Dental pulp stem cell-derived exosome light-cured hydrogel, preparation method thereof and application of hydrogel in treatment of severe periodontitis

Abstract

The invention discloses a dental pulp stem cell-derived exosome photocuring hydrogel and a preparation method and application thereof in treating severe periodontitis, and the dental pulp stem cell-derived exosome photocuring hydrogel has better biocompatibility and capability of promoting periodontal tissue regeneration by utilizing the dental pulp stem cell-derived exosome; the sustained-release effect of the hydrogel is beneficial to continuously providing treatment components and reducing the treatment times; through local administration, systemic side effects are reduced, and the safety and effectiveness of treatment are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and periodontal disease treatment, and relates to a hydrogel prepared by using exosomes derived from dental pulp stem cells and its application in treating severe periodontitis. Background Art

[0002] Periodontitis is a common oral disease, mainly caused by chronic inflammation of the tissues surrounding the teeth. When it progresses to a severe stage, it may lead to serious consequences such as gingival recession, loose teeth, and even tooth loss. The mechanism of severe periodontitis is complex, involving a variety of local and systemic factors. Regenerative repair therapy is an important means of treating severe periodontitis. It restores the function of damaged periodontal tissues through periodontal surgery, bone grafting, guided tissue regeneration, the application of growth factors and bioactive substances, but there is still a lack of effective means. Tissue engineering is a new technology developed in recent years. By combining seed cells, scaffold materials and growth factors, bioactive tissue engineering products are constructed to repair periodontal tissue defects. Although the application of tissue engineering in periodontal regeneration and repair is still in its early stages, it has broad prospects.

[0003] Exosomes are nano-sized vesicles secreted by cells. They play an important role in intercellular communication and can affect the function of receptor cells. In regenerative medicine, exosomes are widely used to treat various tissue injuries, such as heart disease, kidney damage, liver damage, lung damage and wound healing. However, the retention rate, biological activity and stability of exosomes in local and systemic treatments are key to the therapeutic effect. Summary of the invention

[0004] Hydrogel is a polymer material with a three-dimensional network structure. It has good biocompatibility, degradability and porosity, and is suitable for loading exosomes and promoting their release in target tissues. By loading exosomes into hydrogels, it is possible to prevent the exosomes from being cleared prematurely, ensuring that the exosomes maintain a high concentration at or near the target site, thereby providing a sustained and more obvious therapeutic effect.

[0005] How to design and research and develop suitable stem cell-derived exosomes and hydrogel systems based on different disease treatment scenarios, as well as improve the regenerative biological activity, utilization, and clinical applicability are the current research focuses and hotspots. In the treatment of severe periodontitis and periodontal tissue defects, we need to achieve high exosome retention and stability in the exosome-loaded hydrogel system to enhance the therapeutic effect of sustained release. On the other hand, we also need the system to achieve the combined synergistic effect of hydrogels and exosomes and a minimally invasive treatment method suitable for local periodontal operations.

[0006] The purpose of the present invention is to provide a novel photocurable hydrogel loaded with exosomes derived from dental pulp stem cells for treating severe periodontitis. The hydrogel has good biocompatibility, the effect of promoting periodontal tissue regeneration and inhibiting inflammation.

[0007] The present invention provides a dental pulp stem cell-derived exosome hydrogel, the hydrogel consisting of the following weight percentages: 0.1-1% of dental pulp stem cell-derived exosomes, 98-99.9% of a hydrogel matrix, wherein the hydrogel matrix is ​​selected from natural or synthetic biocompatible polymer materials;

[0008] Furthermore, the hydrogel matrix is ​​an acrylated polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer hydrogel.

[0009] Furthermore, the hydrogel further comprises a pharmaceutically acceptable excipient;

[0010] Furthermore, the auxiliary materials include excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, solvents, surfactants or emulsifiers.

