Ultramicro-diameter cerium dioxide nano-enzyme, engineered dental pulp mesenchymal stem cells thereof and method for improving exosome stability
By preparing ultramicrodiameter ceria nanoenzymes and constructing engineered pulp mesenchymal stem cells, the problem of lack of effective application in periodontal soft and hard tissue regeneration is solved, and the periodontal regeneration effect is improved and exosome stability is enhanced.
Patent Information
- Application Number
- CN202411608101.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-12
- Publication Date
- 2025-03-25
AI Technical Summary
The lack of application of cerium oxide engineered dental stem cells in periodontal soft and hard tissue regeneration in the prior art has led to a lack of effective new ideas for periodontal regeneration treatment.
By preparing ultramicrodiameter ceria nanoenzymes, it uses its endocytosis to enter the pulp stem cells, engineered pulp mesenchymal stem cells (E-DPSCs), and the room temperature stability of exosomes is improved through the incubation process.
The osteogenic and cartilage differentiation ability of mesenchymal stem cells was improved, showing good regeneration effect of periodontal soft and hard tissues, and at the same time improving the physical stability of room temperature storage of exosomes.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of nano-biomedicine, and in particular to an ultra-micro-diameter cerium dioxide nanozyme and an engineered dental pulp mesenchymal stem cell thereof and a method for improving the stability of exosomes. Background Art
[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.
[0003] Nanozymes are small molecules that can mimic the activity of natural enzymes. Since the first discovery of ferroferric oxide (Fe 3 O 4 Since the discovery of nanoparticles with peroxidase-like catalytic activity, the research on nanozymes has made more and more progress. Many different types of nanozymes have been discovered or designed, and now they can simulate the activities of dozens of enzymes such as peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and oxidase (OXD). Compared with natural antioxidant enzymes, nanozymes are a new type of artificial enzyme with great potential. They combine the functions of natural enzymes and the characteristics of nanomaterials, and have the advantages of low cost, high stability, modifiability, and multifunctionality. Recent studies have shown that various antioxidant defense strategies based on natural enzymes, nanozymes, and antioxidants can effectively maintain intracellular redox balance and protect cells from oxidative damage. In vitro studies have found that compared with mesenchymal stem cells (MSCs), the stemness, differentiation, migration, antioxidant, and anti-inflammatory properties of engineered mesenchymal stem cells (E-MSCs) obtained by endocytosing cerium oxide are significantly enhanced. However, in the field of periodontal regeneration, there are still few reports on engineered mesenchymal stem cells.
[0004] Cerium (Ce) was discovered in 1803 and is a lanthanide element with an atomic number of 58. Cerium ranks 25th in the earth's crust (more abundant than copper) and is the most abundant lanthanide element. It has a wide range of applications in batteries, catalysts, sensors and other fields. Cerium mainly exists in the form of cerium oxide, which has a unique face-centered cubic fluorite lattice structure. Unlike most lanthanides that are trivalent, cerium exists in both Ce and 3+ and Ce 4+It has two valence states and can be rapidly converted between them, thus acting as both an oxidant and a reductant, showing the mimetic activity of superoxide dismutase and catalase. Cerium oxide nanoparticles have been widely used in anti-radiation, anti-inflammatory, ischemic stroke protection and in the treatment of skin wounds. In the field of periodontal treatment, cerium oxide can enhance the osteogenic differentiation ability of periodontal ligament stem cells, promote the polarization of macrophages to M2 type, and downregulate the expression level of inflammatory factor TNF-α, showing good regeneration effect of periodontal soft and hard tissues.
[0005] Cell engineering strategies and in vivo tracing techniques mainly include gene modification, nanoparticle internalization, nanoparticle adhesion, etc. Compared with traditional stem cells, engineering methods can assist in tracing the survival, migration, differentiation and other processes of stem cell drugs in the body, so as to fully understand the fate of stem cell drugs in the body to ensure their safety and effectiveness in the body. In recent years, there have been a lot of reports on the use of nanoparticles to internalize mesenchymal stem cells to enhance the survival and multidirectional differentiation ability of stem cells in an inflammatory environment.
