Dental pulp stem cell exosome and preparation method and application thereof

Abstract

This invention proposes a method for preparing dental pulp stem cell exosomes and their applications, belonging to the field of exosome technology. Dental pulp stem cells from exfoliated deciduous teeth are seeded into serum-free α-MEM medium containing microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin, and streptomycin for culture. Exosomes are isolated, dispersed in PBS solution, and incubated with microparticles, propylene glycol, and rhodioloside to obtain highly active dental pulp stem cell exosomes. The surface of these exosomes is then modified with shikonin and interferon to obtain dental pulp stem cell exosomes. The dental pulp stem cell exosomes prepared by this invention exhibit good affinity for titanium implant-mediated osteointegration repair materials, demonstrating good anti-inflammatory, immunomodulatory, osteogenic, and damage-reducing effects. Furthermore, the preparation method is simple, yields high output, and operates under mild conditions, showing broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of exosome technology, specifically to a dental pulp stem cell exosome, its preparation method, and its application. Background Technology

[0002] Dental pulp stromal cells (DPSCs), isolated from dental pulp tissue, are a commonly used type of odontogenic mesenchymal cell, applied in various tissue repair and regeneration studies. DPSCs originate from the neural crest and possess high proliferative potential for self-renewal and multi-lineage differentiation. Exosomes (Exo) are small vesicles formed by cells through lysosomal microparticle invagination, containing abundant bioactive substances such as proteins, lipids, mRNA, and miRNA, playing a role in intercellular communication and substance exchange. Recent studies have found that DPSC exosomes (DPSC-Exo) have various therapeutic effects, and their applications in regenerative medicine have also been reported. The cell growth microenvironment influences exosome secretion; regulating the cell growth environment may optimize the composition of exosomes and alter their therapeutic potential.

[0003] Children's exfoliated deciduous tooth pulp stem cells are dental mesenchymal stem cells with strong self-renewal and multi-lineage differentiation potential. However, there are no reports on the osteogenic differentiation of bone marrow mesenchymal stem cells by their exosomes, and research on the bone regeneration capacity of age-related, body-derived BMSCs is limited. Promoting osteointegration repair mediated by titanium implants remains a hot research topic and a significant challenge in the medical field, as bone regeneration is a complex mechanism influenced by multiple factors and involving multiple cells. Although titanium materials possess excellent biological properties, as a foreign substance, implantation typically triggers a series of host immune responses, hindering the formation of an immune microenvironment conducive to damage repair, leading to the development of chronic inflammation, and ultimately resulting in osteointegration failure. Summary of the Invention

[0004] The purpose of this invention is to propose a dental pulp stem cell exosome, its preparation method, and its application. It has good affinity for titanium implant materials mediated by osteointegration repair materials, and exhibits good anti-inflammatory, immunomodulatory, osteogenic, and damage-reducing effects. Moreover, the preparation method is simple, the yield is high, the conditions are mild, and it has broad application prospects.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a method for preparing dental pulp stem cell exosomes. Dental pulp stem cells from exfoliated deciduous teeth are seeded into serum-free α-MEM medium containing microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin, and streptomycin for culture. Exosomes are isolated, dispersed in PBS solution, and incubated with microparticles, propylene glycol, and rhodioloside to obtain highly active dental pulp stem cell exosomes. The surface of these exosomes is then modified with shikonin and interferon to obtain dental pulp stem cell exosomes.

[0007] As a further improvement to the present invention, the following steps are included:

[0008] S1. Preparation of culture medium: Microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin, and streptomycin were added to 8-12% serum-free α-MEM medium to prepare the culture medium;

[0009] S2. Stem cell culture and exosome isolation: Exfoliated deciduous tooth pulp stem cells were revived, inoculated into culture medium, cultured under micro-hypoxia and magnetic field conditions, microparticles were separated by magnet, cell culture medium was collected, and active dental pulp stem cell exosomes were extracted by ultra-high speed centrifugation combined with ultrafiltration tube method.

[0010] S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution, microparticles, propylene glycol, and rhodioloside were added, and the mixture was incubated under a magnetic field. The microparticles were separated by a magnet, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes.

[0011] S4. Modification: Shisonic acid and interferon were added to PBS buffer, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added, the mixture was stirred and activated, highly active dental pulp stem cell exosomes were added, the mixture was stirred and reacted, dialyzed, and freeze-dried to obtain dental pulp stem cell exosomes.

[0012] As a further improvement of the present invention, the content of each component in the culture medium in step S1 is as follows: microparticles 2-4 g / L, insulin-like growth factor 100-120 mg / L, transforming growth factor-β1 50-100 mg / L, penicillin 0.5-1.5 wt%, and streptomycin 0.5-1.5 wt%.

[0013] As a further improvement of the present invention, the seeding amount of deciduous tooth pulp stem cells in step S2 is 10. 3 -10 4 The culture conditions under micro-hypoxia and magnetic field conditions are: 3-5 v / v% O2, magnetic field strength 0.1-0.15 T, and culture time 44-52 h.

