditional bioglasses have limited antioxidant capacity and cannot effectively protect dental pulp cells from oxidative damage, thus affecting the repair effect of dental pulp tissue. Therefore, developing a multifunctional bioglass that can effectively fight bacteria, scavenge reactive oxygen species, induce odontogenic differentiation, and stimulate dental pulp regeneration is of great significance.

[0004] Selenium (Se), an essential trace element for the human body, possesses diverse biological activities. By introducing appropriate amounts of selenium, bioglass not only retains its original biological activity but also further enhances its antioxidant and antibacterial properties. Furthermore, selenium-containing bioglass (SeBG) can continuously release bioactive ions of calcium, silicon, phosphorus, and selenium, which may promote the activity of dental pulp stem cells, thereby effectively improving the regenerative capacity of the dental pulp. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a selenium-containing bioglass operculum material.

[0006] To address the aforementioned problems, this invention provides a selenium-containing bioglass, which is a bioactive glass material prepared primarily using a Se-Ca-Si-P quaternary system prepared by the sol-gel method.

[0007] Preferably, the selenium-containing bioglass has a selenium content of 0.5-3.0 mol%; the selenium source used in the Se-Ca-Si-P quaternary system is at least one of selenite and selenic acid, the calcium source is calcium nitrate tetrahydrate, the silicon source is tetraethyl orthosilicate, and the phosphorus source is triethyl phosphate; the molar ratio of SiO2, CaO, P2O5, and SeO2 in the selenium-containing bioglass is 40-58:20-36:4-6:0.1-5; and the particle diameter in the selenium-containing bioglass is 1.0-5.0 μm.

[0008] Preferably, the selenium-containing bioglass has the ability to rapidly induce bone-like apatite deposition.

[0009] Preferably, the selenium-containing bioglass has antioxidant activity.

[0010] Preferably, the selenium-containing bioglass has antibacterial properties against Escherichia coli, Staphylococcus aureus, and Actinobacillus parasiticus.

[0011] Preferably, the selenium-containing bioglass is biocompatible with human dental pulp stem cells, promotes the proliferation and migration of human dental pulp stem cells, removes intracellular reactive oxygen species, and regulates the expression levels of genes related to odontogenesis differentiation.

[0012] More preferably, the genes regulating odontogenesis include RUNX transcription factor 2, dentin salivary phosphoprotein, and dentin matrix acid phosphoprotein 1.

[0013] The present invention also provides a method for preparing the above-mentioned selenium-containing bioglass, comprising the following steps: preparing a Se-Ca-Si-P quaternary system sol using the sol-gel method, aging it at room temperature in a sealed manner until it becomes gel-like and then drying it; grinding the dried product and calcining it, then grinding the calcined powder a second time and sieving it to obtain the selenium-containing bioglass.

[0014] Preferably, the drying temperature is 120°C; the calcination temperature is 650°C, and the time is 4 hours.

[0015] This invention also provides the application of the above-mentioned selenium-containing bioglass in pulp capping materials for the repair of dental pulp injuries, or in the expression promoters of RUNX transcription factor 2, dentin salivary phosphoprotein, and dentin matrix acid phosphoprotein 1.

[0016] This invention synthesizes bioactive glass (SeBG) via a sol-gel method, with its selenium content controllably adjustable within the range of 0.5-3.0 mol%. This material not only possesses excellent antioxidant and antibacterial activities but also significantly promotes the proliferation and migration of dentin hyperplasia-pregnancy stem cells (HDPSCs) and effectively upregulates the expression of odontogenic differentiation genes RUNX transcription factor 2 (RUNX2), dentin salivary phosphoprotein (DSPP), and dentin matrix acid phosphoprotein 1 (DMP-1). This selenium-containing bioglass can be used as a pulp capping material in pulp injury repair and dentin regeneration.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The selenium-containing bioglass pulp capping material prepared by the present invention is a new type of dental pulp repair material containing selenium bioactive glass. By precisely controlling the amount of selenium source added as a precursor, the selenium content in the bioglass is effectively regulated.

[0018] (2) The bioglasses prepared by this invention with different selenium contents all have excellent ability to rapidly induce bone-like apatite deposition.

[0019] (3) The selenium-containing bioglass capillary material prepared in this invention has excellent antioxidant activity, and it scavenge hydroxyl radicals (OH-). - ), 2,2-Aza-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt cationic radical (ABTS) + The ability of bioglass to react with increasing selenium content is significantly enhanced.

[0020] (4) The selenium-containing bioglass capping pulp material prepared in this invention exhibits good antibacterial properties, and is effective against common oral pathogens such as Escherichia coli (E. coli). E. coli Staphylococcus aureus ( S.aureus It has a significant inhibitory effect on both Actinobacillus actinomycetii (Aa) and its antibacterial effect gradually increases with the increase of selenium content, which can effectively reduce the risk of pulp infection.