[0006] However, there are no reports on the use of cerium oxide engineered dental pulp stem cells for periodontal soft and hard tissue regeneration. In this study, ultra-micro diameter cerium oxide nanoparticles with a diameter of 1-5 nm were prepared, which entered dental pulp stem cells through cellular endocytosis to construct engineered dental pulp mesenchymal stem cells (E-DPSC). The SOD activity of E-DPSC and its osteogenic and chondrogenic differentiation potential were studied to reflect the regeneration effect of periodontal soft and hard tissues. At the same time, we found that the physical stability of exosomes produced in the culture supernatant during incubation was improved at room temperature. Summary of the invention
[0007] The purpose of the present invention is to provide an ultra-micro diameter cerium dioxide nanozyme and its engineered dental pulp mesenchymal stem cells and a method for improving the stability of exosomes in response to the current lack of application of cerium oxide engineered dental pulp stem cells in periodontal soft and hard tissue regeneration, which solves the problem of new application of cerium oxide engineered dental pulp stem cells in periodontal soft and hard tissue regeneration and provides a new idea for periodontal soft and hard tissue regeneration treatment. The prepared ultra-micro cerium dioxide nanozyme particles have a concentrated particle size distribution and dispersed particles. They can form a stable dispersion and enter cells by ultra-micro-nano-scale particle endocytosis to play an enzyme-like role, effectively maintain the intracellular redox balance, and protect cells from oxidative damage. The ability to enhance the osteogenic and chondrogenic differentiation of E-DPSCs is enhanced, showing a good periodontal soft and hard tissue regeneration effect; at the same time, the physical stability of dental pulp mesenchymal stem cell exosomes stored at room temperature is improved.
[0008] The technical solution of the present invention is as follows: On the one hand, the present invention provides a method for preparing a cerium dioxide nanozyme, comprising the following steps: Step S1: mixing citric acid and cerium nitrate hexahydrate according to a mass ratio; Step S2: adding sodium hydroxide solution to the mixed solution, stirring and performing intermittent ultrasonic treatment; Step S3: high-speed centrifugation, washing and drying to obtain cerium dioxide nanozyme.
[0009] The diameter of the prepared ultra-micro-diameter cerium dioxide nanozyme is 1-5nm.
[0010] The prepared ultra-micro-diameter ceria nanozyme has a concentrated particle size distribution and dispersed particles. It can not only maintain a stable dispersion and enter the cell by ultra-micro-nano-particle endocytosis to play an enzyme-like role, effectively maintain the intracellular redox balance, and protect the cells from oxidative damage; it can also enhance the osteogenic and chondrogenic differentiation ability of ceria-engineered dental pulp mesenchymal stem cells, showing a good periodontal soft and hard tissue regeneration effect; at the same time, it can improve the physical stability of dental pulp mesenchymal stem cell exosomes stored at room temperature.
[0011] In the step S1, citric acid and cerium nitrate hexahydrate are respectively dissolved in deionized water, and then the cerium nitrate hexahydrate solution and citric acid are evenly mixed.
[0012] In the step S2, the amount of the sodium hydroxide solution added is: the mass ratio of the mixture of citric acid and cerium nitrate hexahydrate to the sodium hydroxide solution is 1:0.38.
[0013] The conditions for ultrasonic treatment in step S2 are: stirring at room temperature for 24 hours, ultrasonicating for 10-20 seconds and pausing for 20-30 seconds every 4 hours during the stirring process, the ultrasonic frequency is 30-40KHz, and the total time of intermittent ultrasonic treatment is 2-3 minutes; Preferably, in step S3, the ultrasound time is 20 seconds, and the pause is 30 seconds, and the total duration of a single ultrasound and the pause is 3 minutes.
[0014] The step S3 specifically comprises: after the stirring is completed, the obtained transparent yellow nanoparticle solution is centrifuged at 2500-2800g for 30-40 minutes; the supernatant of the centrifuged solution is further purified by ultracentrifugation at 14000g-16000g for 10-15 minutes, and the final nanoparticle solution is suspended in deionized water or PBS.