[0011] Further, the exosome hydrogel is administered orally, by suppository, around the lesion, on the lesion surface, topically, by corneal stroma injection, by subconjunctival injection, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously;

[0012] Further, the exosome hydrogel further comprises a drug selected from anti-apoptotic agents, mitotic inhibitors, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, anti-metabolites, boron-containing agents, chemotherapeutic protectants, hormone agents, anti-hormonal agents, antiviral drugs, photosensitizing therapeutic agents, oligonucleotides, radionuclide agents, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and antibiotic drugs.

[0013] In some experimental schemes, the hydrogel comprises 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% of dental pulp stem cell-derived exosomes. In some embodiments, the hydrogel comprises 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the hydrogel matrix.

[0014] In some embodiments, the biocompatible polymer material includes chondroitin sulfate, chitosan, alginate, hyaluronic acid, cellulose, fibrin, collagen, gelatin, polypeptide, polylactic acid, polycarbonate, polyurethane, polyanhydride, polyphosphazene, polyethylene, polypropylene, polytetrafluoroethylene, polymethyl methacrylate, polydimethylsiloxane, polyethylene terephthalate, polysulfone, polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer.

[0015] The term "exosome" used in the present invention refers to endocytosis-derived nanovesicles secreted by almost all cell types in the body. Exosomes or exosome-like vesicles include proteins, nucleic acids (especially miRNA) and lipids. In practice, exosomes or exosome-like vesicles can be separated and / or purified according to any suitable method known to those skilled in the art, or a method improved therefrom. In certain specific embodiments, the cells secreting exosomes are stem cells, more specifically, the cells are dental pulp stem cells.

[0016] In some embodiments, the "administration" involves the actual physical introduction of the agent into or onto the target biological target (e.g., host and / or subject) (where appropriate). The composition can be applied to cells in vitro or in vivo (e.g., "contacting"). The composition can be applied to a subject in vivo via an appropriate administration route. Any and all methods of introducing the composition into a host are contemplated according to the present invention. The method does not depend on any particular mode of introduction and should not be interpreted as such. The mode of introduction is well known to those skilled in the art and is also exemplified herein. The term includes routes of administration that allow the agent to perform its intended function. Examples of routes of administration that can be used to treat the body include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection may be a bolus or may be a continuous infusion. Depending on the route of administration, the agent may be coated or arranged in a selected material to protect it from natural conditions that may adversely affect its ability to perform its intended function. The agent may be administered alone or in combination with a pharmaceutically acceptable carrier. In the present invention, the agent used is an exosome hydrogel.

[0017] The present invention provides a method for preparing the above-mentioned exosome hydrogel, the method comprising:

[0018] Extraction of exosomes from dental pulp stem cells and preparation of hydrogels;

[0019] The extraction of exosomes derived from dental pulp stem cells comprises the following steps:

[0020] Isolate and culture dental pulp stem cells from the dental pulp of healthy teeth; collect exosomes secreted by dental pulp stem cells by inducing and culturing dental pulp stem cells; purify exosomes by centrifugation, filtration and other methods to obtain exosomes derived from dental pulp stem cells;

[0021] The preparation of the hydrogel comprises the steps of:

[0022] The exosomes derived from dental pulp stem cells are mixed with a hydrogel matrix; the pH value, ionic strength and temperature are adjusted so that the mixture forms a stable hydrogel; the formed hydrogel is sterilized to obtain the exosome hydrogel described above.

[0023] Further, the healthy tooth is an impacted third molar of an adult;

[0024] Furthermore, the adult is a healthy adult aged 18-25 years old;

[0025] Furthermore, the exosomes secreted by dental pulp stem cells were collected and the exosomes used were extracted from dental pulp stem cells of passage 3-5.

[0026] In some embodiments, the dental pulp stem cells derived from the adult impacted third molar are called DPSCs.

[0027] Furthermore, the specific steps of collecting exosomes secreted by dental pulp stem cells include:

[0028] After culturing in osteogenic induction medium for 10 days, the cells were appropriately cultured in serum-free OM for 48 hours for OM-EV isolation. Ultracentrifugation was used to eliminate cell debris and large vesicles, and then ultracentrifuged at 100,000 g for 90 minutes. To eliminate the influence of different cell culture media, the precipitated EXOs were washed twice with pre-cooled PBS and centrifuged again at 100,000 g for 90 minutes. The precipitate was resuspended with cold PBS, and the suspended precipitate was the extracted exosomes.

