Preparation method and application of dental pulp stem cells

Abstract

The application provides a preparation method and application of dental pulp stem cells, and belongs to the technical field of medicines. An alpha-MEM basic culture solution containing a complexing agent, a growth promoting factor and a natural antibacterial agent is added into a surface treated glass culture dish, fresh human dental pulp tissue after cleaning and cutting is added, constant temperature culture is carried out, after primary cells are climbed out, liquid is poured out, the primary cells are collected, part of the primary cells is resuspended in a cell freezing solution, freezing is carried out, the remaining part of the primary cells is added into a culture medium containing nano-activated particles, subculture is carried out under low oxygen, low frequency magnetic field and light conditions, the subculture cells are collected, resuspended in the cell freezing solution and frozen, and the dental pulp stem cells are prepared. The preparation method is simple, the cost is low, industrial application is easy to realize, the culture period is short, the dental pulp stem cells obtained through culture have strong survival ability and good differentiation, can be differentiated into osteoblasts, and lay a foundation for preparing medicines for treating tooth defects and tooth loss.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a preparation method and application of dental pulp stem cells. BACKGROUND

[0002] Dental pulp stem cells were first discovered in 2000. Gronthos et al. found dental pulp stem cells in ex vivo deciduous teeth and wisdom teeth. They are a kind of cells with high proliferation ability, high self-renewal ability and multi-directional differentiation ability. Studies have successfully constructed dental pulp, dentin and tooth tissue using the cells. Dental pulp stem cells (DPSC) have many similarities in biological characteristics with other tissue-derived stem cells. Gronthos et al. studied the clonogenicity of dental pulp stem cells and found that the clonogenicity of DPSC from teeth was significantly higher than that of bone marrow stromal cells (BMSC), which indicated that DPSC had higher proliferation and self-renewal ability than BMSC. Dental pulp stem cells have strong proliferation, high tissue compatibility and convenient storage, which gives modern people the opportunity to store stem cells for the second time.

[0003] Currently, the commonly used methods for preparing dental pulp stem cells include enzyme combined digestion, tissue block culture and tissue block enzyme digestion. Although various methods can obtain dental pulp stem cells, dental pulp stem cells are easily contaminated during preparation and culture due to the complex types of bacteria in the oral cavity. In addition, the number of extracted dental pulp stem cells is small, and the cell growth is slow, which leads to the difficulty in preparing dental pulp stem cells. This is also the biggest problem encountered in storing dental pulp stem cells. Therefore, antibiotics are usually added to inhibit bacterial reproduction during the preparation of dental pulp stem cells. The 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 application of the above antibiotics cannot ensure that dental pulp stem cells are not contaminated. SUMMARY

[0004] The present application aims to provide a preparation method and application of dental pulp stem cells. The preparation method is simple, low in cost and easy to realize industrial application. The culture period is short, and the obtained dental pulp stem cells have strong survival ability and good differentiation ability, and can differentiate into osteoblasts, which lays a foundation for the preparation of drugs for treating dental defects and tooth loss.

[0005] The technical solution of the present application is as follows:

[0006] The application provides a preparation method of dental pulp stem cells, which comprises the following steps: adding alpha-MEM basic culture solution containing a complexing agent, a growth promoting factor and a natural antibacterial agent into a surface treated glass culture dish, adding fresh human dental pulp tissue which is cleaned and cut into pieces, culturing at constant temperature, pouring out the liquid after primary cells grow out, collecting the primary cells, resuspending part of the primary cells in a cell cryopreservation solution, cryopreserving, adding the remaining primary cells into a culture medium containing nano-activated particles, culturing by subculture under the conditions of hypoxia, low frequency magnetic field and light, collecting the subculture cells, resuspending the subculture cells in the cell cryopreservation solution, and cryopreserving to obtain the dental pulp stem cells.

[0007] As a further improvement of the application, the following steps are included:

[0008] S1. Surface treatment of the glass culture dish: the glass culture dish is filled with lye and heated, the lye is poured out, then Tris-HCl solution is added, polydopamine is added, heated and stirred for reaction, the solution is poured out, then deionized water, hydroxyapatite and collagen peptide are added, NHS and EDC are added, ultrasonic treatment is performed, stirring reaction is performed, the liquid is poured out, washed, dried, and the surface treated glass culture dish is obtained;

[0009] S2. Preparation of the primary culture solution: the alpha-MEM basic culture solution is added with the complexing agent, the growth promoting factor and the natural antibacterial agent, and stirred and mixed uniformly to obtain the primary culture solution;

[0010] S3. Preparation of the cell cryopreservation solution: the DMEM culture medium is added with mannose, DMSO, glutathione and bovine serum albumin, and mixed uniformly to obtain the cell cryopreservation solution;

[0011] S4. Culturing: the primary culture solution is added into the surface treated glass culture dish, the fresh human dental pulp tissue which is cleaned and cut into pieces is added, constant temperature culturing is performed, the primary cells grow out, the liquid is poured out, the primary cells are collected, part of the primary cells is resuspended in the cell cryopreservation solution, and cryopreservation is performed;

[0012] S5. Subculture: the remaining primary cells are added into the culture medium containing nano-activated particles, subculture is performed under the conditions of hypoxia, low frequency magnetic field and light, the subculture cells are collected, resuspended in the cell cryopreservation solution, and cryopreservation is performed to obtain the dental pulp stem cells.