[0015] Preferably, in step S3, the centrifugal force is 2600 g and the total centrifugation time is 30 minutes.
[0016] Preferably, in step S4, the centrifugal force is 15000 g and the total centrifugation time is 10 minutes.
[0017] The second aspect of the present invention provides a cerium dioxide nanozyme prepared by the preparation method as described above, wherein the ultrafine cerium dioxide nanozyme is made of citric acid and cerium nitrate hexahydrate; the mass ratio of the citric acid to the cerium nitrate hexahydrate is 2:1.17-3.34. Preferably, the concentration of the cerium nitrate hexahydrate is 54.25-108.5 mg / ml. The concentration of the citric acid is 50-200 mg / ml.
[0018] According to a preferred embodiment, the mass ratio of citric acid to cerium nitrate hexahydrate is preferably 2:2.17; in some examples, the mass ratio of citric acid to cerium nitrate hexahydrate may be, but is not limited to, 2:1.17, 2:2.17 or 2:3.17; The third aspect of the present invention provides an engineered dental pulp mesenchymal stem cell, which is constructed by endocytosing ultrafine ceria nanozyme particles, wherein the nanozyme has a diameter of 1-5 nm. The ultrafine ceria nanozyme is the ultrafine ceria nanozyme prepared as described above.
[0019] According to a preferred embodiment, the method for constructing the engineered dental pulp mesenchymal stem cells is specifically as follows: 5-40 μg of the aforementioned ultramicro-diameter ceria nanozyme is incubated with dental pulp mesenchymal stem cells at 37° C. for 12-24 h to prepare ceria engineered dental pulp mesenchymal stem cells.
[0020] According to a preferred embodiment, when the ultramicro-diameter ceria nanozyme is co-incubated with dental pulp mesenchymal stem cells, the amount of the drug is preferably 20 μg. In some examples, the amount of the ultramicro-diameter ceria nanozyme can be, but is not limited to, 5 μg, 10 μg, 20 μg or 40 μg.
[0021] According to a preferred embodiment, the ultramicro-diameter ceria nanozyme and cells can be incubated together at 37° C. for 12 h, 18 h or 24 h, but are not limited to.
[0022] According to a preferred embodiment, when the ultra-micro-diameter ceria nanozyme is co-incubated with dental pulp mesenchymal stem cells, the cells are cultured at a rate of 6.11×10 5 Density seeding, wait until the cell density in the plate grows to 50%-70%, then add ceria nanozyme.
[0023] The fourth aspect of the present invention provides a method for improving the room temperature stability of exosomes of dental pulp mesenchymal stem cells, comprising the following steps: incubating dental pulp mesenchymal stem cells with ultra-micro-diameter ceria nanozymes to prepare ceria-engineered dental pulp mesenchymal stem cells, and taking the exosomes of ceria-engineered dental pulp mesenchymal stem cells as exosomes with room temperature stability.
[0024] The fifth aspect of the present invention provides the exosomes of the ceria-engineered dental pulp mesenchymal stem cells as described above.