[0029] Furthermore, the osteogenic induction medium was OM, which consisted of α-MEM supplemented with 10% FBS, 1% P / S, 10 mmol / L β-glycerophosphate, 50 μmol / L ascorbic acid, and 100 nmol / L dexamethasone;

[0030] Furthermore, the specific steps of collecting exosomes secreted by dental pulp stem cells include:

[0031] The serum-free conventional culture medium of dental pulp stem cells was collected for CM-EV isolation. Ultracentrifugation was used to eliminate cell debris and large vesicles, and then ultracentrifuged at 100,000 g for 90 minutes. To eliminate the influence of different cell culture media, the precipitated EXOs were washed twice with pre-cooled PBS and centrifuged again at 100,000 g for 90 minutes. The precipitate was resuspended with cold PBS. The suspended precipitate was the extracted exosomes.

[0032] Furthermore, the serum-free conventional culture medium components were α-MEM and 1% P / S;

[0033] Furthermore, the centrifugation conditions used to eliminate cell debris and large vesicles were centrifugation at 300 g for 10 minutes, 2000 g for 15 minutes, and 10,000 g for 30 minutes, respectively.

[0034] Further, the preparation of the hydrogel comprises the steps of:

[0035] Take the photoinitiator powder, add 20 ml PBS to dissolve it according to the instructions to obtain the photoinitiator solution (protect from light when not in use, store at 4°C); take 0.2 g of F127DA, add 4 ml of the dissolved photoinitiator solution, mix evenly at 2-8°C and place for 15 minutes to dissolve, filter through a 0.22 μm filter membrane after dissolution; prepare exosome hydrogels at a ratio of 1:4, 1:9, and 1:19 respectively;

[0036] Further, the exosomes are frozen exosomes with a density of 5E10 / ml or more, and the exosomes need to be heated in a 37°C water bath to dissolve;

[0037] Furthermore, the protein concentration of the exosomes is 0.5 mg / ml.

[0038] The present invention provides the use of the aforementioned exosome hydrogel or the aforementioned method in preparing a product for treating periodontitis;

[0039] Furthermore, the periodontitis includes mild periodontitis and severe periodontitis.

[0040] In some embodiments, the "treatment" refers to the improvement, prevention, or reversal of a disease or condition or at least one distinguishable symptom thereof. In certain specific embodiments, the "treatment" refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, which parameter is not necessarily recognizable in mammals or recognized by mammals. In some embodiments, the "treatment" refers to the inhibition or reduction of a disease or disease process, which can be physical, such as certain distinguishable adverse symptoms; such inhibition or reduction can be physiological, such as stabilizing physiological parameters; such inhibition or reduction can be both.

[0041] The present invention provides the use of the exosome hydrogel described above, or the method described above, in the preparation of a product that promotes the proliferation of periodontal ligament fibroblasts, local periodontal tissue regeneration and repair, regulates the periodontal microenvironment, and provides anti-oxidation or anti-inflammatory effects at periodontitis sites.

[0042] The present invention provides an exosome hydrogel product, which includes the exosome hydrogel described above, and an auxiliary laminating material;

[0043] Further, the auxiliary lamination material includes a patch;

[0044] Furthermore, the preparation materials of the patch include non-woven fabrics, glass fibers, polyester films, glass fiber films, hydrophilic polyethylene materials, and olefinic acid fibers.

[0045] In some embodiments, the auxiliary bonding material can be in the form of a scaffold, which serves to anchor the hydrogel product to the cavity surface or the outer surface, and is used to fix the hydrogel so that it is stabilized at a fixed position to release exosomes.

