Preparation method and application of dental pulp stem cells

Abstract

The invention provides a preparation method and application of dental pulp stem cells, and belongs to the technical field of medicines. The method comprises the following steps: adding an alpha-MEM basic culture solution containing a complexing agent, a growth promoting factor and a natural antibacterial agent into a glass culture dish subjected to surface treatment, adding cleaned and cut fresh human dental pulp tissues, performing constant-temperature culture, after primary cells climb out, pouring out liquid, collecting the primary cells, re-suspending part of the primary cells in a cell cryopreservation solution, and performing cryopreservation, thereby obtaining the human dental pulp tissue culture medium. And adding the remaining primary cells into a culture medium containing nano activated particles, subculturing under the conditions of low oxygen, low-frequency magnetic field and illumination, collecting subcultured cells, resuspending the subcultured cells in a cell cryopreservation solution, and cryopreserving to obtain the dental pulp stem cells. The preparation method is simple, the cost is low, industrial application is easy to achieve, the culture period is short, the dental pulp stem cells obtained through culture are high in viability and good in differentiation performance and can be differentiated into osteoblasts, and a foundation is laid for preparing medicine for treating tooth defects and tooth loss.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing dental pulp stem cells and their application. Background Technology

[0002] Dental pulp stem cells (DPSCs) were first discovered in 2000. Gronthos et al. found DPSCs in both deciduous teeth and wisdom teeth. These cells possess high proliferative capacity, strong self-renewal ability, and multi-lineage differentiation capacity. Studies have successfully utilized these cells to construct dental pulp, dentin, and tooth tissue. DPSCs share many biological characteristics with stem cells from other tissues. Gronthos et al. studied the clonogenic rate of DPSCs and compared them with bone marrow stromal cells (BMSCs), finding that the clonogenic rate of tooth-derived DPSCs was significantly higher than that of bone marrow-derived BMSCs. This indicates that DPSCs have higher proliferative and self-renewal capacity than BMSCs. The strong proliferative capacity, high tissue compatibility, and ease of storage of DPSCs offer modern humans a second opportunity to store stem cells.

[0003] Currently, common methods for preparing dental pulp stem cells include enzymatic digestion, tissue block culture, and tissue block enzymatic digestion. While all these methods can yield dental pulp stem cells, the diverse range of bacteria in the oral cavity makes them highly susceptible to contamination during preparation and culture. Furthermore, the number of isolated and extracted dental pulp stem cells is relatively small, and cell growth is slow, making the preparation of dental pulp stem cells difficult. This is currently the biggest challenge in storing dental pulp stem cells. Therefore, antibiotics are usually added during the preparation of dental pulp stem cells to inhibit bacterial growth. Commonly used antibiotics in stem cell culture include: penicillin and streptomycin for inhibiting bacteria; amphotericin B and nystatin for inhibiting fungi; and gentamicin, tetracycline, and erythromycin for inhibiting mycoplasma contamination. However, the use of these antibiotics cannot guarantee that dental pulp stem cells will not be contaminated. Summary of the Invention

[0004] The purpose of this invention is to propose a method for preparing dental pulp stem cells and its application. The preparation method is simple, low-cost, and easy to industrialize. The culture cycle is short, and the dental pulp stem cells obtained from the culture have strong viability and good differentiation, and can differentiate into osteoblasts, laying the foundation for the preparation of drugs for treating tooth defects and tooth loss.

[0005] The technical solution of this invention is implemented as follows: This invention provides a method for preparing dental pulp stem cells. The method involves adding α-MEM basal culture medium containing a complexing agent, growth factor, and natural antibacterial agent to a surface-treated glass culture dish, adding cleaned and minced fresh human dental pulp tissue, and culturing at a constant temperature. After the primary cells emerge, the liquid is poured off, and the primary cells are collected. A portion is resuspended in a cell cryopreservation solution and cryopreserved. The remaining primary cells are added to a culture medium containing nano-activating particles and passaged under hypoxia, low-frequency magnetic field, and light conditions. The passaged cells are collected, resuspended in a cell cryopreservation solution, and cryopreserved to obtain dental pulp stem cells.

[0006] As a further improvement to the present invention, the following steps are included: S1. Surface treatment of glass petri dishes: Alkali solution is placed in a glass petri dish and heated. The alkali solution is then poured out. Tris-HCl solution is added, followed by polydopamine. The mixture is heated and stirred to react. The solution is then poured out. Deionized water, hydroxyapatite, and collagen peptides are then added. NHS and EDC are added. The mixture is sonicated and stirred to react. The liquid is then poured out, washed, and dried to obtain a surface-treated glass petri dish. S2. Preparation of primary culture medium: Add complexing agent, growth factor and natural antibacterial agent to α-MEM basic culture medium, stir and mix evenly to obtain primary culture medium; S3. Preparation of cell cryopreservation solution: Add mannose, DMSO, glutathione and bovine serum albumin to DMEM medium and mix well to prepare cell cryopreservation solution; S4. Culture: Add the primary culture medium to a surface-treated glass culture dish, add the cleaned and minced fresh human dental pulp tissue, and incubate at a constant temperature. After the primary cells have emerged, pour off the liquid, collect the primary cells, and partially resuspend them in cell cryopreservation solution for cryopreservation. S5. Passaging: The remaining primary cells were added to a culture medium containing nano-activating particles and passaged under hypoxia, low-frequency magnetic field and light conditions. The passaged cells were collected, resuspended in cell cryopreservation solution, and cryopreserved to obtain dental pulp stem cells.