[0013] As a further improvement of the present application, the alkali solution in step S1 is a 20-25wt% NaOH or KOH solution, the temperature of the heating treatment is 50-60℃, the pH value of the Tris-HCl solution is 8.5-9.5, the temperature of the heating and stirring reaction is 40-50℃, the time is 1h, the mass ratio of the deionized water, hydroxyapatite, collagen peptide, NHS and EDC is 80-100:1:2-4:1-2:1-2, the power of the ultrasonic treatment is 1000-2000W, the time is 5-10min, and the time of the stirring reaction is 10-12h.

[0014] As a further improvement of the present application, the mass ratio of the α-MEM basic culture solution, complexing agent, growth promoting factor and natural antibacterial agent in step S2 is 150-200:1:0.3-0.5:1-2, the complexing agent is at least one selected from citric acid, sodium citrate, disodium EDTA, EDTA and phytic acid, the growth promoting factor includes heparin sodium and interleukin transforming growth factor-β, the mass ratio is 2-3:0.2-0.5, and the natural antibacterial agent includes ferulic acid and allicin, the mass ratio is 2-3:5-7; the mass ratio of the DMEM culture medium, mannose, DMSO, glutathione and bovine serum albumin in step S3 is 70-90:5-10:20-30:2-3:1-3.

[0015] As a further improvement of the present application, the temperature of the constant temperature culture in step S4 is 35-37℃, and the time is 2-4d; in step S5, the oxygen content in the low oxygen, low frequency magnetic field and light irradiation conditions is 3-6v / v%, the magnetic field strength of the low frequency magnetic field is 0.1-0.3T, the light intensity of the light irradiation is 3000-5000Lux, and the temperature of the subculture is 35-37℃.

[0016] As a further improvement of the present application, the content of the nano-activated particles in the culture medium in step S5 is 5-10wt%, and the preparation method of the nano-activated particles is as follows:

[0017] T1. The bulk black phosphorus is added into N-methyl pyrrolidone, ultrasonic treatment is carried out in ice bath under light shielding, centrifugation, washing and drying are carried out, the product is added into Tris-HCl solution, dopamine hydrochloride is added, heating and stirring reaction is carried out, centrifugation, washing and drying are carried out, and modified black phosphorus nanosheet is prepared;

[0018] T2. The modified black phosphorus nanosheet and sodium molybdate are added into water, the pH value of the solution is adjusted, glutathione is added, mixing and stirring are carried out, hydrothermal reaction is carried out, cooling, centrifugation, washing and drying are carried out, and N-doped MoS2 quantum dots@modified black phosphorus nanosheet is prepared;

[0019] T3. Add the N-doped MoS2 quantum dots modified black phosphorus nanosheet into water, and add ferric chloride and ferrous chloride under inert gas protection, dropwise add ammonia water, heat and stir to react, centrifuge, wash, dry, and obtain the nano-activated particle.

[0020] As a further improvement of the application, the power of the light-proof ice bath ultrasonic in step T1 is 300-500W, the time is 1-3d, the method of centrifugation is centrifuged at 3000-5000r / min for 10-20min, and after collecting the supernatant, centrifuged at 10000-12000r / min for 15-25min, and the precipitate is collected, the pH value of the Tris-HCl solution is 8.5-9.5, the mass ratio of the blocky black phosphorus and dopamine hydrochloride is 10:1, the temperature of the heating and stirring reaction is 40-50℃, and the time is 2-4h.

[0021] As a further improvement of the application, the mass ratio of the modified black phosphorus nanosheet, sodium molybdate and glutathione in step T2 is 10:3-4:5-7, the pH value of the solution is adjusted to 6-6.5, the temperature of the hydrothermal reaction is 190-210℃, and the time is 10-15h.

[0022] As a further improvement of the application, the mass ratio of the N-doped MoS2 quantum dots modified black phosphorus nanosheet, ferric chloride and ferrous chloride in step T3 is 10-12:3.24:1.26, the pH value is adjusted to 9-10 by dropwise adding ammonia water, the temperature of the heating and stirring reaction is 80-90℃, and the time is 1h.

[0023] The application further protects the use of the dental pulp stem cells prepared by the above preparation method in the preparation of a drug for treating tooth defects and tooth loss.

[0024] The application has the following beneficial effects:

[0025] The alkali solution is used to treat the inner surface of the glass culture dish, and the alkali can react with the glass silicon dioxide, so that the inner surface of the glass is roughened to generate a large number of active groups, and then a polydopamine layer is formed on the surface to adhere nano-hydroxyapatite, has good biological activity, can promote the adhesion, proliferation and differentiation of odontoblast stem cells, and is coupled with collagen peptides to provide a growth environment close to the in-vivo environment for cells, promote the adhesion, proliferation and differentiation of cells, the surface-treated glass culture dish can enhance the cell adhesion capacity, shorten the primary cell outgrowth time, reduce the cost and be easy to realize industrial application.