[0021] (5) The selenium-containing bioglass operculum material prepared by the present invention has the ability to promote the proliferation and migration of HDPSCs.

[0022] (6) The selenium-containing bioglass capping material prepared by the present invention can effectively remove intracellular hydrogen peroxide.

[0023] (7) The selenium-containing bioglass pulp capping material prepared in this invention can effectively regulate the expression levels of odontoblast differentiation-related genes RUNX2, DSPP, and DMP-1, and significantly upregulate the expression of these genes, thereby efficiently inducing HDPSCs to differentiate into odontoblasts and promoting dentin regeneration and repair. Attached Figure Description

[0024] Figure 1 XRD patterns of different selenium-containing bioactive glass operculum materials prepared in Example 1 in simulated body fluid-induced bone-like hydroxyapatite; Figure 2 Scanning electron microscope images of different selenium-containing bioactive glass thalassium materials prepared in Example 1 on the surface of hydroxyapatite induced by simulated body fluids; Figure 3 The graph shows the test results of the free radical scavenging ability of the selenium-containing bioglass operculum material prepared in Example 1; Figure 4 The selenium-containing bioglass operculum material prepared in Example 1 E. coli, S. aureus, Aa Photos of the antibacterial performance test results and corresponding antibacterial data graphs; Figure 5 The following are data graphs showing the effects of the selenium-containing bioglass capping material prepared in Example 1 on HDPSCs. In this graph, A shows the results of the cell proliferation behavior of HDPSCs affected by the selenium-containing bioglass capping material prepared in Example 1; B shows the fluorescence staining of the cell proliferation of HDPSCs affected by the selenium-containing bioglass capping material prepared in Example 1; and C shows the test results of the effect of the selenium-containing bioglass capping material prepared in Example 1 on the migration behavior of HDPSCs. Figure 6 The graph shows the test results of the ability of the selenium-containing bioglass operculum material prepared in Example 1 to remove hydrogen peroxide from HDPSCs. Figure 7 The figure shows the test results of the effect of selenium-containing bioglass pulp capping material on the expression of odontogenic differentiation genes in Example 1. Detailed Implementation

[0025] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0026] Example 1 A selenium-containing bioglass operculum material, the preparation method of which is as follows: Step 1: The specific process for preparing selenium-containing bioglass SeBG by sol-gel method is as follows: 200 mL H2O, 20 mL 2MHNO3 and 129.115 mL tetraethyl orthosilicate (TEOS) are stirred at room temperature for 30 min-1 h.

[0027] Add 20.391 mL of triethyl phosphate (TEP) and stir for 1 h. Then add 82.7 g or 77.9 g of calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and stir until completely dissolved. Next, add 1.7 g or 5.2 g of sodium selenite (Na2SeO3) to obtain bioglass with different selenium contents and stir until completely dissolved.

[0028] Step 2: After sealing and aging at room temperature, the sol is dried in a 120℃ forced-air drying oven for 2 days. The dried 1SeBG / 3SeBG is then ground in a grinder and placed in a muffle furnace, heated to 650℃ at a heating rate of 2℃ / min, and held at that temperature for 4 hours.

[0029] Example 2: Experiment on inducing bone-like hydroxyapatite formation in simulated body fluid (SBF) using selenium-containing bioglass. The material prepared in Example 1 was mixed with SBF and cultured on a shaker for 24 hours. The microstructure of the generated hydroxyapatite and its morphology after immersion in SBF were observed using SEM.

[0030] like Figure 1 , 2 As shown, the incorporation of Se did not affect the ability of BG-like bone apatite to form, but with the increase of Se doping, it had a significant impact on the morphology of the generated bone apatite, changing it from nanoneedle-like to honeycomb-like.

[0031] Example 3: Test of free radical scavenging ability of selenium-containing bioglass (1) ABTS cationic free radical (ABTS) + Measurement: Step 1: First, prepare the ABTS cationic free radical stock solution. Add the prepared 7.0 mmol / L ABTS... + The solution and 2.45 mmol / L potassium persulfate solution were mixed thoroughly at a volume ratio of 1:1 and allowed to stand in the dark at room temperature for 16 hours to obtain ABTS. + Stock solution.

[0032] Step 2: Dilute the stock solution with deionized water to make ABTS + The absorbance of the stock solution at a wavelength of 734 nm is 0.70 ± 0.02.

[0033] Step 3: Add 1 mL of the extract of different bioglasses to ABTS + In the solution, shake well to ensure complete reaction, and after standing for 12 hours, measure its absorbance at 734 nm.

[0034] Formula 1; In Formula 1: A1 represents different selenium-containing bioglass materials and ABTS. + The absorbance of the solution; A0 is ABTS + The absorbance of the solution; A2 uses deionized water instead of ABTS. + The absorbance value.