[0014] As a further improvement of the present invention, in step S3, the content of microparticles, propylene glycol, and rhodioloside in the system is 1-2 g / L, the content of propylene glycol is 10-20 mmol / L, and the content of rhodioloside is 20-40 mmol / L. The incubation conditions under the magnetic field are 0.1-0.15T magnetic field for 5-7 hours.

[0015] As a further improvement of the present invention, the mass ratio of shikonin, interferon, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and highly active dental pulp stem cell exosomes in step S4 is 3-5:1-3:2-4:2-4:17-22.

[0016] As a further improvement of the present invention, the method for preparing the microparticles is as follows:

[0017] T1. Preparation of magnetic microparticles: Ferric chloride and ferrous chloride were dissolved in water, and ammonia was added dropwise under inert gas protection. The mixture was heated and stirred to react, centrifuged, washed, and dried to obtain magnetic microparticles.

[0018] T2. TiO2 deposition: Magnetic microparticles are added to water, tetrabutyl titanate is added, the pH of the solution is adjusted, the reaction is stirred, the particles are separated by magnets, washed, dried, and calcined to obtain TiO2 deposited magnetic microparticles;

[0019] T3. Graphene oxide coating: TiO2-deposited magnetic microparticles are added to water, graphene oxide is added, the mixture is stirred and mixed evenly, and then spray-dried to obtain graphene oxide / TiO2-deposited magnetic microparticles;

[0020] T4. Reduction: Magnetic microparticles deposited with graphene oxide / TiO2 are reduced by hydrazine hydrate vapor to obtain microparticles.

[0021] As a further improvement of the present invention, in step T1, the mass ratio of ferric chloride to ferrous chloride is 3.24:1.26, the heating and stirring reaction temperature is 75-85℃, and the time is 3-5h; in step T2, the mass ratio of magnetic microparticles to tetrabutyl titanate is 10:3-4, the calcination temperature is 400-500℃, and the time is 1-2h; in step T3, the mass ratio of TiO2 deposited magnetic microparticles to graphene oxide is 10:2-3; and in step T4, the hydrazine hydrate vapor reduction time is 7-10h.

[0022] This invention further protects a dental pulp stem cell exosome prepared by the above-described preparation method.

[0023] This invention further protects the application of the above-mentioned dental pulp stem cell exosomes in the preparation of osteointegrative repair and protective materials mediated by titanium implant materials.

[0024] The present invention has the following beneficial effects:

[0025] This invention prepares microparticles with magnetic iron oxide (Fe3O4) as the core, coated with a TiO2 layer and a graphene layer. On one hand, the graphene / TiO2 composite layer improves the utilization efficiency of TiO2 for visible light, enabling efficient release of far-infrared rays and oxygen free radicals at low temperatures (e.g., 36°C), promoting the secretion of more highly active exosomes by stem cell culture. On the other hand, the magnetic iron oxide, under the influence of a magnetic field, affects the cell membrane or intracellular proteins by controlling the intensity, frequency, and exposure time of the electromagnetic field, thereby influencing the secretory activity of cells towards exosomes. Simultaneously, during exosome incubation, the magnetic field influences the exosome membrane morphology, forming micro-openings that facilitate the entry of the active component, rhodioloside, into the exosomes, improving the substances within them. Propylene glycol also enhances permeability, further promoting incubation and improvement. After entering the exosomes, rhodioloside increases the expression of osteogenic differentiation-related genes by activating the bone morphogenetic protein signaling pathway, improving the bone integration and repair effect mediated by titanium implant materials. Furthermore, the magnetic properties of these microparticles facilitate separation and reuse, reducing production costs.

[0026] Exosomes from exfoliated deciduous tooth pulp stem cells possess similar biological potentials, including promoting angiogenesis and new bone formation, inhibiting inflammatory responses, and promoting osteogenic processes, showing promising translational applications. In this invention, exosomes secreted by exfoliated deciduous tooth pulp stem cells, after special culture, can increase growth factor secretion by regulating the expression of related intracellular genes, thereby promoting tissue regeneration, directed differentiation, and ultimately restoring the function of damaged tissues. Transforming growth factor-β1 (TGF-β1) and insulin-like growth factor (IGF) are added to the stem cell culture medium. IGF-β1 promotes stem cell exosome secretion through the c-Jun N-terminal kinase and extracellular regulatory protein kinase signaling pathways; TGF-β1 regulates cell function by binding to specific receptors on the cell membrane. The synergistic effect of these two factors significantly promotes exosome secretion, and the secreted exosomes exhibit good osteogenic repair and anti-inflammatory effects. Microhypoxic conditions can also induce a stress response in stem cells, thereby increasing the amount of exosome secreted.

[0027] This invention couples shikonin and interferon to the surface of highly active dental pulp stem cell exosomes. Shikonin is a vitamin K backbone containing abundant hydroxyl structures, and interferon also has good immunomodulatory effects. This makes the prepared dental pulp stem cell exosomes more compatible with the osteointegration repair material mediated by titanium implants, allowing them to be stably adsorbed on their surface. This results in better anti-inflammatory, immunomodulatory, osteogenic, and damage-reducing effects.