[0025] Compared with the prior art, the present invention has the following beneficial effects: 1. An ultra-micro-diameter cerium dioxide nanozyme and its engineered dental pulp mesenchymal stem cells. The ultra-micro-diameter cerium dioxide nanozyme prepared by improving the raw material ratio and preparation process is encapsulated by citric acid to produce a stable dispersion to stabilize and show excellent enzyme-like activity. The ultra-micro-scale nanozyme particles prepared after the improved preparation method have a diameter of only 1-5nm, which is smaller than the average particle size prepared by the original preparation method, and the distribution is more concentrated, and the particle dispersion is higher. The engineered dental pulp mesenchymal stem cells constructed by the nanozyme have higher osteogenic and chondrogenic differentiation capabilities than the engineered dental pulp mesenchymal stem cells constructed by the original preparation method, and show good periodontal soft and hard tissue regeneration effects; 2. An ultra-micro-diameter ceria nanozyme and its engineered dental pulp mesenchymal stem cells and a method for improving the stability of exosomes. The cell activity of the engineered dental pulp mesenchymal stem cells (E-DPSC) constructed by the method of the present application is not affected, and the physical stability of the exosomes extracted from the culture supernatant produced under this culture condition is effectively improved at room temperature; 3. An ultra-micro-diameter cerium dioxide nanozyme and its engineered dental pulp mesenchymal stem cells and a method for improving the stability of exosomes. The raw materials and preparation process used in the present invention are simple and efficient, overcoming the problem of the cumbersome original preparation process, with good repeatability and conducive to industrial production; no toxic organic reagents are contacted during the operation, and the safety is high. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the transmission electron microscopy result of the ultrafine cerium dioxide nanozyme prepared in Example 2; Figure 2 The cell viability results in Experimental Examples 6-10 of the invention are shown in FIG. Figure 3 The results of SOD activity of DPSCs incubated in Example 6 and E-DPSCs constructed in Example 9 are shown in FIG. Figure 4 The results of qualitative and semi-quantitative analysis of the osteogenic differentiation ability of DPSC, E-DPSC-1 and E-DPSC-2 in Example 11 and Example 12 are shown; Figure 5 This is the result of surface marker identification of dental pulp mesenchymal stem cells by flow cytometry; Figure 6 The diameter distribution results of Examples 11-14 and the electron microscope results of Examples 11 and 12 are shown. DETAILED DESCRIPTION
[0027] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.
[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.
[0029] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.
[0030] Example 1-5 A method for preparing ultra-micro diameter cerium dioxide nanozyme The specific steps include: Step S1: Weigh cerium nitrate hexahydrate and citric acid according to the mass ratio, dissolve them in deionized water, and mix the cerium nitrate hexahydrate solution and the citric acid; The amounts of cerium nitrate hexahydrate and citric acid added are as follows:
[0031] Step S2: adding the mixture of step S1 into 0.4M sodium hydroxide solution according to the mass ratio of citric acid, cerium nitrate hexahydrate mixture and sodium hydroxide solution of 1:0.38; Step S3: stirring at room temperature for 24 hours, ultrasonicating for 10 seconds and pausing for 20 seconds every 4 hours during the stirring process, the ultrasonic frequency is 30 kHz, and the total time of a single intermittent ultrasonic treatment is 3 minutes. After the stirring is completed, the obtained transparent yellow nanoparticle solution is centrifuged at 2600g for 30 minutes; Step S4: The supernatant of the centrifuged solution was further purified by ultracentrifugation at 15,000 g for 10 min, and the final nanoparticle solution was suspended in deionized water or PBS.
[0032] The transmission electron microscopy results of the ultra-micro diameter ceria nanozyme prepared in Example 2 are shown in FIG. Figure 1 shown.
[0033] The ultramicro-diameter cerium dioxide nanozymes prepared in Examples 1-3 have a high recovery rate, among which Example 2 has the highest recovery rate, and the particle diameter of the cerium dioxide nanozymes prepared in Example 2 is in the ultramicro-diameter range of 1-5 nm.
[0034] Example 6-10 Construction of Ceria-engineered Dental Pulp Mesenchymal Stem Cells The ultra-micro-diameter ceria nanozymes prepared in Example 2 were taken and incubated together with dental pulp mesenchymal stem cells at different dosages.
[0035] Identification of dental pulp mesenchymal stem cells: Figure 5 As shown, the dental pulp mesenchymal stem cells used were identified by flow cytometry surface markers. This stem cell population highly expressed mesenchymal stem cell surface markers CD73 and CD44, and did not express hematopoietic stem cell surface markers CD34 and CD45.
[0036] The specific dosage of ceria nanozyme during incubation is as follows:
[0037] The specific steps are as follows: S1: 2-4 generations of dental pulp mesenchymal stem cells (DPSC) in the logarithmic growth phase were selected, and ceria nanoparticles were added to a 96-well plate in which cells were plated overnight. After incubation with different concentrations of ceria nanozymes for 24 hours, the activity of dental pulp mesenchymal stem cells was detected using the CCK-8 method. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that after Example 9 was co-incubated with 20 μg of ultra-micro-diameter cerium dioxide nanozyme and dental pulp mesenchymal stem cells, the survival rate of the dental pulp mesenchymal stem cells was the highest.