[0007] As a further improvement of the present invention, the alkaline solution in step S1 is a 20-25 wt% NaOH or KOH solution, the heating treatment temperature is 50-60℃, the pH value of the Tris-HCl solution is 8.5-9.5, the heating and stirring reaction temperature is 40-50℃, and the time is 1 h, the mass ratio of deionized water, hydroxyapatite, collagen peptides, NHS and EDC is 80-100:1:2-4:1-2:1-2, the ultrasonic treatment power is 1000-2000W, the time is 5-10 min, and the stirring reaction time is 10-12 h.

[0008] As a further improvement of the present invention, in step S2, the mass ratio of α-MEM basic culture medium, complexing agent, growth factor and natural antibacterial agent is 150-200:1:0.3-0.5:1-2. The complexing agent is selected from at least one of citric acid, sodium citrate, disodium EDTA, EDTA and phytic acid. The growth factor includes heparin sodium and interleukin transforming growth factor-β, with a mass ratio of 2-3:0.2-0.5. The natural antibacterial agent includes ferulic acid and allicin, with a mass ratio of 2-3:5-7. In step S3, the mass ratio of DMEM culture medium, mannose, DMSO, glutathione and bovine serum albumin is 70-90:5-10:20-30:2-3:1-3.

[0009] As a further improvement of the present invention, the isothermal culture temperature in step S4 is 35-37℃ and the time is 2-4 days; the oxygen content in the low oxygen, low frequency magnetic field and light conditions in step S5 is 3-6v / v%, the magnetic field strength of the low frequency magnetic field is 0.1-0.3T, the light intensity is 3000-5000Lux, and the subculture temperature is 35-37℃.

[0010] As a further improvement of the present invention, the content of nano-activating particles in the culture medium containing nano-activating particles in step S5 is 5-10 wt%, and the preparation method of the nano-activating particles is as follows: T1. Add block black phosphorus to N-methylpyrrolidone, sonicate in an ice bath in the dark, centrifuge, wash, dry, add the product to Tris-HCl solution, add dopamine hydrochloride, heat and stir to react, centrifuge, wash, dry, and obtain modified black phosphorus nanosheets. T2. Modified black phosphorus nanosheets and sodium molybdate were added to water, the pH of the solution was adjusted, glutathione was added, the mixture was stirred, hydrothermal reaction was carried out, cooled, centrifuged, washed, and dried to obtain N-doped MoS2 quantum dots@modified black phosphorus nanosheets. T3. Add N-doped MoS2 quantum dots@modified black phosphorus nanosheets to water, add ferric chloride and ferrous chloride under inert gas protection, add ammonia dropwise, heat and stir the reaction, centrifuge, wash, and dry to obtain nano-activated particles.

[0011] As a further improvement of the present invention, the power of the light-shielded ice bath ultrasound in step T1 is 300-500W, the time is 1-3 days, the centrifugation method is to centrifuge at 3000-5000 r / min for 10-20 min, collect the supernatant, centrifuge at 10000-12000 r / min for 15-25 min, collect the precipitate, the pH value of the Tris-HCl solution is 8.5-9.5, the mass ratio of the block black phosphorus to dopamine hydrochloride is 10:1, the temperature of the heating and stirring reaction is 40-50℃, and the time is 2-4 h.

[0012] As a further improvement of the present invention, the mass ratio of the modified black phosphorus nanosheets, sodium molybdate, and glutathione in step T2 is 10:3-4:5-7, the pH value of the adjusted solution is 6-6.5, the temperature of the hydrothermal reaction is 190-210℃, and the time is 10-15h.

[0013] As a further improvement of the present invention, in step T3, the mass ratio of N-doped MoS2 quantum dots@modified black phosphorus nanosheets, ferric chloride and ferrous chloride is 10-12:3.24:1.26, the pH value is adjusted to 9-10 by adding ammonia water, and the temperature of the heating and stirring reaction is 80-90℃ for 1 hour.

[0014] This invention further protects the application of dental pulp stem cells prepared by the above-mentioned preparation method in the preparation of drugs for treating tooth defects and tooth loss.

[0015] The present invention has the following beneficial effects: This invention employs alkaline treatment of the inner surface of glass culture dishes. The alkali reacts with the silica in the glass, roughening the inner surface and generating numerous active groups. A polydopamine layer then forms on the surface, enabling the adhesion of nano-hydroxyapatite. This process exhibits excellent biological activity, promoting the attachment, proliferation, and differentiation of dental pulp stem cells. Furthermore, it couples with collagen peptides, providing a growth environment close to that in vivo, further enhancing cell attachment, proliferation, and differentiation. This surface-treated glass culture dish enhances cell adhesion, significantly shortens the time required for primary cells to migrate out, reduces costs, and facilitates industrial application.