[0035] 1 mL of extracts from different bioglasses was added to ABTS. +The free radical working solution was reacted in the dark for 5 min, 180 min, and 360 min.

[0036] as follows Figure 3 As shown in Figure A, selenium-containing bioglass is effective against ABTS. + The free radical scavenging ability exhibits concentration-dependent and time-dependent characteristics, and is positively correlated with selenium content.

[0037] (2) Hydroxyl radical (OH) - Measurement: Step 1: Add 1 mL of the extraction solution of different bioglasses, 1 mL of 9.0 mmol / L ferrous sulfate solution, 1 mL of 9.0 mmol / L salicylic acid-ethanol solution, and 1 mL of 8.8 mmol / L hydrogen peroxide solution to the test tube respectively. After mixing thoroughly, react in a water bath at 37℃ for 12 h and then measure the absorbance at 510 nm.

[0038] Formula 2; In Equation 2: A1 is the absorbance of samples with different hydrogels added; A0 is the absorbance of the mixed solution; A2 is the absorbance of the mixed solution when deionized water is used instead.

[0039] The results are as follows Figure 3 The results showed that there was almost no difference between the BG group and the control group, while the 1SeBG group doped with Se had a significant impact on OH-. - The scavenging effect was 32.85±2.67%, and the scavenging effect of 3SeBG on OH- was 78.05±0.54%.

[0040] Example 4: Antibacterial Experiment of Selenium-Containing Bioglass In vitro antibacterial experiments were conducted on *Escherichia coli*, *Staphylococcus aureus*, and *A. spectroscopy* using the plate count method according to GB / T 21510-2008. The initial bacterial concentrations of the samples were 10⁻⁶ from left to right and from top to bottom. 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 CFU / mL. Results are as follows: Figure 4 As shown, Figure 4 The image in central AC shows the macroscopic effect of selenium-containing bioglass pulp capping material on dental bacteria. With increasing selenium content, the inhibition rate against Escherichia coli, Staphylococcus aureus, and Actinobacillus actinomycetii gradually increases. Figure 4The DF diagram shows the 24-hour inhibition rate of selenium-containing bioglass capping pulp material against *Escherichia coli*, *Staphylococcus aureus*, and *Actinomyces actinomycetes*. Significant differences existed between the experimental and control groups. After 24 hours of culture, the effects of BG, 1SeBG, and 3SeBG on… E. coli The antibacterial rates were 50.89±4.73%, 95.89±1.17%, and 99.26±0.16%, respectively; [the text abruptly ends here, likely due to an incomplete sentence or missing information.] S.aureus The antibacterial rates were 50.34±5.06%, 99.35±0.10%, and 99.90±0.01, respectively; the antibacterial rates against Aa were 40.74±5.13%, 52.59±8.89%, and 76.30±7.84%, respectively.

[0041] Example 5: Effects of selenium-containing bioglass on the cell proliferation and migration of HDPSCs Cell proliferation assays were performed using the CCK-8 assay at preset time points of 6h, 24h, 48h, and 72h, with absorbance values ​​measured at 450nm to assess cell proliferation at concentrations of 1 / 2048-1 / 256 of selenium-containing bioglass.

[0042] Cell proliferation was observed in materials containing selenium-containing bioglass at concentrations of 1 / 2048 to 1 / 256, all of which promoted the growth of HDPSCs.

[0043] like Figure 5 As shown in Figure A, the results of selenium-containing bioglass at a concentration of 1 / 2048 indicate that selenium-containing bioglass pulp capping material promotes the growth of dental pulp stem cells. Figure 5 As shown in Figure B, the fluorescence staining images of HDPSCs by selenium-containing bioglass BG and 1SeBG at 24h, 48h, and 72h indicate the effect of selenium-containing bioglass medullary capping material on cell proliferation.

[0044] Step 3: HDPSCs at concentrations of 1 / 512 and 1 / 1024 were subjected to a cell scratch assay to detect the effect of the selenium-containing bioglass extract prepared in Example 1 on the proliferation and migration ability of HDPSCs. The results of the cell scratch assay are as follows: Figure 5 The results from the C-value indicate that the selenium-containing bioglass extract promotes the proliferation and migration of HDPSCs.

[0045] Example 6: Determination of the intracellular reactive oxygen species scavenging capacity of selenium-containing bioglass To evaluate the effect of selenium-containing bioglass on the scavenging of hydrogen peroxide in HDPSCs, the following experiments were conducted: Step 1: Cell Culture: HDPSCs were extracted using an enzymatic digestion method and cultured to the logarithmic growth phase for subsequent experiments. Preparation of Selenium-Containing Bioglass Extract: Selenium-containing bioglass extracts (BG, 1SeBG, 3SeBG) of different concentrations were prepared according to the method in Example 2.