[0038] S2: According to the cell viability test results obtained in step S1, select 20 μg of ceria nanozyme and add it to a 96-well plate where cells were plated overnight in advance, and incubate for 12-24 hours.
[0039] S3: Using the commercial WST-8 total SOD kit, the absorbance of the 96-well plate was measured at 240 nm to detect the SOD enzyme activity of dental pulp mesenchymal stem cells. Figure 3 As shown. Figure 3 It can be seen that the SOD activity of the ceria-engineered dental pulp mesenchymal stem cells constructed by co-incubating 20 μg of ultra-micro-diameter ceria nanozymes with dental pulp mesenchymal stem cells in Example 9 is enhanced compared with the dental pulp mesenchymal stem cells in Control Example 6.
[0040] In step S1 and step S2, the cells were cultured at 6.11×10 5 When the cell density in the plate reaches 70%, ultra-micro-diameter cerium dioxide nanozymes are added and incubated together.
[0041] Example 11-14 A method for preparing ultra-micro diameter cerium dioxide nanozyme Embodiments 11-14 are further improvements on Embodiment 2. Except for step S3, the remaining steps are exactly the same as those of Embodiment 2.
[0042] The specific stirring method of step S3 of Examples 11-14 is as follows:
[0043] Comparison of the nanoparticle diameter and aggregation of the ultra-micro-diameter ceria nanozymes prepared in Examples 11-14 shows that Figure 6 As shown. Figure 6 It can be seen that the particle size of the ultramicro-diameter cerium dioxide nanozyme prepared in Example 12 is smaller than the average diameter of other groups, and the particle size distribution is more concentrated, and the particle dispersion is higher; by comparing the electron microscope photos of Example 11 and Example 12, it can be seen that the agglomeration phenomenon can be effectively improved by intermittent ultrasound.
[0044] Comparing Example 2 and Example 12, the intermittent ultrasonic treatment with different ultrasonic and pause times and the single ultrasonic and pause times were changed, and there was no significant difference in the results.
[0045] Experimental Example 1 Study on the osteogenic and chondrogenic potential of cerium dioxide engineered dental pulp mesenchymal stem cells The ultra-micro-diameter ceria nanozymes prepared in Example 11 and Example 12 were co-incubated with dental pulp mesenchymal stem cells at a dosage of 20 μg to construct E-DPSC-1 and E-DPSC-2; an equal volume of PBS buffer was co-incubated with dental pulp mesenchymal stem cells as a control group of DPSCs.
[0046] DPSC, E-DPSC-1 and E-DPSC-2 were induced to form osteoblasts and chondrocytes to determine their osteoblastic and chondrogenic potentials and differences: Determination of osteogenic potential: DPSC, E-DPSC-1 and E-DPSC-2 were cultured in osteogenic differentiation medium and stained with Alizarin red. The formation of calcium nodules was observed to determine whether they had the potential for osteoblast differentiation. The Alcian blue staining solution was eluted with guanidine hydrochloride solution, and the guanidine hydrochloride solution was used as a blank control. The OD value was measured at 600 mm using an enzyme marker.
[0047] Determination of chondrogenic potential: After DPSC, E-DPSC-1 and E-DPSC-2 were cultured in induced chondrogenic differentiation medium, they were stained with Alcian blue. The staining of cell aggregates was observed to determine whether they had the potential for chondrogenic differentiation. 10% cetylpyridinium chloride solution was used to elute the Alizarin Red S staining solution. 10% cetylpyridinium chloride solution was used as a blank control. The OD value was measured at 550 mm using a microplate reader.
[0048] Qualitative and semi-quantitative results such as Figure 4 As shown in the figure, it can be seen that the E-PDSC-2 constructed by incubating the ultramicro-diameter cerium dioxide nanozyme prepared in Example 12 with dental pulp mesenchymal stem cells has stronger osteogenic and chondrogenic abilities than DPSC and E-DPSC-1 constructed by incubating the ultramicro-diameter cerium dioxide nanozyme prepared in Example 11 with dental pulp mesenchymal stem cells.