[0046] The term "scaffold" is used in the present invention to refer to molecules that can be used to anchor a target payload or any other compound (e.g., ASO) to an EV on the luminal surface or on the outer surface of an EV (extracellular vesicle, e.g., exosome). In some aspects, the scaffold comprises a synthetic molecule. In some aspects, the scaffold comprises a non-polypeptide portion. In other aspects, the scaffold comprises a lipid, carbohydrate, or protein that is naturally present in an EV. In some aspects, the scaffold comprises a lipid, carbohydrate, or protein that is not naturally present in an EV. Non-limiting examples of other scaffolds that can be used in embodiments include: aminopeptidase N (CD13); enkephalinase, AKA membrane metalloendopeptidase (MME); ectonucleotide pyrophosphatase / phosphodiesterase family member 1 (ENPP1); neuropilin-1 (NRP1); CD9, CD63, CD81, PDGFR, GPI anchor protein, lactadherin (MFGE8), LAMP2, and LAMP2B.

[0047] Advantages and beneficial effects of the present invention:

[0048] This patent designs a photocurable hydrogel loaded with exosomes derived from dental pulp stem cells, which has excellent biocompatibility and biodegradability, can significantly promote the proliferation of periodontal ligament fibroblasts and local periodontal tissue regeneration and repair, and has great application potential in the treatment of periodontitis. Although the pathogenesis of severe periodontitis is complex and involves the combined effects of multiple factors. In terms of regenerative repair treatment, in addition to traditional periodontal surgery and bone grafting, the application of growth factors and bioactive substances, laser treatment, and the development of new technologies such as tissue engineering provide new options for the treatment of severe periodontitis. In the future, the development direction of tissue engineering repair treatment for severe periodontitis will focus on the following aspects: first, the development of more efficient and safe seed cell sources and derivatives, such as induced pluripotent stem cells, and the stable acquisition of stem cell derivative exosomes; second, the development of scaffold materials with better biocompatibility and controllable degradation rate; third, the exploration of more precise growth factor delivery systems to improve the treatment effect. In addition, combined with advanced technologies such as gene editing and biological 3D printing, it is expected to achieve personalized periodontal tissue engineering repair treatment and bring better treatment effects to patients with severe periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is the particle size distribution, particle number measurement and negative staining electron microscopy analysis results of OM-EV;

[0050] Figure 2 It is the particle size distribution, particle number measurement and negative staining electron microscopy analysis results of CM-EV;

[0051] Figure 3 It is the WB result image of CM-EV and OM-EV;

[0052] Figure 4 It is a statistical graph of cell growth after using CM-EV and OM-EV;

[0053] Figure 5 This is the gelling performance result diagram of F127DA-DPSC-EXO;

[0054] Figure 6 This is the thermosensitive / photosensitive performance result diagram of the copolymer hydrogel of F127DA-DPSC-EXO;

[0055] Figure 7 This is a real picture of the F127DA-DPSC-EXO bracket adsorption;

[0056] Figure 8 This is a statistical chart of the sustained release results of F127DA-DPSC-EXO gel;

[0057] Fig. 9 This is the restoration result diagram using F127DA-DPSC-EXO as restoration material. DETAILED DESCRIPTION

[0058] As used herein, the terms "a", "an" and "the" include singular and plural references unless the context clearly indicates otherwise. The term "consisting essentially of" is used to define a lipid composition that includes the listed elements but excludes other elements, that is, the term lipid composition is used to define a composition that is essentially composed only of lipids. "Consisting of" thus means excluding elements that exceed trace amounts of other elements. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0059] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, apparatus.

[0060] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, so that those skilled in the art can understand and implement the present invention and further recognize the advantages of the present invention.