[0028] The dental pulp stem cell exosomes prepared by this invention have good affinity for titanium implant materials mediated bone integration repair materials, and exhibit good anti-inflammatory, immunomodulatory, osteogenic, and damage-reducing effects. Moreover, the preparation method is simple, the yield is high, the conditions are mild, and it has broad application prospects. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a TEM image of dental pulp stem cell exosomes obtained in Example 1 of the present invention;

[0031] Figure 2 This is a SEM image of the nanosphere mixture obtained from the dental pulp stem cell exocrine preparation in Example 1 of the present invention. Detailed Implementation

[0032] 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.

[0033] Preparation Example 1: Microparticles

[0034] The preparation method is as follows:

[0035] T1. Preparation of magnetic microparticles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 200 mL of water. Under nitrogen protection, ammonia was added dropwise to adjust the pH of the solution to 9. The solution was heated to 75 °C and stirred for 5 h. After centrifugation, washing and drying, magnetic microparticles were obtained.

[0036] T2. TiO2 deposition: 1g of magnetic microparticles were added to 200mL of water, 0.3g of tetrabutyl titanate was added, the pH of the solution was adjusted to 9.5, the reaction was stirred for 5h, the magnets were separated, washed, dried, and calcined at 400℃ for 2h to obtain TiO2 deposited magnetic microparticles.

[0037] T3. Graphene oxide coating: 1g of TiO2 deposited magnetic microparticles were added to 200mL of water, 0.2g of graphene oxide was added, the mixture was stirred and mixed evenly, and spray dried to obtain graphene oxide / TiO2 deposited magnetic microparticles;

[0038] T4. Reduction: Magnetic microparticles deposited with graphene oxide / TiO2 were reduced by hydrazine hydrate vapor for 7 hours to obtain microparticles.

[0039] Preparation Example 2: Microparticles

[0040] The preparation method is as follows:

[0041] T1. Preparation of magnetic microparticles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 200 mL of water. Under nitrogen protection, ammonia was added dropwise to adjust the pH of the solution to 10. The solution was heated to 85 °C and stirred for 3 h. After centrifugation, washing and drying, magnetic microparticles were obtained.

[0042] T2. TiO2 deposition: 1g of magnetic microparticles were added to 200mL of water, 0.4g of tetrabutyl titanate was added, the pH of the solution was adjusted to 9.5, the reaction was stirred for 7h, the magnets were separated, washed, dried, and calcined at 500℃ for 1h to obtain TiO2 deposited magnetic microparticles.

[0043] T3. Graphene oxide coating: 1g of TiO2 deposited magnetic microparticles were added to 200mL of water, 0.3g of graphene oxide was added, the mixture was stirred and mixed evenly, and spray dried to obtain graphene oxide / TiO2 deposited magnetic microparticles;

[0044] T4. Reduction: Magnetic microparticles deposited with graphene oxide / TiO2 were reduced by hydrazine hydrate vapor for 10 hours to obtain microparticles.

[0045] Preparation Example 3: Microparticles

[0046] The preparation method is as follows:

[0047] T1. Preparation of magnetic microparticles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 200 mL of water. Under nitrogen protection, ammonia was added dropwise to adjust the pH of the solution to 9.5. The solution was heated to 80 °C and stirred for 4 h. After centrifugation, washing and drying, magnetic microparticles were obtained.

[0048] T2. TiO2 deposition: 1g of magnetic microparticles were added to 200mL of water, 0.35g of tetrabutyl titanate was added, the pH of the solution was adjusted to 9.5, the reaction was stirred for 6h, the magnets were separated, washed, dried, and calcined at 450℃ for 1.5h to obtain TiO2 deposited magnetic microparticles.

[0049] T3. Graphene oxide coating: 1g of TiO2 deposited magnetic microparticles were added to 200mL of water, 0.25g of graphene oxide was added, the mixture was stirred and mixed evenly, and spray dried to obtain graphene oxide / TiO2 deposited magnetic microparticles;

[0050] T4. Reduction: Magnetic microparticles deposited with graphene oxide / TiO2 were reduced by hydrazine hydrate vapor for 8 hours to obtain microparticles.

[0051] Comparative Preparation Example 1

[0052] The difference compared to preparation example 3 is that step T2 was not performed.

[0053] Specifically as follows:

[0054] T1. Preparation of magnetic microparticles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 200 mL of water. Under nitrogen protection, ammonia was added dropwise to adjust the pH of the solution to 9.5. The solution was heated to 80 °C and stirred for 4 h. After centrifugation, washing and drying, magnetic microparticles were obtained.

[0055] T2. Graphene oxide coating: 1g of magnetic microparticles were added to 200mL of water, 0.25g of graphene oxide was added, the mixture was stirred and mixed evenly, and then spray-dried to obtain graphene oxide-coated magnetic microparticles.

[0056] T3. Reduction: Magnetic microparticles coated with graphene oxide were reduced by hydrazine hydrate vapor for 8 hours to obtain microparticles.

[0057] Comparative Preparation Example 2

[0058] The difference compared to Preparation Example 3 is that steps T3 and T4 were not performed.