[0016] This invention adds a complexing agent, growth factors, and a natural antibacterial agent to the α-MEM basal culture medium in the primary culture medium. The complexing agent disrupts intercellular calcium adhesion, and combined with mechanical vibration to separate cells, making it suitable for enzyme-sensitive dental pulp tissue samples. The added growth factors include sodium heparin and transforming growth factor-β. Sodium heparin can bind to transforming growth factor-β, enhancing its stability and preventing degradation. It also promotes the binding of transforming growth factor-β to its receptor, enhancing signal transduction. Sodium heparin can also interact with proteoglycans in the extracellular matrix, altering the structure and properties of the extracellular matrix and providing a more favorable microenvironment for stem cell adhesion, migration, and proliferation, thereby influencing stem cell growth and differentiation. Sodium heparin has anticoagulant properties, preventing blood clotting and improving blood supply to tissues and cells, thus indirectly providing better nutrition and oxygen supply for stem cell growth, promoting stem cell growth and survival. Transforming growth factor-β promotes the growth and proliferation of dental pulp stem cells through signaling pathway activation, gene expression regulation, extracellular matrix remodeling, and anti-inflammatory and immunomodulatory effects.

[0017] The added natural antibacterial agents include ferulic acid and allicin. The lipid-soluble structure of ferulic acid can insert into the lipid bilayer of the bacterial cell membrane, altering membrane fluidity and permeability, leading to changes in intracellular electrolytes (such as potassium). + Allicin, through its lipid-soluble sulfur groups, can insert into the bacterial cell membrane, increasing membrane permeability and inhibiting the function of membrane proteins (such as transport proteins), leading to leakage of intracellular substances. It also disrupts the biofilms of drug-resistant bacteria by degrading extracellular polysaccharides in the biofilm matrix, preventing bacterial adhesion and colonization. The synergistic effect of allicin inhibits bacterial contamination and promotes osteogenic differentiation by activating the Wnt / β-catenin pathway.

[0018] The cell cryopreservation solution of this invention contains mannose, DMSO, glutathione, and bovine serum albumin. DMSO penetrates into the cells, lowering the intracellular freezing point and reducing ice crystal formation; it also regulates the osmotic pressure inside and outside the cells, preventing cell dehydration or swelling. Mannose creates a high osmotic pressure environment outside the cells, reducing water leaching and indirectly inhibiting ice crystal growth. Bovine serum albumin coats the cell surface, reducing mechanical damage during cryopreservation, providing nutrients, and maintaining cell viability. Glutathione reduces oxidative stress damage during cryopreservation and thawing, maintaining cell metabolic function. DMEM medium provides electrolytes, a buffer system, and basic nutrients, maintaining stable cell osmotic pressure and pH.

[0019] This invention incorporates nano-activating particles into the culture medium during subculture. The core is polydopamine-modified black phosphorus nanosheets. These black phosphorus nanosheets have a large specific surface area and extremely strong surface adsorption capacity. The lone pairs of electrons in phosphorus combine with the empty orbitals of heavy metals, giving the black phosphorus nanosheets a strong chelating ability for heavy metal ions. This allows for the rapid adsorption of large quantities of heavy metal ions and the removal of reactive oxygen species, thereby improving stem cell viability and promoting proliferation. The surface is loaded with MoS2 quantum dots. Due to the presence of polydopamine, the quantum dots are doped with nitrogen, enabling them to form heterojunctions with black phosphorus. Utilizing the photothermal conversion of black phosphorus and the photodynamic effect of MoS2 quantum dots, under light irradiation... Simultaneously generating heat and singlet oxygen promotes stem cell migration and angiogenesis. Nitrogen doping reduces the band gap of black phosphorus, improving photodynamic effects by regulating electronic structure and enhancing interfacial coupling, thus promoting stem cell growth and survival. In addition, the surface is deposited with magnetic iron oxide, making the nano-activating particles not only magnetic and separable, easy to recycle and reuse, but also able to generate trace amounts of heat energy under the action of a magnetic field. This can activate heat-sensitive proteins on the mitochondrial membrane, promote oxidative phosphorylation efficiency, reduce lactate production, maintain a low-acid microenvironment, which is conducive to stem cell self-renewal. The trace amounts of heat energy promote the secretion of matrix metalloproteinases by stem cells, providing a more suitable microenvironment for cell migration and differentiation.

[0020] The method for preparing dental pulp stem cells in this invention is simple, low-cost, and easy to industrialize. It has a short culture cycle, and the dental pulp stem cells obtained from the culture have strong viability and good differentiation, and can differentiate into osteoblasts, laying the foundation for the preparation of drugs to treat tooth defects and tooth loss. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide.