[0026] The primary culture solution of the application adds complexing agents, growth promoting factors and natural antibacterial agents to the alpha-MEM basic culture solution, uses the complexing agents to destroy the calcium adhesion between cells, and combines mechanical vibration to separate the cells, which is suitable for enzyme-sensitive dental pulp sample tissues; the added growth promoting factors include heparin sodium and transforming growth factor-beta, wherein the heparin sodium can be combined with the transforming growth factor-beta to enhance the stability thereof, prevent degradation thereof, promote the combination of the transforming growth factor-beta with its receptor, enhance signal transmission, and interact with proteoglycans in the extracellular matrix to change the structure and properties of the extracellular matrix, provide a more favorable microenvironment for the adhesion, migration and proliferation of stem cells, and further affect the growth and differentiation of stem cells. The heparin sodium has anticoagulation properties, can prevent blood coagulation, improve the blood supply of tissues and cells, thereby indirectly providing better nutrition and oxygen supply for the growth of stem cells, promoting the growth and survival of stem cells, and the transforming growth factor-beta promotes the growth and proliferation of dental pulp stem cells through signal pathway activation, gene expression regulation, extracellular matrix remodeling and anti-inflammatory and immune regulation.

[0027] The added natural antibacterial agents include ferulic acid and allicin, the lipid-soluble structure of ferulic acid can be inserted into the lipid bilayer of bacterial cell membranes to change the fluidity and permeability of the membranes, leading to leakage of intracellular electrolytes (such as K + ) and proteins, and ultimately inhibiting bacterial growth, and the lipid-soluble sulfur group of allicin can be inserted into bacterial cell membranes to increase membrane permeability and inhibit the function of membrane proteins (such as transport proteins), leading to leakage of intracellular substances. It has a destructive effect on biofilms of drug-resistant bacteria, prevents bacterial adhesion and colonization by degrading extracellular polysaccharides in the biofilm matrix, and the synergistic effect of the two inhibits bacterial contamination and promotes osteogenic differentiation by activating the Wnt / β-catenin pathway.

[0028] The cell cryopreservation solution of the application adds mannose, DMSO, glutathione and bovine serum albumin, wherein DMSO penetrates into cells to reduce the freezing point in cells and reduce the formation of ice crystals; adjust the osmotic pressure inside and outside the cells to avoid cell dehydration or swelling, mannose forms a high-osmotic pressure environment outside the cells to reduce water permeation and indirectly inhibit the growth of ice crystals; bovine serum albumin wraps the cell surface to reduce mechanical damage during the freezing process, provides nutrients and maintains cell activity; glutathione reduces oxidative stress damage during the freezing and thawing processes and maintains cell metabolic function; DMEM medium provides electrolytes, buffer systems and basic nutrients to maintain cell osmotic pressure and pH stability.

[0029] The nanometer activating particle is added to the culture medium in the subculture process, the polydopamine modified black phosphorus nanosheet is used as a core, the specific surface area of the black phosphorus nanosheet is large, has strong surface adsorption capacity, the lone pair of the phosphorus element is combined with the empty orbital of the heavy metal element, so that the black phosphorus nanosheet has strong heavy metal ion chelation capacity, can adsorb a large amount of heavy metal ions in a short time, and removes active oxygen, so that the survival capacity of stem cells can be improved, proliferation is promoted, the surface is loaded with MoS2 quantum dots, due to the presence of polydopamine, the N element is doped in the quantum dots, so that a heterojunction is formed with the black phosphorus, under light irradiation, heat and singlet oxygen are generated at the same time by using the photothermal conversion of the black phosphorus and the photodynamic effect of the MoS2 quantum dots, stem cell migration and angiogenesis are promoted, the N doping can reduce the band gap of the black phosphorus, the photodynamic effect is improved by regulating the electronic structure and enhancing the interface coupling, the growth and survival of stem cells are promoted, in addition, the magnetic ferroferric oxide is deposited on the surface, so that the nanometer activating particle not only has magnetism and can be separated and recycled, but also can generate trace heat under the action of a magnetic field, can activate heat-sensitive proteins on the mitochondrial membrane, promote the efficiency of oxidative phosphorylation, reduce lactic acid generation, maintain a low acidic microenvironment, is conducive to stem cell self-renewal, trace heat promotes stem cells to secrete matrix metalloproteinase, and provides a more suitable microenvironment for cell migration and differentiation.

[0030] The preparation method of the dental pulp stem cells is simple, low in cost, easy to realize industrial application, short in culture period, high in survival capacity of the obtained dental pulp stem cells, and good in differentiation, and the dental pulp stem cells can be differentiated into osteoblasts, which lays a foundation for preparing drugs for treating tooth defects and tooth loss. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below, obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0032] EDC, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide; NHS, N-hydroxysuccinimide.