[0059] Specifically as follows:

[0060] T1. Preparation of magnetic microparticles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 200 mL of water. Under nitrogen protection, ammonia was added dropwise to adjust the pH of the solution to 9.5. The solution was heated to 80 °C and stirred for 4 h. After centrifugation, washing and drying, magnetic microparticles were obtained.

[0061] T2. TiO2 deposition: 1g of magnetic microparticles were added to 200mL of water, 0.35g of tetrabutyl titanate was added, the pH of the solution was adjusted to 9.5, the reaction was stirred for 6h, the magnets were separated, washed, dried, and calcined at 450℃ for 1.5h to obtain TiO2 deposited magnetic microparticles, which are microparticles.

[0062] Comparative preparation example 3

[0063] The difference compared to preparation example 3 is that step T4 was not performed.

[0064] Specifically as follows:

[0065] T1. Preparation of magnetic microparticles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 200 mL of water. Under nitrogen protection, ammonia was added dropwise to adjust the pH of the solution to 9.5. The solution was heated to 80 °C and stirred for 4 h. After centrifugation, washing and drying, magnetic microparticles were obtained.

[0066] T2. TiO2 deposition: 1g of magnetic microparticles were added to 200mL of water, 0.35g of tetrabutyl titanate was added, the pH of the solution was adjusted to 9.5, the reaction was stirred for 6h, the magnets were separated, washed, dried, and calcined at 450℃ for 1.5h to obtain TiO2 deposited magnetic microparticles.

[0067] T3. Graphene oxide coating: Add 1g of TiO2 deposited magnetic microparticles to 200mL of water, add 0.25g of graphene oxide, stir and mix evenly, spray dry to obtain graphene oxide / TiO2 deposited magnetic microparticles, which are microparticles.

[0068] Comparative preparation example 4

[0069] The difference from preparation example 3 is that step T1 was not performed.

[0070] Specifically as follows:

[0071] T1. TiO2 particles: 1.35g tetrabutyl titanate was added to 200mL of water, the pH of the solution was adjusted to 9.5, the mixture was stirred for 6h, separated by magnet, washed, dried, and calcined at 450℃ for 1.5h to obtain TiO2 particles.

[0072] T2. Graphene oxide coating: Add 1g of TiO2 particles to 200mL of water, add 0.25g of graphene oxide, stir and mix evenly, spray dry to obtain graphene oxide / TiO2 particles;

[0073] T3. Reduction: Graphene oxide / TiO2 particles were reduced by hydrazine hydrate vapor for 8 hours to obtain microparticles.

[0074] Example 1: This example provides a method for preparing dental pulp stem cell exosomes, including the following steps:

[0075] S1. Preparation of culture medium: Microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin and streptomycin obtained in Preparation Example 1 were added to 8% serum-free α-MEM culture medium to prepare the culture medium;

[0076] The culture medium contains the following components: microparticles 2 g / L, insulin-like growth factor 100 mg / L, transforming growth factor-β1 50 mg / L, penicillin 0.5 wt%, and streptomycin 0.5 wt%.

[0077] S2. Stem cell culture and exosome isolation: Resuscitate deciduous tooth pulp stem cells and inoculate them into culture medium at a density of 102. 3 -10 4 Cells / mL were cultured at 3 v / v% O2 and a magnetic field strength of 0.1 T for 44 h. Microparticles were separated by magnets, and the cell culture medium was collected. Active dental pulp stem cell exosomes were extracted by ultra-high speed centrifugation combined with ultrafiltration tube method.

[0078] The ultracentrifugation combined with ultrafiltration tube method is as follows: Cell supernatant is centrifuged at 700 rpm for 15 min at 4°C to remove cells, then centrifuged at 3500 rpm for 20 min at 4°C to remove cell debris, etc. The supernatant is concentrated using an ultrafiltration tube, centrifuged at 13000 rpm for 30 min at 4°C to remove large vesicles and proteins, the supernatant is filtered through a 0.22 μm filter membrane, and finally centrifuged at 30000 rpm for 60 min at 4°C. The precipitate is resuspended in PBS at pH 7, and centrifuged again at 30000 rpm for 60 min at 4°C. The resulting precipitate is the exosome, which is resuspended in PBS solution at pH 7. Protein quantification is performed according to the BCA kit steps. The final exosome solution with a concentration of 100 μg / mL is obtained through calculation and stored at -80°C.

[0079] S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution at pH=7 to obtain a concentration of 70 μg / mL. Microparticles, propylene glycol, and rhodioloside prepared in Preparation Example 1 were added. The concentrations of microparticles, propylene glycol, and rhodioloside in the system were 1 g / L, 10 mmol / L, and 20 mmol / L, respectively. The mixture was incubated under a 0.1T magnetic field for 5 h. The microparticles were separated by magnetism, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes.

[0080] S4. Modification: 3g of shikonin and 1g of interferon were added to 200mL of PBS buffer (pH=7), followed by 2g of N-hydroxysuccinimide and 2g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred at 0℃ for 30min to activate the reaction. Then, 17g of highly active dental pulp stem cell exosomes were added, and the reaction was stirred for 12h. The mixture was dialyzed through an 8000Da dialysis bag for 24h, and then freeze-dried to obtain dental pulp stem cell exosomes. TEM images are shown below. Figure 1 Its particle size is between 180-220nm.