[0023] Preparation Example 1: Preparation of Nanoparticles The method is as follows: T1. 10 mg of blocky black phosphorus was added to 100 mL of N-methylpyrrolidone, and the mixture was placed in an ice bath in the dark and sonicated at 300 W for 1 day. After centrifugation at 3000 r / min for 10 min, the supernatant was collected and then centrifuged at 10000 r / min for 15 min. The precipitate was collected, washed, and dried. The product was added to 50 mL of Tris-HCl solution with a pH of 8.5, and 3 mg of dopamine hydrochloride was added. The mixture was heated to 40 °C and stirred for 2 h. After centrifugation, washing, and drying, modified black phosphorus nanosheets were obtained. T2. Add 1g of modified black phosphorus nanosheets and 0.3g of sodium molybdate to 50mL of water, adjust the pH of the solution to 6, add 0.5g of glutathione, mix and stir for 5min, hydrothermally react at 190℃ for 10h, cool, centrifuge, wash, and dry to obtain N-doped MoS2 quantum dots@modified black phosphorus nanosheets. T3. Add 1g of N-doped MoS2 quantum dots@modified black phosphorus nanosheets to 150mL of water. Under nitrogen protection, add 0.324g of ferric chloride and 0.126g of ferrous chloride. Adjust the pH to 9 by adding ammonia dropwise. Heat to 80℃ and stir for 3h. Centrifuge, wash, and dry to obtain nano-activated particles.

[0024] Preparation Example 2: Preparation of Nanoparticles The method is as follows: T1. 10 mg of blocky black phosphorus was added to 100 mL of N-methylpyrrolidone, and the mixture was placed in an ice bath in the dark and sonicated at 500 W for 3 days. After centrifugation at 5000 r / min for 20 min, the supernatant was collected and centrifuged at 12000 r / min for 25 min. The precipitate was collected, washed, and dried. The product was added to 50 mL of Tris-HCl solution with a pH of 9.5, and 5 mg of dopamine hydrochloride was added. The mixture was heated to 50 °C and stirred for 4 h. After centrifugation, washing, and drying, modified black phosphorus nanosheets were obtained. T2. Add 1g of modified black phosphorus nanosheets and 0.4g of sodium molybdate to 50mL of water, adjust the pH of the solution to 6.5, add 0.7g of glutathione, mix and stir for 5min, hydrothermally react at 210℃ for 15h, cool, centrifuge, wash, and dry to obtain N-doped MoS2 quantum dots@modified black phosphorus nanosheets. T3. 1.2 g of N-doped MoS2 quantum dots@modified black phosphorus nanosheets were added to 150 mL of water. Under nitrogen protection, 0.324 g of ferric chloride and 0.126 g of ferrous chloride were added. Ammonia was added dropwise to adjust the pH to 10. The mixture was heated to 90 °C and stirred for 5 h. After centrifugation, washing, and drying, nano-activated particles were obtained.

[0025] Preparation Example 3: Preparation of Nanoparticles The method is as follows: T1. 10 mg of blocky black phosphorus was added to 100 mL of N-methylpyrrolidone, and the mixture was placed in an ice bath in the dark and sonicated at 400 W for 2 days. After centrifugation at 4000 r / min for 15 min, the supernatant was collected and then centrifuged at 11000 r / min for 20 min. The precipitate was collected, washed, and dried. The product was added to 50 mL of Tris-HCl solution with a pH of 9, and 4 mg of dopamine hydrochloride was added. The mixture was heated to 45 °C and stirred for 3 h. After centrifugation, washing, and drying, modified black phosphorus nanosheets were obtained. T2. Add 1g of modified black phosphorus nanosheets and 0.35g of sodium molybdate to 50mL of water, adjust the pH of the solution to 6.2, add 0.6g of glutathione, mix and stir for 5min, hydrothermally react at 200℃ for 12h, cool, centrifuge, wash, and dry to obtain N-doped MoS2 quantum dots@modified black phosphorus nanosheets. T3. 1.1 g of N-doped MoS2 quantum dots@modified black phosphorus nanosheets were added to 150 mL of water. Under nitrogen protection, 0.324 g of ferric chloride and 0.126 g of ferrous chloride were added. Ammonia was added dropwise to adjust the pH to 9.5. The mixture was heated to 85 °C and stirred for 4 h. After centrifugation, washing, and drying, nano-activated particles were obtained.

[0026] Comparative Preparation Example 1 The difference compared to preparation example 3 is that step T2 was not performed.

[0027] Specifically as follows: T1. 10 mg of blocky black phosphorus was added to 100 mL of N-methylpyrrolidone, and the mixture was placed in an ice bath in the dark and sonicated at 400 W for 2 days. After centrifugation at 4000 r / min for 15 min, the supernatant was collected and then centrifuged at 11000 r / min for 20 min. The precipitate was collected, washed, and dried. The product was added to 50 mL of Tris-HCl solution with a pH of 9, and 4 mg of dopamine hydrochloride was added. The mixture was heated to 45 °C and stirred for 3 h. After centrifugation, washing, and drying, modified black phosphorus nanosheets were obtained. T2. Add 1.1g of modified black phosphorus nanosheets to 150mL of water. Under nitrogen protection, add 0.324g of ferric chloride and 0.126g of ferrous chloride. Adjust the pH to 9.5 by adding ammonia dropwise. Heat to 85℃ and stir for 4h. Centrifuge, wash, and dry to obtain nano-activated particles.