[0046] Step 2: Use a hydrogen peroxide (H2O2) detection kit for testing. For example... Figure 6 As shown, selenium-containing bioglasses possess the ability to scavenge hydrogen peroxide. 1SeBG and 3SeBG exhibit significantly better scavenging effects on intracellular hydrogen peroxide than BG.

[0047] Example 7: The effect of selenium-containing bioglass on the odontogenic differentiation ability of HDPSCs To investigate the effect of selenium-containing bioglass on HDPSCs, quantitative real-time PCR experiments with concentrations of 1 / 512 and 1 / 1024 were designed according to Example 1 to assess the expression levels of odontogenic differentiation-related markers in cells treated with BG and 1SeBG. The gene expression levels of RUNX2, DSPP, and DMP-1 in stem cells were also examined. To investigate the osteogenic properties of SeBG bioglass on HDPSCs, an alkaline phosphatase (ALP) chromogenic kit from Beyotime was used. HDPSCs were passaged at 5 × 10⁶ cells / year. 3 The plates were seeded in 48-well plates. After adhesion, the experimental group was treated with a screening concentration of extract, while the control group was treated with α-MEM medium. The medium was changed every 3 days, and the plates were cultured for 3 and 7 days. The medium was discarded and the plates were washed with PBS. 200 μL of pre-cooled paraformaldehyde was added to each well for fixation for 30 min. At the same time, ALP staining reagent was prepared at a ratio of 1:2:300. After washing with ultrapure water, 100 μL of staining reagent was added to each well, and the plates were incubated in the dark for 30 min. After washing, the plates were photographed under a microscope.

[0048] Quantitative real-time PCR results showed that selenium-containing bioglass significantly promoted the expression of dentin formation-related proteins RUNX2, DSPP, and DMP-1. Figure 7 As shown in Figure AC, BG and 1SeBG treatment significantly increased the mRNA expression levels of RUNX2, DSPP, and DMP-1. Figure 7 As shown in Figure D, the ALP staining images are obtained after culturing 1BG512 extract with HDPSCs for 3 and 7 days. It can be seen that 1SeBG512 has a more significant effect on ALP production.

Claims

1. A selenium-containing bioglass, characterized in that, The selenium-containing bioglass is a bioactive glass material prepared mainly by the Se-Ca-Si-P quaternary system prepared by the sol-gel method.

2. The selenium-containing bioglass as described in claim 1, characterized in that, The selenium-containing bioglass has a selenium content of 0.5-3.0 mol%; the selenium source used in the Se-Ca-Si-P quaternary system is at least one of selenite and selenic acid, the calcium source is calcium nitrate tetrahydrate, the silicon source is tetraethyl orthosilicate, and the phosphorus source is triethyl phosphate; the molar ratio of SiO2, CaO, P2O5, and SeO2 in the selenium-containing bioglass is 40-58:20-36:4-6:0.1-5; and the particle diameter in the selenium-containing bioglass is 1.0-5.0 μm.

3. The selenium-containing bioglass as described in claim 1, characterized in that, The selenium-containing bioglass has the ability to rapidly induce bone-like apatite deposition.

4. The selenium-containing bioglass as described in claim 1, characterized in that, The selenium-containing bioglass has antioxidant activity.

5. The selenium-containing bioglass as described in claim 1, characterized in that, The selenium-containing bioglass has antibacterial properties against Escherichia coli, Staphylococcus aureus, and Actinobacillus parasiticus.

6. The selenium-containing bioglass as described in claim 1, characterized in that, The selenium-containing bioglass is biocompatible with human dental pulp stem cells, promotes the proliferation and migration of human dental pulp stem cells, removes intracellular reactive oxygen species, and regulates the expression levels of genes related to odontogenesis differentiation.

7. The selenium-containing bioglass as described in claim 6, characterized in that, The genes involved in regulating odontogenesis include RUNX transcription factor 2, dentin salivary phosphoprotein, and dentin matrix acid phosphoprotein 1.

8. The method for preparing selenium-containing bioglass according to any one of claims 1-7, characterized in that, Includes the following steps: A Se-Ca-Si-P quaternary sol was prepared by the sol-gel method, aged at room temperature in a sealed manner until it reached a gel state and was then dried. The dried product was ground and calcined, and the calcined powder was ground a second time and sieved to obtain selenium-containing bioglass.

9. The preparation method according to claim 8, characterized in that, The drying temperature is 120℃; the calcination temperature is 650℃ and the time is 4 hours.

10. The use of the selenium-containing bioglass according to any one of claims 1-7 in pulp capping materials for repairing dental pulp injuries, or in the expression promoters of RUNX transcription factor 2, dentin salivary phosphoprotein, and dentin matrix acid phosphoprotein 1.

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