[0081] Example 2: This example provides a method for preparing dental pulp stem cell exosomes, including the following steps:

[0082] S1. Preparation of culture medium: Microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin and streptomycin obtained in Preparation Example 2 were added to 12% serum-free α-MEM medium to prepare the culture medium;

[0083] The culture medium contains the following components: microparticles 4 g / L, insulin-like growth factor 120 mg / L, transforming growth factor-β1 100 mg / L, penicillin 1.5 wt%, and streptomycin 1.5 wt%.

[0084] S2. Stem cell culture and exosome isolation: Resuscitate deciduous tooth pulp stem cells and inoculate them into culture medium at a density of 102. 3 -10 4 Cells / mL were cultured at 5 v / v% O2 and a magnetic field strength of 0.15 T for 52 h. Microparticles were separated by magnets, and the cell culture medium was collected. Active dental pulp stem cell exosomes were extracted by ultra-high speed centrifugation combined with ultrafiltration tube method.

[0085] The ultracentrifugation combined with ultrafiltration tube method is as follows: Cell supernatant is centrifuged at 700 rpm for 15 min at 4°C to remove cells, then centrifuged at 3500 rpm for 20 min at 4°C to remove cell debris, etc. The supernatant is concentrated using an ultrafiltration tube, centrifuged at 13000 rpm for 30 min at 4°C to remove large vesicles and proteins, the supernatant is filtered through a 0.22 μm filter membrane, and finally centrifuged at 30000 rpm for 60 min at 4°C. The precipitate is resuspended in PBS at pH 7, and centrifuged again at 30000 rpm for 60 min at 4°C. The resulting precipitate is the exosome, which is resuspended in PBS solution at pH 7. Protein quantification is performed according to the BCA kit steps. The final exosome solution with a concentration of 100 μg / mL is obtained through calculation and stored at -80°C.

[0086] S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution at pH=7 to obtain a concentration of 90 μg / mL. Microparticles, propylene glycol, and rhodioloside prepared in Preparation Example 2 were added. The concentrations of microparticles, propylene glycol, and rhodioloside in the system were 2 g / L, 20 mmol / L, and 40 mmol / L, respectively. The mixture was incubated in a 0.15T magnetic field for 7 h. The microparticles were separated by magnetization, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes.

[0087] S4. Modification: Add 5g of shikonin and 3g of interferon to 200mL of PBS buffer (pH=7), add 4g of N-hydroxysuccinimide and 4g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir and activate at 0℃ for 30min, add 22g of highly active dental pulp stem cell exosomes, stir and react for 12h, dialyze using a dialysis bag with a pore size of 10000Da for 24h, freeze-dry to obtain dental pulp stem cell exosomes.

[0088] Example 3: This example provides a method for preparing dental pulp stem cell exosomes, including the following steps:

[0089] S1. Preparation of culture medium: Microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin and streptomycin obtained in Preparation Example 3 were added to 10% serum-free α-MEM culture medium to prepare the culture medium;

[0090] The culture medium contains the following components: microparticles 3 g / L, insulin-like growth factor 110 mg / L, transforming growth factor-β1 70 mg / L, penicillin 1 wt%, and streptomycin 1 wt%.

[0091] S2. Stem cell culture and exosome isolation: Resuscitate deciduous tooth pulp stem cells and inoculate them into culture medium at a density of 102. 3 -10 4 Cells / mL were cultured at 4 v / v% O2 and a magnetic field strength of 0.12 T for 48 h. Microparticles were separated by magnets, and the cell culture medium was collected. Active dental pulp stem cell exosomes were extracted by ultra-high speed centrifugation combined with ultrafiltration tube method.

[0092] The ultracentrifugation combined with ultrafiltration tube method is as follows: Cell supernatant is centrifuged at 700 rpm for 15 min at 4°C to remove cells, then centrifuged at 3500 rpm for 20 min at 4°C to remove cell debris, etc. The supernatant is concentrated using an ultrafiltration tube, centrifuged at 13000 rpm for 30 min at 4°C to remove large vesicles and proteins, the supernatant is filtered through a 0.22 μm filter membrane, and finally centrifuged at 30000 rpm for 60 min at 4°C. The precipitate is resuspended in PBS at pH 7, and centrifuged again at 30000 rpm for 60 min at 4°C. The resulting precipitate is the exosome, which is resuspended in PBS solution at pH 7. Protein quantification is performed according to the BCA kit steps. The final exosome solution with a concentration of 100 μg / mL is obtained through calculation and stored at -80°C.

[0093] S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution at pH=7 to obtain a concentration of 80 μg / mL. Microparticles, propylene glycol, and rhodioloside prepared in Preparation Example 3 were added. The concentrations of microparticles, propylene glycol, and rhodioloside in the system were 1.5 g / L, 15 mmol / L, and 30 mmol / L, respectively. The mixture was incubated under a magnetic field of 0.12 T for 6 h. The microparticles were separated by magnetism, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes.