[0033] Preparation Example 1 Preparation of nanometer activating particle

[0034] The method is as follows:

[0035] T1. 10 mg bulk black phosphorus was added into 100 mL N-methyl pyrrolidone, ice bath, 300 W ultrasonic for 1 d, centrifuged at 3000 r / min for 10 min, the supernatant was collected and centrifuged at 10000 r / min for 15 min, the precipitate was collected, washed, dried, the product was added into 50 mL Tris-HCl solution with pH value of 8.5, 3 mg dopamine hydrochloride was added, heated to 40℃, stirred for 2 h, centrifuged, washed, dried, and modified black phosphorus nanosheets were prepared;

[0036] T2. 1 g of modified black phosphorus nanosheets and 0.3 g of sodium molybdate were added to 50 mL of water, the pH value of the solution was adjusted to 6, 0.5 g of glutathione was added, mixed and stirred for 5 min, and hydrothermal reaction was carried out at 190℃ for 10 h, cooled, centrifuged, washed, and dried to obtain N-doped MoS2 quantum dots@modified black phosphorus nanosheets;

[0037] T3. 1 g of N-doped MoS2 quantum dots@modified black phosphorus nanosheets was added to 150 mL of water, 0.324 g of ferric chloride and 0.126 g of ferrous chloride were added under nitrogen protection, ammonia water was added dropwise to adjust the pH value to 9, heated to 80℃, stirred for 3 h, centrifuged, washed, and dried to obtain nano-activated particles.

[0038] Preparation of nano-activated particles

[0039] The method is as follows:

[0040] T1. 10 mg bulk black phosphorus was added into 100 mL N-methyl pyrrolidone, ice bath, 300 W ultrasonic for 1 d, centrifuged at 3000 r / min for 10 min, the supernatant was collected and centrifuged at 10000 r / min for 15 min, the precipitate was collected, washed, dried, the product was added into 50 mL Tris-HCl solution with pH value of 8.5, 3 mg dopamine hydrochloride was added, heated to 40℃, stirred for 2 h, centrifuged, washed, dried, and modified black phosphorus nanosheets were prepared;

[0041] T2. 1 g of modified black phosphorus nanosheets and 0.3 g of sodium molybdate were added to 50 mL of water, the pH value of the solution was adjusted to 6, 0.5 g of glutathione was added, mixed and stirred for 5 min, and hydrothermal reaction was carried out at 190℃ for 10 h, cooled, centrifuged, washed, and dried to obtain N-doped MoS2 quantum dots@modified black phosphorus nanosheets;

[0042] T3. 1.2 g of N-doped MoS2 quantum dots@modified black phosphorus nanosheets was added to 150 mL of water, 0.324 g of ferric chloride and 0.126 g of ferrous chloride were added under nitrogen protection, ammonia water was added dropwise to adjust the pH value to 9, heated to 80℃, stirred for 3 h, centrifuged, washed, and dried to obtain nano-activated particles.

[0043] Preparation of nano-activated particles

[0044] The method is as follows:

[0045] T1. 10 mg of bulk black phosphorus was added to 100 mL of N-methylpyrrolidone, and ice-bath ultrasonic treatment was performed in the dark for 2 d at 400 W. Centrifugation was performed at 4000 r / min for 15 min, and the supernatant was collected and centrifuged at 11000 r / min for 20 min. The precipitate was collected, washed, and dried. The product was added to 50 mL of a Tris-HCl solution with a pH value of 9, and 4 mg of dopamine hydrochloride was added. The solution was heated to 45℃, and stirring was performed for 3 h. Centrifugation, washing, and drying were performed to obtain modified black phosphorus nanosheets;

[0046] T2. 1 g of modified black phosphorus nanosheets and 0.35 g of sodium molybdate were added to 50 mL of water, and the pH value of the solution was adjusted to 6.2. 0.6 g of glutathione was added, and stirring was performed for 5 min. Hydrothermal reaction was performed at 200℃ for 12 h. After cooling, centrifugation, washing, and drying were performed to obtain N-doped MoS2 quantum dots@modified black phosphorus nanosheets;

[0047] T3. 1.1 g of N-doped MoS2 quantum dots@modified black phosphorus nanosheets was added to 150 mL of water, and 0.324 g of ferric chloride and 0.126 g of ferrous chloride were added under nitrogen protection. Ammonia water was added dropwise to adjust the pH value to 9.5. The solution was heated to 85℃, and stirring was performed for 4 h. Centrifugation, washing, and drying were performed to obtain nano-activated particles.

[0048] Comparative Preparation Example 1

[0049] Compared with Preparation Example 3, the difference is that step T2 is not performed.

[0050] The specific process is as follows:

[0051] T1. 10 mg of bulk black phosphorus was added to 100 mL of N-methylpyrrolidone, and ice-bath ultrasonic treatment was performed in the dark for 2 d at 400 W. Centrifugation was performed at 4000 r / min for 15 min, and the supernatant was collected and centrifuged at 11000 r / min for 20 min. The precipitate was collected, washed, and dried. The product was added to 50 mL of a Tris-HCl solution with a pH value of 9, and 4 mg of dopamine hydrochloride was added. The solution was heated to 45℃, and stirring was performed for 3 h. Centrifugation, washing, and drying were performed to obtain modified black phosphorus nanosheets;

[0052] T2. 1.1 g of modified black phosphorus nanosheets was added to 150 mL of water, and 0.324 g of ferric chloride and 0.126 g of ferrous chloride were added under nitrogen protection. Ammonia water was added dropwise to adjust the pH value to 9.5. The solution was heated to 85℃, and stirring was performed for 4 h. Centrifugation, washing, and drying were performed to obtain nano-activated particles.