[0028] Comparative Preparation Example 2 The difference from Preparation Example 3 is that dopamine hydrochloride was not added in step T1.

[0029] Specifically as follows: T1. Add 10 mg of blocky black phosphorus to 100 mL of N-methylpyrrolidone, incubate in an ice bath in the dark, sonicate at 400 W for 2 days, centrifuge at 4000 r / min for 15 min, collect the supernatant, centrifuge at 11000 r / min for 20 min, collect the precipitate, wash, dry, and obtain black phosphorus nanosheets. T2. Add 1g of black phosphorus nanosheets and 0.35g of sodium molybdate to 50mL of water, adjust the pH of the solution to 6.2, add 0.6g of glutathione, mix and stir for 5min, hydrothermally react at 200℃ for 12h, cool, centrifuge, wash, and dry to obtain N-doped MoS2 quantum dots@black phosphorus nanosheets. T3. 1.1 g of N-doped MoS2 quantum dots@black phosphorus nanosheets were added to 150 mL of water. Under nitrogen protection, 0.324 g of ferric chloride and 0.126 g of ferrous chloride were added. Ammonia was added dropwise to adjust the pH to 9.5. The mixture was heated to 85 °C and stirred for 4 h. After centrifugation, washing, and drying, nano-activated particles were obtained. Example 1

[0030] This embodiment provides a method for preparing dental pulp stem cells, including the following steps: S1. Surface treatment of glass petri dishes: Place 20wt% NaOH solution in a glass petri dish, heat to 50℃ for 2 min, pour out the liquid, then add Tris-HCl solution with pH value of 8.5, add 3g polydopamine, heat to 40℃ and stir for 3 h, pour out the solution, then add 80mL deionized water, 3g hydroxyapatite and 2g collagen peptide, add 1g NHS and 1g EDC, sonicate at 1000W for 5 min, stir for 10 h, pour out the liquid, wash, dry, and obtain the surface-treated glass petri dish; S2. Preparation of primary culture medium: Add 3g citric acid, 0.3g growth factor and 1g natural antibacterial agent to 150g α-MEM basic culture medium, stir and mix evenly to obtain primary culture medium; The growth-promoting factors include sodium heparin and interleukin-transforming growth factor-β in a mass ratio of 2:0.2; the natural antibacterial agents include ferulic acid and allicin in a mass ratio of 2:5. S3. Preparation of cell cryopreservation solution: Add 5g mannose, 20g DMSO, 2g glutathione and 1g bovine serum albumin to 70g DMEM medium and mix well to prepare cell cryopreservation solution; S4. Culture: Add the primary culture medium to a surface-treated glass culture dish, add the cleaned and minced fresh human dental pulp tissue, and incubate at 35°C for 2 days. After the primary cells have emerged, pour off the liquid, collect the primary cells, and partially resuspend them in cell cryopreservation solution for cryopreservation. S5. Passaging: The remaining primary cells were added to DMEM medium containing the nano-activating particles prepared in Example 1. The nano-activating particles in the medium were 5 wt%. Under hypoxia, low-frequency magnetic field and light conditions, the oxygen content was 3 v / v%, the magnetic field strength of the low-frequency magnetic field was 0.1 T, and the light intensity was 3000 Lux. The cells were passaged at 35°C. The nano-activating particles were separated by magnets. The passaged cells were collected, resuspended in cell cryopreservation solution, and cryopreserved to obtain dental pulp stem cells. Example 2

[0031] This embodiment provides a method for preparing dental pulp stem cells, including the following steps: S1. Surface treatment of glass petri dishes: Place 25wt% KOH solution in a glass petri dish, heat to 60℃ for 4 min, pour out the liquid, then add Tris-HCl solution with pH 9.5, add 5g polydopamine, heat to 50℃ and stir for 5 h, pour out the solution, then add 100mL deionized water, 5g hydroxyapatite and 4g collagen peptide, add 2g NHS and 2g EDC, sonicate at 2000W for 10 min, stir for 12 h, pour out the liquid, wash, and dry to obtain the surface-treated glass petri dish; S2. Preparation of primary culture medium: Add 5g of disodium EDTA, 0.5g of growth factor and 2g of natural antibacterial agent to 200g of α-MEM basic culture medium, stir and mix evenly to obtain the primary culture medium; The growth-promoting factors include sodium heparin and interleukin-transforming growth factor-β in a mass ratio of 3:0.5, and the natural antibacterial agents include ferulic acid and allicin in a mass ratio of 3:7. S3. Preparation of cell cryopreservation solution: Add 10g mannose, 30g DMSO, 3g glutathione and 3g bovine serum albumin to 90g DMEM medium and mix well to prepare cell cryopreservation solution; S4. Culture: Add the primary culture medium to a surface-treated glass culture dish, add the cleaned and minced fresh human dental pulp tissue, and incubate at 37°C for 4 days. After the primary cells have emerged, pour off the liquid, collect the primary cells, and partially resuspend them in cell cryopreservation solution for cryopreservation. S5. Passaging: The remaining primary cells were added to DMEM medium containing the nano-activating particles prepared in Preparation Example 2. The nano-activating particles in the medium were 10 wt%. Under hypoxia, low-frequency magnetic field and light conditions, the oxygen content was 6 v / v, the magnetic field strength of the low-frequency magnetic field was 0.3 T, and the light intensity was 5000 Lux. The cells were passaged at 37°C. The nano-activating particles were separated by magnets. The passaged cells were collected, resuspended in cell cryopreservation solution, and cryopreserved to obtain dental pulp stem cells. Example 3