[0049] Experimental Example 2 Analysis of the room temperature stability of cerium dioxide engineered dental pulp mesenchymal stem cells The ultramicro-diameter cerium dioxide nanozyme prepared in Example 12 was co-incubated with dental pulp mesenchymal stem cells at a dosage of 20 μg to construct E-DPSC, and an equal volume of PBS buffer was co-incubated with dental pulp mesenchymal stem cells as a control group. The culture supernatant was taken, and the exosomes were extracted by ultracentrifugation, stored at room temperature, and the zeta potential, PDI value, and pH value were detected on days 0, 1, 3, 5, 7, and 9, respectively. The results are shown in the following table.
[0050]
[0051]
[0052]
[0053] As can be seen from the above table, the PDI of ceria engineered dental pulp mesenchymal stem cell exosomes (E-DPSC-exos) is smaller than that of ordinary dental pulp mesenchymal stem cell exosomes (DPSC-exos) in the control group, and the system distribution is more uniform; the absolute value of zeta potential is larger, the repulsive force between system particles is greater, and the system stability is improved; the room temperature physical stability is improved compared with DPSC-exos; the pH value is slightly reduced and remains basically consistent within 9 days. It can be seen that the room temperature storage stability of E-DPSC-exos is improved.
[0054] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.
Claims
1. An engineered dental pulp mesenchymal stem cell, characterized in that: The ultra-micro-diameter ceria nanozyme 5-40 μg was co-incubated with dental pulp mesenchymal stem cells at 37°C for 12-24 h; The ultra-micro diameter ceria nanozyme is prepared by the following preparation method: Step S1: mixing citric acid and cerium nitrate hexahydrate according to a mass ratio; Step S2: adding sodium hydroxide solution to the mixed solution, stirring and performing intermittent ultrasonic treatment; Step S3: high-speed centrifugation, washing and drying to obtain cerium dioxide nanozyme.
2. The engineered dental pulp mesenchymal stem cells according to claim 1, characterized in that: The diameter of the prepared ultra-micro nanozyme particles is 1-5nm.
3. The engineered dental pulp mesenchymal stem cells according to claim 1, characterized in that: In step S1, the mass ratio of citric acid to cerium nitrate hexahydrate is 2:1.17-3.
34.
4. The engineered dental pulp mesenchymal stem cells according to claim 3, characterized in that: In step S1, the mass ratio of citric acid to cerium nitrate hexahydrate is preferably 2:1.17, 2:2.17 or 2:3.
17.
5. The engineered dental pulp mesenchymal stem cells according to claim 1, characterized in that: When the ultramicro-diameter ceria nanozyme is co-incubated with cells, the amount of the ultramicro-diameter ceria nanozyme can be, but is not limited to, 5 μg, 10 μg, 20 μg or 40 μg.
6. The engineered dental pulp mesenchymal stem cells according to claim 1, characterized in that: The conditions for ultrasonic treatment in step S2 are: stirring at room temperature for 24 hours, ultrasonicating for 10-20 seconds and pausing for 20-30 seconds every 4 hours during the stirring process, the ultrasonic frequency is 30-40KHz, and the total time of a single intermittent ultrasonic treatment is 2-3 minutes.
7. The engineered dental pulp mesenchymal stem cells according to claim 1, characterized in that: The step S3 specifically comprises: after the stirring is completed, the obtained transparent yellow nanoparticle solution is centrifuged at 2500-2800g for 30-40 minutes; the supernatant of the centrifuged solution is further purified by ultracentrifugation at 14000g-16000g for 10-15 minutes, and the final nanoparticle solution is suspended in deionized water or PBS.
8. The exosomes secreted by the engineered dental pulp mesenchymal stem cells according to any one of claims 1 to 7.
9. A method for improving the room temperature stability of dental pulp mesenchymal stem cell exosomes, characterized in that: The steps include: Step S1: preparing dental pulp mesenchymal stem cells into an engineered dental pulp mesenchymal stem cell as described in any one of claims 1 to 7.