[0053] Comparative Preparation Example 2

[0054] The difference compared with Preparation Example 3 is that no dopamine hydrochloride is added in step T1.

[0055] The details are as follows:

[0056] T1. 10 mg of bulk black phosphorus was added into 100 mL of N-methylpyrrolidone, and the mixture was subjected to ultrasonic treatment in dark for 2 days in an ice bath at 400 W, centrifuged at 4000 r / min for 15 min, and then the supernatant was collected and centrifuged at 11000 r / min for 20 min, and the precipitate was collected, washed, and dried to obtain black phosphorus nanosheets;

[0057] T2. 1 g of black phosphorus nanosheets and 0.35 g of sodium molybdate were added into 50 mL of water, the pH value of the solution was adjusted to 6.2, 0.6 g of glutathione was added, and the mixture was stirred for 5 min, and then subjected to hydrothermal reaction at 200℃ for 12 h, and then cooled, centrifuged, washed, and dried to obtain N-doped MoS2 quantum dots@black phosphorus nanosheets;

[0058] T3. 1.1 g of N-doped MoS2 quantum dots@black phosphorus nanosheets were added into 150 mL of water, and then 0.324 g of ferric chloride and 0.126 g of ferrous chloride were added under nitrogen protection, and the pH value was adjusted to 9.5 by dropwise addition of ammonia water, and then the mixture was heated to 85℃ and stirred for 4 h, and then centrifuged, washed, and dried to obtain nano-activated particles. Example 1

[0059] The present embodiment provides a preparation method of dental pulp stem cells, comprising the following steps:

[0060] S1. Surface treatment of a glass culture dish: 20 wt% NaOH solution was placed in a glass culture dish, heated to 50℃ for 2 min, and then the liquid was poured out, and then a Tris-HCl solution with a pH value of 8.5 was added, 3 g of polydopamine was added, heated to 40℃ and stirred for 3 h, and then the solution was poured out, and then 80 mL of deionized water, 3 g of hydroxyapatite and 2 g of collagen peptide were added, 1 g of NHS and 1 g of EDC were added, 1000 W ultrasonic treatment was performed for 5 min, and then stirring reaction was performed for 10 h, and then the liquid was poured out, washed, and dried to obtain a surface-treated glass culture dish;

[0061] S2. Preparation of a primary culture solution: 3 g of citric acid, 0.3 g of a growth promoting factor and 1 g of a natural antibacterial agent were added into 150 g of α-MEM basic culture solution, and then the mixture was stirred and mixed uniformly to obtain a primary culture solution;

[0062] The growth promoting factor comprises heparin sodium and interleukin transforming growth factor-β, and the mass ratio is 2:0.2, and the natural antibacterial agent comprises ferulic acid and allicin, and the mass ratio is 2:5;

[0063] S3. Preparation of cell cryopreservation solution: 5g of mannose, 20g of DMSO, 2g of glutathione, and 1g of bovine serum albumin were added to 70g of DMEM medium, and the mixture was uniformly mixed to prepare the cell cryopreservation solution;

[0064] S4. Cultivation: The primary culture solution was added to the surface-treated glass culture dish, and the washed and cut fresh human dental pulp tissue was added. The mixture was incubated at 35°C for 2d. After the primary cells were climbed out, the liquid was poured out, and the primary cells were collected. Part of the primary cells was resuspended in the cell cryopreservation solution and cryopreserved.

[0065] S5. Subculture: The remaining primary cells were added to the DMEM medium containing the nano-activated particles prepared in Preparation Example 1. The content of the nano-activated particles in the medium was 5wt%. The medium was incubated under low-oxygen, low-frequency magnetic field, and light conditions. The oxygen content was 3v / v%, the magnetic field strength of the low-frequency magnetic field was 0.1T, the light intensity was 3000Lux, and the incubation temperature was 35°C. The nano-activated particles were separated by a magnet, and the subcultured cells were collected, resuspended in the cell cryopreservation solution, and cryopreserved to obtain the dental pulp stem cells. Example 2

[0066] The present embodiment provides a method for preparing dental pulp stem cells, which comprises the following steps:

[0067] S1. Surface treatment of glass culture dish: 25wt% KOH solution was placed in a glass culture dish and heated to 60°C for 4min. The liquid was then poured out, and a Tris-HCl solution with a pH value of 9.5 was added. Then, 5g of polydopamine was added, and the mixture was stirred and reacted at 50°C for 5h. The solution was then poured out, and 100mL of deionized water, 5g of hydroxyapatite, and 4g of collagen peptide were added. Then, 2g of NHS and 2g of EDC were added, and the mixture was ultrasonically treated at 2000W for 10min. The mixture was stirred and reacted for 12h, and then the liquid was poured out, washed, and dried to obtain the surface-treated glass culture dish.