[0032] This embodiment provides a method for preparing dental pulp stem cells, including the following steps: S1. Surface treatment of glass petri dishes: 22wt% NaOH solution was placed in a glass petri dish and heated to 55℃ for 3 min. The liquid was poured out, and then Tris-HCl solution with pH 9 was added. 4g of polydopamine was added, and the mixture was heated to 45℃ and stirred for 4 h. The solution was poured out, and then 90mL of deionized water, 4g of hydroxyapatite and 3g of collagen peptides were added. 1.5g of NHS and 1.5g of EDC were added, and the mixture was sonicated at 1500W for 7 min. The mixture was stirred for 11 h, and the liquid was poured out. The petri dishes were washed and dried to obtain the surface-treated glass petri dishes. S2. Preparation of primary culture medium: Add 4g EDTA, 0.4g growth factor and 1.5g natural antibacterial agent to 170g α-MEM basic culture medium, stir and mix evenly to obtain primary culture medium; The growth-promoting factors include sodium heparin and interleukin-transforming growth factor-β in a mass ratio of 2.5:0.35, and the natural antibacterial agents include ferulic acid and allicin in a mass ratio of 2.5:6. S3. Preparation of cell cryopreservation solution: Add 7g mannose, 25g DMSO, 2.5g glutathione and 2g bovine serum albumin to 80g DMEM medium and mix well to prepare cell cryopreservation solution; S4. Culture: Add the primary culture medium to a surface-treated glass culture dish, add the cleaned and minced fresh human dental pulp tissue, and incubate at 36°C for 3 days. After the primary cells have emerged, pour off the liquid, collect the primary cells, and partially resuspend them in cell cryopreservation solution for cryopreservation. S5. Passaging: The remaining primary cells were added to DMEM medium containing the nano-activating particles prepared in Example 3. The nano-activating particles in the medium were 7 wt%. Under hypoxia, low-frequency magnetic field and light conditions, the oxygen content was 5 v / v, the magnetic field strength of the low-frequency magnetic field was 0.2 T, and the light intensity was 4000 Lux. The cells were passaged at 36℃, the nano-activating particles were separated by magnet, the passaged cells were collected, resuspended in cell cryopreservation solution, and cryopreserved to obtain dental pulp stem cells.

[0033] Comparative Example 1 The difference from Example 3 is that the nano-activated particles were prepared by Comparative Preparation Example 1.

[0034] Comparative Example 2 The difference from Example 3 is that the nano-activating particles were prepared by Comparative Preparation Example 2.

[0035] Comparative Example 3 The difference compared to Example 3 is that no nano-activating particles were added.

[0036] Specifically as follows: S5. Passaging: Add the remaining primary cells to DMEM medium and passage them at 36°C under hypoxic conditions (oxygen content 5v / v%). Collect the passaged cells, resuspend them in cell cryopreservation solution, and freeze them to obtain dental pulp stem cells.

[0037] Comparative Example 4 The difference from Example 3 is that step S1 was not performed.

[0038] Specifically as follows: S1. Preparation of primary culture medium: Add 4g EDTA, 0.4g growth factor and 1.5g natural antibacterial agent to 170g α-MEM basic culture medium, stir and mix evenly to obtain primary culture medium; The growth-promoting factors include sodium heparin and interleukin-transforming growth factor-β in a mass ratio of 2.5:0.35, and the natural antibacterial agents include ferulic acid and allicin in a mass ratio of 2.5:6. S2. Preparation of cell cryopreservation solution: Add 7g mannose, 25g DMSO, 2.5g glutathione and 2g bovine serum albumin to 80g DMEM medium and mix well to prepare cell cryopreservation solution; S3. Culture: Add the primary culture medium to a glass culture dish, add the cleaned and minced fresh human dental pulp tissue, and incubate at 36°C for 3 days. After the primary cells have emerged, pour off the liquid, collect the primary cells, and partially resuspend them in cell cryopreservation solution for cryopreservation. S4. Passaging: The remaining primary cells were added to DMEM medium containing the nano-activating particles prepared in Example 3. The nano-activating particles in the medium were 7 wt%. Under hypoxia, low-frequency magnetic field and light conditions, the oxygen content was 5 v / v, the magnetic field strength of the low-frequency magnetic field was 0.2 T, and the light intensity was 4000 Lux. The cells were passaged at 36℃, the nano-activating particles were separated by magnet, the passaged cells were collected, resuspended in cell cryopreservation solution, and cryopreserved to obtain dental pulp stem cells.

[0039] Comparative Example 5 The difference from Example 3 is that no growth-promoting factor was added in step S2.