[0061] Example 1 Cultivation of human DPSCs and isolation, purification and identification of exosomes

[0062] DPSCs were obtained from impacted third molars of patients aged 18-25 years. The cell morphology of DPSCs at P0 and P3 was observed using an inverted microscope (Zeiss AxioObserver, Oberkochen, Germany). The proliferation capacity of DPSCs was identified using a colony formation method. To examine MSCs-specific markers, cells were appropriately stained with antibodies (CD34, CD44, CD45, CD73, CD90, and CD105, Biolegend, China) and analyzed by flow cytometry (Beckman Coulter, USA). To determine the multipotency of DPSCs, cells were treated with osteogenic induction medium for 14 days and then stained with Alizarin red solution (Cyagen Biosciences Inc., China). Adipogenic induction medium of DPSCs was used to induce adipogenic differentiation for 21 days, and lipid droplet formation was assessed using Oil red O staining.

[0063] Example 2 Isolation and purification of EVs

[0064] The cell culture supernatant of DPSCs from P3-P5 was collected for EV isolation. For EXO isolation, DPSCs were cultured in osteogenic induction medium (OM, α-MEM supplemented with 10% FBS, 1% P / S, 10 mmol / L β-glycerophosphate, 50 μmol / L ascorbic acid, and 100 nmol / L dexamethasone) for 10 days, and then cultured appropriately in serum-free OM for 48 hours. Serum-free regular medium (CM, α-MEM with 1% P / S) of DPSCs was collected for CM-EV isolation, and ultracentrifugation (10 min at 300 g, 15 min at 2000 g, and 30 min at 10,000 g) was used to eliminate cell debris and large vesicles, followed by ultracentrifugation at 100,000 g for 90 min (using ultracentrifuge Beckman Coulter Optima L-100XP, USA). To eliminate the effect of different cell culture media, the precipitated EXOs were washed twice with pre-cooled PBS and centrifuged again at 100,000 g for 90 min. The precipitate was resuspended with cold PBS. The protein concentration of EXOs was quantified using a micro BCA protein assay kit (CWbio, Shanghai, China).

[0065] The particle size distribution, particle number measurement and negative staining electron microscopy analysis results of OM-EVs are shown in Figure 2. Figure 1 The particle size distribution, particle number measurement and negative staining electron microscopy analysis results of CM-EV are shown in Figure 2 shown.

[0066] Example 3 Identification and characterization of CM-EVs and OM-EVs

[0067] Appropriate identification of OM-EVs and CM-EVs was performed as recommended by the International Society for Extracellular Vesicles (ISEV). The morphology of EVs was observed by transmission electron microscopy (TEM, Hitachi H-7650, Japan) and atomic force microscopy (AFM, Bruker, Karlsruhe, Germany). For atomic force microscopy (AFM), EVs in PBS were incubated on a clean glass slide. MLCT-F cantilevers with a nominal tip radius of 20 nm and a spring constant of 0.6 N / m were used to perform the required measurements. The size and particle concentration of EVs were determined by nanoparticle tracking analysis (NTA) using a ZetaView nanoparticle tracker (Particle Metrix, Germany). EV markers CD63 and TSG 101 (Zen-bio, China) were determined by Western blotting to identify the purity of EVs.

[0068] The WB results of CM-EV and OM-EV are as follows Figure 3 As shown, each marker protein corresponds to two measurement groups, CM-EV on the left and OM-EV on the right. The growth of indicator cells after co-incubation with exosomes was detected by impedance method. The higher the impedance value, the better the growth state of the indicator cells. The test results are shown in Figure 4 As shown, the indicator cells are Hela, the positive group is Hela+10% FBS, the negative group is Hela, experimental group 1 is Hela+OM-EV, and experimental group 2 is Hela+CM-EV.

[0069] Example 4 F127DA-DPSC-EXO loading and performance experiment

[0070] 1. Verification of gelling properties

[0071] A. Preparation of exosomes and hydrogel solution:

[0072] Take 0.2g of F127DA in a 10ml centrifuge tube, add 4ml of dissolved photoinitiator solution, dissolve at 2-8℃, and take exosomes with a frozen density above 5E10 / ml to melt for use (protein concentration is about 0.5mg / ml).