[0068] S2. Preparation of primary culture solution: 5g of EDTA disodium, 0.5g of growth-promoting factor, and 2g of natural antibacterial agent were added to 200g of α-MEM basic culture solution, and the mixture was uniformly stirred and mixed to obtain the primary culture solution.

[0069] The growth-promoting factor comprises heparin sodium and interleukin transforming growth factor-β, and the mass ratio is 3:0.5. The natural antibacterial agent comprises ferulic acid and allicin, and the mass ratio is 3:7.

[0070] S3. Preparation of cell cryopreservation solution: 5g of mannose, 20g of DMSO, 2g of glutathione, and 1g of bovine serum albumin were added to 70g of DMEM medium, and the mixture was uniformly mixed to prepare the cell cryopreservation solution;

[0071] S4. Culturing: the primary culture solution is added to the surface-treated glass culture dish, fresh human dental pulp tissue after washing and cutting is added, and the culture is incubated at 37℃ for 4 days. After the primary cells grow out, the liquid is poured out, the primary cells are collected, and part of them is resuspended in the cell freezing solution for freezing;

[0072] S5. Subculture: the remaining primary cells are added to the DMEM medium containing the nano-activated particles prepared in Preparation Example 2, the content of the nano-activated particles in the medium is 10wt%, and the subculture is carried out under the conditions of hypoxia, low-frequency magnetic field and light. The oxygen content is 6v / v%, the magnetic field strength of the low-frequency magnetic field is 0.3T, the light intensity of the light is 5000Lux, and the subculture is carried out at 37℃. The nano-activated particles are separated by a magnet, the subculture cells are collected, resuspended in the cell freezing solution, frozen, and the dental pulp stem cells are prepared. Example 3

[0073] The present embodiment provides a method for preparing dental pulp stem cells, comprising the following steps:

[0074] S1. Surface treatment of glass culture dish: 22wt% NaOH solution is placed in the glass culture dish, heated to 55℃ for 3min, and then the liquid is poured out. Then, Tris-HCl solution with pH value of 9 is added, 4g polydopamine is added, heated to 45℃ and stirred for 4h of reaction. The solution is poured out, then 90mL deionized water, 4g hydroxyapatite and 3g collagen peptide are added, 1.5g NHS and 1.5g EDC are added, 1500W ultrasonic treatment is carried out for 7min, and stirring reaction is carried out for 11h. The liquid is poured out, washed, dried, and the surface-treated glass culture dish is prepared;

[0075] S2. Preparation of primary culture solution: 4g EDTA, 0.4g growth promoting factor and 1.5g natural antibacterial agent are added to 170g α-MEM basic culture solution, and stirred and mixed uniformly to prepare the primary culture solution;

[0076] The growth promoting factor includes sodium heparin and interleukin transforming growth factor-β, and the mass ratio is 2.5:0.35. The natural antibacterial agent includes ferulic acid and allicin, and the mass ratio is 2.5:6;

[0077] S3. Preparation of cell freezing solution: 7g mannose, 25g DMSO, 2.5g glutathione and 2g bovine serum albumin are added to 80g DMEM medium, and mixed uniformly to prepare the cell freezing solution;

[0078] S4. Culturing: the primary culture solution is added to the surface-treated glass culture dish, fresh human dental pulp tissue after washing and cutting is added, and the culture is incubated at 36℃ for 3 days. After the primary cells grow out, the liquid is poured out, the primary cells are collected, and part of them is resuspended in the cell freezing solution for freezing;

[0079] S5. Subculture: the remaining primary cells are added to DMEM medium containing the nano-activated particles prepared in Preparation Example 3, the content of the nano-activated particles in the medium is 7wt%, under the conditions of hypoxia, low-frequency magnetic field and light, the oxygen content is 5v / v%, the magnetic field strength of the low-frequency magnetic field is 0.2T, the light intensity of the light is 4000Lux, and the subculture is carried out at 36℃, the nano-activated particles are separated by a magnet, the subculture cells are collected, resuspended in a cell freezing solution, frozen, and dental pulp stem cells are prepared.

[0080] Comparative Example 1

[0081] Compared with Example 3, the difference is that the nano-activated particles are prepared in Comparative Preparation Example 1.

[0082] Comparative Example 2

[0083] Compared with Example 3, the difference is that the nano-activated particles are prepared in Comparative Preparation Example 2.

[0084] Comparative Example 3

[0085] Compared with Example 3, the difference is that no nano-activated particles are added.

[0086] The details are as follows:

[0087] S5. Subculture: the remaining primary cells are added to DMEM medium, under the condition of hypoxia, the oxygen content is 5v / v%, and the subculture is carried out at 36℃, the subculture cells are collected, resuspended in a cell freezing solution, frozen, and dental pulp stem cells are prepared.

[0088] Comparative Example 4

[0089] Compared with Example 3, the difference is that step S1 is not performed.