[0040] Specifically as follows: S2. Preparation of primary culture medium: Add 4g EDTA and 1.5g natural antibacterial agent to 170g α-MEM basic culture medium, stir and mix evenly to obtain primary culture medium; The natural antibacterial agent comprises ferulic acid and allicin in a mass ratio of 2.5:6.

[0041] Comparative Example 6 The difference from Example 3 is that no natural antibacterial agent was added in step S2.

[0042] Specifically as follows: S2. Preparation of primary culture medium: Add 4g EDTA and 0.4g growth factor to 170g α-MEM basic culture medium, stir and mix evenly to obtain primary culture medium; The growth-promoting factors include heparin sodium and interleukin-transforming growth factor-β in a mass ratio of 2.5:0.35.

[0043] Comparative Example 7 The difference from Example 3 is that hypoxic culture was not performed in step S5.

[0044] Specifically as follows: S5. Passaging: The remaining primary cells were added to DMEM medium containing the nano-activating particles prepared in Example 3. The content of nano-activating particles in the medium was 7 wt%. The cells were passaged at 36°C under low-frequency magnetic field and light conditions. The magnetic field strength of the low-frequency magnetic field was 0.2 T and the light intensity was 4000 Lux. The nano-activating particles were separated by magnet. The passaged cells were collected, resuspended in cell cryopreservation solution, and cryopreserved to obtain dental pulp stem cells.

[0045] Test Example 1 Following the methods in Examples 1-3 or Comparative Examples 1-7, during the first passage, cell concentration was determined using trypan blue staining. Dental pulp stem cells in the culture dishes were treated with 0.5% disodium EDTA solution (10,000 primary dental pulp stem cells were initially seeded in each culture dish), washed with PBS, and then stained with 0.4% trypan blue solution. The number of viable cells in each culture dish was counted (three parallel experiments).

[0046] The results are shown in Table 1.

[0047] Table 1

[0048] As can be seen from the table above, the methods in Examples 1-3 of this invention produce a large number of live dental pulp stem cells. Flow cytometry analysis shows that each group of dental pulp stem cells exhibits marker characteristics of dental pulp stem cells.

[0049] Test Example 2 Dental pulp stem cells obtained from Examples 1-3 or Comparative Examples 1-7, which were in good growth and nearly confluent at the third generation, were treated with 0.5% disodium EDTA solution and then suspended in the primary culture medium prepared for each experimental group before counting. The cell density was adjusted to 1×10⁻⁶. 5 Add 2 mL / well to each well of a 6-well plate. When the cells reach 80% confluence, discard the culture medium and replace it with osteogenic induction medium (containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / mL gentamicin, 50 μg / mL L-ascorbic acid 2-phosphate, 10 nmol / L dexamethasone, and 10 mmol / L β-glycerophosphate). Induce continuously, changing the induction medium every 3 days. Stop induction on day 28, collect cells, lyse them to extract proteins, and measure alkaline phosphatase. Measure osteocalcin using the culture medium (using a commercially available kit, with 5 samples per group and the average value).

[0050] The results are shown in Table 2.

[0051] Table 2

[0052] As can be seen from the table above, the dental pulp stem cells obtained in Examples 1-3 of this invention have good osteogenic differentiation ability.

[0053] Test Example 3 Dental pulp stem cells obtained in Examples 1-3 or Comparative Examples 1-7 with good third-generation growth and near-confluence were seeded at a cell density of 8000 / well in 96-well plates. After culturing for 24 hours, osteogenic induction medium (containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / mL gentamicin, 50 μg / mL L-ascorbic acid 2-phosphate, 10 nmol / L dexamethasone, and 10 mmol / L β-glycerol phosphate) was added. After culturing for 5 days, the culture medium was discarded, and a culture medium was prepared according to the ratio of culture medium to CCK-8 assay reagent of 9:1 and added to the wells to be tested. After culturing at 37°C for 1.5 hours, the absorbance value at 450 nm was measured.

[0054] The results are shown in Table 3.

[0055] Table 3

[0056] As can be seen from the table above, the dental pulp stem cells obtained in Examples 1-3 of this invention have better proliferative activity.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing dental pulp stem cells, characterized in that, Add α-MEM basal culture medium containing complexing agents, growth factors and natural antibacterial agents to a surface-treated glass culture dish, add cleaned and minced fresh human dental pulp tissue, and incubate at a constant temperature. After the primary cells emerge, pour off the liquid, collect the primary cells, partially resuspend them in cell cryopreservation solution and freeze them. Add the remaining primary cells to a culture medium containing nano-activating particles, and passage them under hypoxia, low-frequency magnetic field and light conditions. Collect the passaged cells, resuspend them in cell cryopreservation solution and freeze them to obtain dental pulp stem cells.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Surface treatment of glass petri dishes: Alkali solution is placed in a glass petri dish and heated. The alkali solution is then poured out. Tris-HCl solution is added, followed by polydopamine. The mixture is heated and stirred to react. The solution is then poured out. Deionized water, hydroxyapatite, and collagen peptides are then added. NHS and EDC are added. The mixture is sonicated and stirred to react. The liquid is then poured out, washed, and dried to obtain a surface-treated glass petri dish. S2. Preparation of primary culture medium: Add complexing agent, growth factor and natural antibacterial agent to α-MEM basic culture medium, stir and mix evenly to obtain primary culture medium; S3. Preparation of cell cryopreservation solution: Add mannose, DMSO, glutathione and bovine serum albumin to DMEM medium and mix well to prepare cell cryopreservation solution; S4. Culture: Add the primary culture medium to a surface-treated glass culture dish, add the cleaned and minced fresh human dental pulp tissue, and incubate at a constant temperature. After the primary cells have emerged, pour off the liquid, collect the primary cells, and partially resuspend them in cell cryopreservation solution for cryopreservation. S5. Passaging: The remaining primary cells were added to a culture medium containing nano-activating particles and passaged under hypoxia, low-frequency magnetic field and light conditions. The passaged cells were collected, resuspended in cell cryopreservation solution, and cryopreserved to obtain dental pulp stem cells.