[0094] S4. Modification: Add 4g of shikonin and 2g of interferon to 200mL of PBS buffer (pH=7), add 3g of N-hydroxysuccinimide and 3g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir and activate at 0℃ for 30min, add 20g of highly active dental pulp stem cell exosomes, stir and react for 12h, dialyze using a dialysis bag with a pore size of 9000Da for 24h, freeze-dry to obtain dental pulp stem cell exosomes.

[0095] Comparative Example 1

[0096] The difference from Example 3 is that the microparticles were prepared by Comparative Preparation Example 1.

[0097] Comparative Example 2

[0098] The difference from Example 3 is that the microparticles were prepared by Comparative Preparation Example 2.

[0099] Comparative Example 3

[0100] The difference from Example 3 is that the microparticles were prepared by Comparative Preparation Example 3.

[0101] Comparative Example 4

[0102] The difference from Example 3 is that the microparticles were prepared by Comparative Preparation Example 4.

[0103] Comparative Example 5

[0104] The difference from Example 3 is that no microparticles were added in step S1.

[0105] Specifically as follows:

[0106] S1. Preparation of culture medium: Insulin-like growth factor, transforming growth factor-β1, penicillin, and streptomycin were added to 10% serum-free α-MEM medium to prepare the culture medium;

[0107] The contents of each component in the culture medium are as follows: insulin-like growth factor 110 mg / L, transforming growth factor-β 170 mg / L, penicillin 1 wt%, and streptomycin 1 wt%.

[0108] Comparative Example 6

[0109] The difference from Example 3 is that insulin-like growth factor and transforming growth factor-β1 were not added in step S1.

[0110] Specifically as follows:

[0111] S1. Preparation of culture medium: The microparticles, penicillin, and streptomycin obtained in Preparation Example 3 were added to 10% serum-free α-MEM culture medium to prepare the culture medium;

[0112] The culture medium contains the following components: microparticles 3 g / L, penicillin 1 wt%, and streptomycin 1 wt%.

[0113] Comparative Example 7

[0114] The difference from Example 3 is that rhodioloside was not added in step S3.

[0115] Specifically as follows:

[0116] S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution at pH=7 to obtain a concentration of 80 μg / mL. Microparticles prepared in Preparation Example 3 and propylene glycol were added. The content of microparticles in the system was 1.5 g / L and the content of propylene glycol was 15 mmol / L. The mixture was incubated under a magnetic field of 0.12T for 6 h. The microparticles were separated by magnet, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes.

[0117] Comparative Example 8

[0118] The difference from Example 3 is that no microparticles were added in step S3.

[0119] Specifically as follows:

[0120] S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution at pH=7 to obtain a concentration of 80 μg / mL. Propylene glycol and rhodioloside were added, with the concentrations of propylene glycol (15 mmol / L) and rhodioloside (30 mmol / L) in the system. The mixture was incubated under a magnetic field of 0.12T for 6 h. The microparticles were separated by magnetization, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes.

[0121] Comparative Example 9

[0122] The difference from Example 3 is that propylene glycol was not added in step S3.

[0123] S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution at pH=7 to obtain a concentration of 80 μg / mL. Microparticles and rhodioloside prepared in Preparation Example 3 were added. The content of microparticles in the system was 1.5 g / L and the content of rhodioloside was 30 mmol / L. The mixture was incubated under a magnetic field of 0.12T for 6 h. The microparticles were separated by magnetism, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes.

[0124] Comparative Example 10

[0125] The difference from Example 3 is that step S4 was not performed.

[0126] Specifically as follows:

[0127] S1. Preparation of culture medium: Microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin and streptomycin obtained in Preparation Example 3 were added to 10% serum-free α-MEM culture medium to prepare the culture medium;

[0128] The culture medium contains the following components: microparticles 3 g / L, insulin-like growth factor 110 mg / L, transforming growth factor-β1 70 mg / L, penicillin 1 wt%, and streptomycin 1 wt%.

[0129] S2. Stem cell culture and exosome isolation: Resuscitate deciduous tooth pulp stem cells and inoculate them into culture medium at a density of 102. 3 -10 4 Cells / mL were cultured at 4 v / v% O2 and a magnetic field strength of 0.12 T for 48 h. Microparticles were separated by magnets, and the cell culture medium was collected. Active dental pulp stem cell exosomes were extracted by ultra-high speed centrifugation combined with ultrafiltration tube method.

[0130] The ultracentrifugation combined with ultrafiltration tube method is as follows: Cell supernatant is centrifuged at 700 rpm for 15 min at 4°C to remove cells, then centrifuged at 3500 rpm for 20 min at 4°C to remove cell debris, etc. The supernatant is concentrated using an ultrafiltration tube, centrifuged at 13000 rpm for 30 min at 4°C to remove large vesicles and proteins, the supernatant is filtered through a 0.22 μm filter membrane, and finally centrifuged at 30000 rpm for 60 min at 4°C. The precipitate is resuspended in PBS at pH 7, and centrifuged again at 30000 rpm for 60 min at 4°C. The resulting precipitate is the exosome, which is resuspended in PBS solution at pH 7. Protein quantification is performed according to the BCA kit steps. The final exosome solution with a concentration of 100 μg / mL is obtained through calculation and stored at -80°C.