[0090] The details are as follows:

[0091] S1. Preparation of primary culture solution: 4g EDTA, 0.4g growth promoting factor and 1.5g natural antibacterial agent are added to 170g α-MEM base culture solution, and stirred and mixed uniformly to prepare a primary culture solution;

[0092] The growth promoting factor includes heparin sodium and interleukin transforming growth factor-β, and the mass ratio is 2.5:0.35, and the natural antibacterial agent includes ferulic acid and allicin, and the mass ratio is 2.5:6;

[0093] S2. Preparation of cell freezing solution: 7g mannose, 25g DMSO, 2.5g glutathione and 2g bovine serum albumin are added to 80g DMEM medium, and mixed uniformly to prepare a cell freezing solution;

[0094] S3. Culturing: the primary culture solution is added into a glass culture dish, and fresh human dental pulp tissue after washing and cutting is added, and the mixture is cultured at 36°C for 3 days. After the primary cells grow out, the liquid is poured out, and the primary cells are collected. Part of the primary cells is resuspended in a cell freezing solution and frozen.

[0095] S4. Subculture: the remaining primary cells are added into a DMEM culture medium containing the nano-activated particles prepared in Preparation Example 3. The content of the nano-activated particles in the culture medium is 7 wt%. The culture is carried out under low-oxygen, low-frequency magnetic field and light conditions. The oxygen content is 5 v / v%, the magnetic field strength of the low-frequency magnetic field is 0.2 T, the light intensity of the light is 4000 Lux, and the culture is carried out at 36°C. The nano-activated particles are separated by a magnet, and the subcultured cells are collected, resuspended in a cell freezing solution, frozen, and dental pulp stem cells are prepared.

[0096] Comparative Example 5

[0097] Compared with Example 3, the difference is that no growth-promoting factor is added in step S2.

[0098] Specifically as follows:

[0099] S2. Preparation of the primary culture solution: 4 g of EDTA and 1.5 g of natural antibacterial agent are added into 170 g of α-MEM basic culture solution, and the mixture is stirred and mixed uniformly to prepare the primary culture solution.

[0100] The natural antibacterial agent includes ferulic acid and allicin, and the mass ratio is 2.5:6.

[0101] Comparative Example 6

[0102] Compared with Example 3, the difference is that no natural antibacterial agent is added in step S2.

[0103] Specifically as follows:

[0104] S2. Preparation of the primary culture solution: 4 g of EDTA, 0.4 g of growth-promoting factor are added into 170 g of α-MEM basic culture solution, and the mixture is stirred and mixed uniformly to prepare the primary culture solution.

[0105] The growth-promoting factor includes heparin sodium and interleukin transforming growth factor-β, and the mass ratio is 2.5:0.35.

[0106] Comparative Example 7

[0107] Compared with Example 3, the difference is that no low-oxygen culture is carried out in step S5.

[0108] Specifically as follows:

[0109] S5. Subculture: the remaining primary cells were added into DMEM medium containing the nano-activated particles prepared in Preparation Example 3, the content of the nano-activated particles in the medium was 7 wt%, and the subculture was carried out at 36°C under the conditions of low-frequency magnetic field with a magnetic field strength of 0.2 T and light with an intensity of 4000 Lux. The nano-activated particles were separated by a magnet, and the subcultured cells were collected, resuspended in a cell freezing medium, frozen, and stored to obtain dental pulp stem cells.

[0110] Test Example 1

[0111] According to the method in Examples 1-3 or Comparative Examples 1-7, at the first subculture, cell concentration determination was performed by trypan blue staining, and the dental pulp stem cells in the culture dish (10000 primary dental pulp stem cells were originally plated in each culture dish) were treated with a 0.5% EDTA disodium solution, washed with PBS, and then stained with a 0.4% trypan blue solution. The number of viable cells in each culture dish was calculated (three parallel tests were performed).

[0112] The results are shown in Table 1.

[0113] Table 1

[0114]

[0115] As shown in the above table, the number of viable cells of the dental pulp stem cells prepared by the method in Examples 1-3 is larger, and the dental pulp stem cells in each group exhibit the marker characteristics of dental pulp stem cells by flow cytometry detection.

[0116] Test Example 2

[0117] The dental pulp stem cells obtained in Examples 1-3 or Comparative Examples 1-7 that grew well and were close to confluence in the third generation were taken, counted after being treated with a 0.5% EDTA disodium solution and prepared into a cell suspension with the primary culture solution prepared in each experimental group. The cell density was adjusted to 1×10 5 / mL, and was added into a 6-well plate at a dose of 2 mL / well. When the cells grew to 80% confluence, the culture solution was discarded, and an osteogenic induction culture solution (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) was used. Continuous induction was performed, the induction solution was replaced every 3 days, and the induction was stopped on the 28th day. The cells were collected, lysed, and extracted to determine alkaline phosphatase, and the culture medium was aspirated to determine osteocalcin (determined according to the method of a commercially available kit, 5 samples were determined in parallel for each group, and the average value was taken).

[0118] The results are shown in Table 2.