3. The preparation method according to claim 2, characterized in that, In step S1, the alkaline solution is a 20-25 wt% NaOH or KOH solution, the heating treatment temperature is 50-60℃, the pH value of the Tris-HCl solution is 8.5-9.5, the heating and stirring reaction temperature is 40-50℃, and the time is 1 h. The mass ratio of deionized water, hydroxyapatite, collagen peptides, NHS and EDC is 80-100:1:2-4:1-2:1-2. The ultrasonic treatment power is 1000-2000W, and the time is 5-10 min. The stirring reaction time is 10-12 h.

4. The preparation method according to claim 2, characterized in that, In step S2, the mass ratio of α-MEM basal culture medium, complexing agent, growth factor, and natural antibacterial agent is 150-200:1:0.3-0.5:1-2. The complexing agent is selected from at least one of citric acid, sodium citrate, disodium EDTA, EDTA, and phytic acid. The growth factor includes heparin sodium and interleukin-transforming growth factor-β, with a mass ratio of 2-3:0.2-0.

5. The natural antibacterial agent includes ferulic acid and allicin, with a mass ratio of 2-3:5-7. In step S3, the mass ratio of DMEM culture medium, mannose, DMSO, glutathione, and bovine serum albumin is 70-90:5-10:20-30:2-3:1-3.

5. The preparation method according to claim 2, characterized in that, The isothermal culture in step S4 is carried out at a temperature of 35-37℃ for 2-4 days; the oxygen content in the low-oxygen, low-frequency magnetic field, and light conditions in step S5 is 3-6 v / v%, the magnetic field strength of the low-frequency magnetic field is 0.1-0.3T, the light intensity is 3000-5000 Lux, and the subculture temperature is 35-37℃.

6. The preparation method according to claim 2, characterized in that, The content of nano-activating particles in the culture medium containing nano-activating particles mentioned in step S5 is 5-10 wt%, and the preparation method of the nano-activating particles is as follows: T1. Add block black phosphorus to N-methylpyrrolidone, sonicate in an ice bath in the dark, centrifuge, wash, dry, add the product to Tris-HCl solution, add dopamine hydrochloride, heat and stir to react, centrifuge, wash, dry, and obtain modified black phosphorus nanosheets. T2. Modified black phosphorus nanosheets and sodium molybdate were added to water, the pH of the solution was adjusted, glutathione was added, the mixture was stirred, hydrothermal reaction was carried out, cooled, centrifuged, washed, and dried to obtain N-doped MoS2 quantum dots@modified black phosphorus nanosheets. T3. Add N-doped MoS2 quantum dots@modified black phosphorus nanosheets to water, add ferric chloride and ferrous chloride under inert gas protection, add ammonia dropwise, heat and stir the reaction, centrifuge, wash, and dry to obtain nano-activated particles.

7. The preparation method according to claim 6, characterized in that, In step T1, the power of the light-shielded ice bath ultrasound is 300-500W, and the time is 1-3 days. The centrifugation method is to centrifuge at 3000-5000 r / min for 10-20 min, collect the supernatant, and then centrifuge at 10000-12000 r / min for 15-25 min to collect the precipitate. The pH value of the Tris-HCl solution is 8.5-9.

5. The mass ratio of the block black phosphorus to dopamine hydrochloride is 10:

1. The temperature of the heating and stirring reaction is 40-50℃, and the time is 2-4 h.

8. The preparation method according to claim 6, characterized in that, In step T2, the mass ratio of modified black phosphorus nanosheets, sodium molybdate, and glutathione is 10:3-4:5-7, the pH of the solution is adjusted to 6-6.5, the hydrothermal reaction temperature is 190-210℃, and the time is 10-15h.

9. The preparation method according to claim 6, characterized in that, In step T3, the mass ratio of N-doped MoS2 quantum dots@modified black phosphorus nanosheets, ferric chloride, and ferrous chloride is 10⁻¹². 3.24:1.26, wherein the pH value is adjusted to 9-10 by adding ammonia water, and the temperature of the heating and stirring reaction is 80-90℃, and the time is 1h.

10. The use of dental pulp stem cells prepared by the preparation method according to any one of claims 1-9 in the preparation of a drug for treating tooth defects and tooth loss.

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