[0131] S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution at pH=7 to obtain a concentration of 80 μg / mL. Microparticles, propylene glycol, and rhodioloside prepared in Preparation Example 3 were added. The concentrations of microparticles, propylene glycol, and rhodioloside in the system were 1.5 g / L, 15 mmol / L, and 30 mmol / L, respectively. The mixture was incubated under a magnetic field of 0.12 T for 6 h. The microparticles were separated by magnetization, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes, which are dental pulp stem cell exosomes.

[0132] Test Example 1

[0133] The dental pulp stem cell exosomes obtained in Examples 1-3 and Comparative Examples 1-10 were embedded in a chitosan shell. The preparation method is as follows: Chitosan was dissolved in a 2 wt% acetic acid solution to obtain a chitosan solution with a concentration of 1 mg / mL. Dental pulp stem cell exosomes were added to this solution at a concentration of 0.05 mg / mL. The mixture was magnetically stirred for 30 min in an ice-water bath to obtain a suspension. The suspension was then allowed to stand at 4°C for 2 h. An equal volume of 1 mg / mL sodium tripolyphosphate solution was added dropwise, and the mixture was stirred for 1 h. After centrifugation, the precipitate was collected, washed, and resuspended in ultrapure water to obtain a 1 mg / mL nanosphere mixture. The SEM image of the nanosphere mixture obtained from the dental pulp stem cell exosomes prepared in Example 1 is shown below. Figure 2 Its particle size is between 500-700nm.

[0134] The control group was prepared as follows: Chitosan was dissolved in 2wt% acetic acid solution to obtain a chitosan solution with a concentration of 1 mg / mL. The solution was magnetically stirred at 500 r / min for 30 min under ice-water bath conditions to obtain a suspension. The suspension was allowed to stand for 2 h at 4℃. An equal volume of 1 mg / mL sodium tripolyphosphate solution was added dropwise, and the mixture was stirred. The mixture was centrifuged, the precipitate was collected, washed, and resuspended in ultrapure water to obtain a 1 mg / mL nanosphere mixture.

[0135] PLA and PEG were dissolved in dichloromethane at a mass ratio of 100:5 and magnetically stirred at room temperature for 12 hours to prepare separate mixed solutions. After defoaming by ultrasonic oscillation for 30 minutes, PLA / PEG fibers were prepared using an electrospinning apparatus. Spinning parameters: needle diameter 0.72 mm, spinning speed 0.25 mm / min, negative voltage 10 kV, positive voltage 20 kV, collection distance 20 cm, room temperature 25 °C, relative humidity 50%.

[0136] PLA / PEG fibers were wound and loaded onto the surface of a titanium sheet specimen (15 mm in diameter and 1.5 mm in thickness). A nanosphere mixture was uniformly dropped onto the surface of the titanium sheet specimen and incubated at 4°C for 24 hours. After washing three times with ultrapure water and drying, the experiment was conducted.

[0137] 1. CCK-8 test

[0138] Periodontal ligament stem cells were used at a rate of 2×10 5 Cells were seeded at a density of cells / well on titanium plates in 12-well plates and co-cultured at 37°C, 5% CO2, and 95% humidity. After 3 days, the old cell culture medium was removed, and fresh culture medium was added, followed by CCK-8 solution at a volume ratio of 9:1. The cells were incubated at 37°C for 2 hours, and the OD value was measured at 450 nm using a microplate reader. The 24-hour cell proliferation capacity of each group was compared. The results are shown in Table 1.

[0139] Table 1

[0140]

[0141] As shown in the table above, the dental pulp stem cell exosomes prepared in Examples 1-3 of this invention have a good effect on promoting cell proliferation.

[0142] Scratch test

[0143] Periodontal ligament stem cells were routinely counted via digestion, adjusted to 2 × 10⁻⁶. 5 PDLSCs were seeded at a density of cells / well on titanium sheets in 12-well plates and incubated according to the group. Microscopic images were taken at 0 and 24 h. Cell migration rates of the two groups were calculated and analyzed using the cell scratch assay and ImageJ. The results are shown in Table 2.

[0144] 24h cell migration rate = (0h scratch width - 24h scratch width) / 0h scratch width × 100%

[0145] Table 2

[0146]

[0147] As shown in the table above, the dental pulp stem cell exosomes prepared in Examples 1-3 of this invention have a good effect on promoting cell migration.

[0148] 3. Promotes the transformation of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype.

[0149] Macrophages were 2 × 10 5Macrophages were seeded at a density of cells / well on titanium sheets in 12-well plates and co-cultured for 3 days at 37°C, 5% CO2, and 95% humidity. Total RNA was extracted from macrophages co-cultured with the fiber coating using Trizol reagent and reverse transcribed into cDNA. The reaction program was SYBRONLY 25 μL, with the following steps: pre-denaturation phase: 95°C, 30 s; PCR amplification phase: 95°C, 5 s, 60°C, annealing for 30 s, repeated for 40 cycles. GAPDH was used as an internal reference gene, and the relative expression level of the target gene was calculated and analyzed using the 2-ΔΔct method. The primer sequences used are shown in Table 3. The relative expression levels of each gene group are shown in Table 4.