[0119] Table 2

[0120]

[0121] From the above table, it can be seen that the dental pulp stem cells prepared in Examples 1-3 have better osteogenic differentiation ability.

[0122] Test Example 3

[0123] The dental pulp stem cells obtained in Examples 1-3 or Comparative Examples 1-7 in the third generation and growing well and close to confluence were seeded in 96-well plates at a cell density of 8000 / well, and after 24 h of culture, 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) was added. After 5 d of culture, the culture medium was discarded, and the culture medium was prepared according to the ratio of 9:1 of culture medium and CCK-8 detection reagent and added to the wells to be detected. After 1.5 h of 37°C culture, the absorbance value at 450 nm was detected.

[0124] The results are shown in Table 3.

[0125] Table 3

[0126]

[0127] From the above table, it can be seen that the dental pulp stem cells prepared in Examples 1-3 have better proliferation activity.

[0128] The above description is merely preferred embodiments of the present application, but not to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing dental pulp stem cells, characterized by, Comprising the following steps: S1. Surface treatment of glass culture dish: Put the glass culture dish in the alkali solution, heat treatment, pour out the alkali solution, then add Tris-HCl solution, add polydopamine, heat stirring reaction, pour out the solution, then add deionized water, hydroxyapatite and collagen peptide, add NHS and EDC, ultrasonic treatment, stirring reaction, pour out the liquid, wash, dry, and prepare the surface treated glass culture dish; S2. Preparation of primary culture solution: Add complexing agent, growth factor and natural antibacterial agent to the alpha-MEM base culture solution, stir and mix uniformly to prepare the primary culture solution; The mass ratio of the alpha-MEM base culture solution, 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-beta, the mass ratio is 2-3:0.2-0.5, and the natural antibacterial agent includes ferulic acid and allicin, the mass ratio is 2-3:5-7; S3. Preparation of cell freezing solution: Add mannose, DMSO, glutathione and bovine serum albumin to the DMEM medium and mix uniformly to prepare the cell freezing solution; S4. Culture: Add the primary culture solution to the surface treated glass culture dish, add the fresh human dental pulp tissue after washing and cutting, and incubate at a constant temperature. After the primary cells crawl out, pour out the liquid, collect the primary cells, resuspend part of the primary cells in the cell freezing solution, and freeze; S5. Subculture: Add the remaining part of the primary cells to the culture medium containing nano-activated particles, subculture under the conditions of low oxygen, low frequency magnetic field and light, collect the subculture cells, resuspend in the cell freezing solution, freeze, and prepare the dental pulp stem cells; The preparation method of the nano-activated particles is as follows: T1. Add the blocky black phosphorus to N-methyl pyrrolidone, avoid light, ice bath ultrasonic, centrifuge, wash, dry, add the product to Tris-HCl solution, add dopamine hydrochloride, heat stirring reaction, centrifuge, wash, dry, and prepare modified black phosphorus nanosheet; T2. Add the modified black phosphorus nanosheet and sodium molybdate to water, adjust the pH value of the solution, add glutathione, mix and stir, hydrothermal reaction, cool, centrifuge, wash, dry, and prepare N-doped MoS2 quantum dots@modified black phosphorus nanosheet; T3. Add the N-doped MoS2 quantum dots@modified black phosphorus nanosheet to water, add ferric chloride and ferrous chloride under inert gas protection, drop ammonia water, heat stirring reaction, centrifuge, wash, dry, and prepare nano-activated particles.

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

3. The preparation method according to claim 1, characterized in that, The mass ratio of the DMEM medium, mannose, DMSO, glutathione and bovine serum albumin in step S3 is 70-90:5-10:20-30:2-3:1-3.

4. The method of claim 1, wherein, The temperature of the constant temperature culture in step S4 is 35-37℃, and the time is 2-4d; in step S5, the oxygen content in the low-oxygen, low-frequency magnetic field and light conditions is 3-6v / v%, the magnetic field strength of the low-frequency magnetic field is 0.1-0.3T, the light intensity of the light is 3000-5000Lux, and the temperature of the subculture is 35-37℃.

5. The preparation method according to claim 1, characterized in that, The content of the nano-activated particles in the medium containing nano-activated particles in step S5 is 5-10wt%.

6. The method of claim 1, wherein, The power of the light-proof ice bath ultrasonic in step T1 is 300-500W, and the time is 1-3d; the method of centrifugation is centrifugation at 3000-5000r / min for 10-20min, and after collecting the supernatant, centrifugation at 10000-12000r / min for 15-25min, and collecting the precipitate; the pH value of the Tris-HCl solution is 8.5-9.5; the mass ratio of the bulk black phosphorus and dopamine hydrochloride is 10:1; and the temperature of the heating and stirring reaction is 40-50℃, and the time is 2-4h.

7. The preparation method according to claim 1, characterized in that, The mass ratio of the modified black phosphorus nanosheet, sodium molybdate and glutathione in step T2 is 10:3-4:5-7, the pH value of the adjusting solution is 6-6.5, the temperature of the hydrothermal reaction is 190-210℃, and the time is 10-15h.

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

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