[0150] Table 3

[0151]

[0152] Table 4

[0153]

[0154] As shown in the table above, the dental pulp stem cell exosomes prepared in Examples 1-3 of this invention have a good effect on promoting the transformation of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype.

[0155] 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 cell exosomes, characterized in that, Dental pulp stem cells from exfoliated deciduous teeth were seeded into serum-free α-MEM medium containing microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin, and streptomycin and cultured. Exosomes were isolated, dispersed in PBS solution, and incubated with microparticles, propylene glycol, and rhodioloside to obtain highly active dental pulp stem cell exosomes. The surface of these exosomes was modified with shikonin and interferon to prepare dental pulp stem cell exosomes. The method for preparing the microparticles is as follows: T1. Preparation of magnetic microparticles: Ferric chloride and ferrous chloride were dissolved in water, and ammonia was added dropwise under inert gas protection. The mixture was heated and stirred to react, centrifuged, washed, and dried to obtain magnetic microparticles. T2. TiO2 deposition: Magnetic microparticles are added to water, tetrabutyl titanate is added, the pH of the solution is adjusted, the reaction is stirred, the mixture is separated by magnets, washed, dried, and calcined to obtain TiO2 deposited magnetic microparticles; T3. Graphene oxide coating: TiO2-deposited magnetic microparticles are added to water, graphene oxide is added, the mixture is stirred and mixed evenly, and then spray-dried to obtain graphene oxide / TiO2-deposited magnetic microparticles; T4. Reduction: Magnetic microparticles deposited with graphene oxide / TiO2 are reduced by hydrazine hydrate vapor to obtain microparticles.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of culture medium: Microparticles, insulin-like growth factor, transforming growth factor-β1, penicillin, and streptomycin were added to 8-12% serum-free α-MEM medium to prepare the culture medium; S2. Stem cell culture and exosome isolation: The exfoliated deciduous tooth pulp stem cells were revived, inoculated into culture medium, cultured under micro-hypoxia and magnetic field conditions, microparticles were separated by magnet, the cell culture medium was collected, and active dental pulp stem cell exosomes were extracted by ultra-high speed centrifugation combined with ultrafiltration tube method. S3. Incubation: Active dental pulp stem cell exosomes were dispersed in PBS solution, microparticles, propylene glycol, and rhodioloside were added, and the mixture was incubated under a magnetic field. The microparticles were separated by a magnet, centrifuged, and the precipitate was collected to obtain highly active dental pulp stem cell exosomes. S4. Modification: Shisonic acid and interferon were added to PBS buffer, along with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred and activated, and highly active dental pulp stem cell exosomes were added. The mixture was stirred and reacted, then dialyzed and freeze-dried to obtain dental pulp stem cell exosomes.

3. The preparation method according to claim 2, characterized in that, The culture medium described in step S1 contains the following components: microparticles 2-4 g / L, insulin-like growth factor 100-120 mg / L, transforming growth factor-β1 50-100 mg / L, penicillin 0.5-1.5 wt%, and streptomycin 0.5-1.5 wt%.

4. The preparation method according to claim 2, characterized in that, The seeding amount of deciduous tooth pulp stem cells in step S2 is 10. 3 -10 4 The culture conditions under micro-hypoxia and magnetic field conditions are: 3-5 v / v% O2, magnetic field strength 0.1-0.15 T, and culture time 44-52 h.

5. The preparation method according to claim 2, characterized in that, In step S3, the addition of microparticles, propylene glycol, and rhodioloside to the system results in a microparticle content of 1-2 g / L, a propylene glycol content of 10-20 mmol / L, and a rhodioloside content of 20-40 mmol / L. The incubation conditions under the magnetic field are 0.1-0.15T magnetic field for 5-7 hours.

6. The preparation method according to claim 2, characterized in that, The mass ratio of shikonin, interferon, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and highly active dental pulp stem cell exosomes in step S4 is 3-5:1-3:2-4:2-4:17-22.

7. The preparation method according to claim 1, characterized in that, In step T1, the mass ratio of ferric chloride to ferrous chloride is 3.24:1.26, and the heating and stirring reaction temperature is 75-85℃ for 3-5 hours; in step T2, the mass ratio of magnetic microparticles to tetrabutyl titanate is 10:3-4, and the calcination temperature is 400-500℃ for 1-2 hours; in step T3, the mass ratio of TiO2 deposited magnetic microparticles to graphene oxide is 10:2-3; and in step T4, the hydrazine hydrate vapor reduction time is 7-10 hours.

8. A dental pulp stem cell exosome prepared by the preparation method according to any one of claims 1-7.

9. The use of dental pulp stem cell exosomes as described in claim 8 in the preparation of osteointegrative repair and protective materials mediated by titanium implant materials.

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