[0073] B. Prepare exosome hydrogels by mixing the melted exosomes and filtered hydrogel containing photoinitiator at a ratio of 1:4, 1:9, and 1:19 (exosome: hydrogel). Drop 200 μl of the hydrogel-exosome mixture onto a sterile anti-adhesive film and perform light curing in a biosafety cabinet. Figure 5 As shown in the figure, the lower the hydrogel content, the greater the deformation before gel formation and the lower the hardness after gel formation. However, all three concentrations can be successfully cured.

[0074] 2. Verification of thermosensitive / photosensitive properties of copolymer hydrogel loaded with F127DA-DPSC-EXO

[0075] After the 24-well plate was coated with low adsorption coating solution at 4°C overnight, the coating solution was discarded for later use. Take the photoinitiator powder, add 20ml PBS to dissolve it according to the instructions, and obtain the photoinitiator solution (keep away from light when not in use and store at 4°C); take F127DA0.2g, add 4ml dissolved photoinitiator solution, mix evenly at 2-8°C and place for 15min to dissolve, and filter through a 0.22μm filter membrane after dissolution; prepare exosome hydrogel by the ratio of exosome: hydrogel at 1:9 (taking this as an example), and mix the final volume of 1000μl in a sterile EP tube. Take 200μl of each exosome hydrogel in a 24-well plate and add 200μl PBS. Repeat 3 wells for each condition, store in an incubator after light curing, and sample the supernatant for detection at 1h and 5h, respectively, sampling 20μl each time and supplementing with the same volume of PBS.

[0076] The growth of indicator cells after co-incubation with exosomes was detected by impedance method. The higher the impedance value, the better the growth of indicator cells. Take 100 μl of buffer solution released from F127DA hydrogel for 1-5 hours, add it to the detection system, and observe the change of cell impedance.

[0077] The results are as follows Figure 6 As shown, Figure 6 Indicator cells: HSF (human skin fibroblasts), positive group is HSF + 10% FBS, negative group is HSF, experimental group 1 is HSF + CM-EV-1h, experimental group 2 is HSF + CM-EV-5h, experimental group 3 is HSF + CM-EV-1h, experimental group 4 is HSF + CM-EV-5h. Through the skin simulation experiment, the copolymer hydrogel loaded with F127DA-DPSC-EXO has good thermosensitive / photosensitive gelation performance. And it can significantly promote the growth of periodontal fibroblasts.

[0078] 3. Bracket adsorption and orifice plate gelation

[0079] 200 μl of the hydrogel-exosome mixture was dripped onto the scaffold on the sterile anti-adhesive film and light-cured in a biosafety cabinet. After light-irradiation, it was successfully cured and separated from the anti-adhesive film using a sterile blade, transferred to a well plate, and 200 μl of culture medium was added, and marked as "scaffold group".

[0080] 200 μl of the hydrogel-exosome mixture was dropped into the well plate and light-cured in a biosafety cabinet. After light-curing, it was successfully solidified and 200 μl of culture medium was added.

[0081] The well plate was placed in an incubator, or under other conditions specified in the protocol, to conduct an exosome secretion test. After the F127DA exosome hydrogel solidified, it was placed in an equal volume of PBS. The protein content in PBS was detected by BCA at different time points to analyze the sustained release ability of the exosome hydrogel.

[0082] After testing, it was found that a certain concentration of protein in the system could be maintained within 5 days. The specific results are as follows: Figure 7 , Figure 8 And as shown in Table 1.

[0083] Table 1

[0084] Incubation time EXO gel group (μg / mL) Blank gel group (μg / mL) 3h 55.31 9.14 5h 55.24 7.79 2d 36.74 7.01 5d 42.47 13.272

[0085] Example 5 Evaluation of in vitro biological activity of F127DA-DPSC-EXO

[0086] 1. Evaluation of the proliferation capacity of periodontal ligament fibroblasts and dental pulp stem cells

[0087] To evaluate the effect of F127DA-DPSC-EXO on the proliferation of periodontal ligament fibroblasts and dental pulp stem cells, a CCK-8 kit (Dojindo, Kumamoto, Japan) was used. To this end, periodontal ligament fibroblasts and dental pulp stem cells were seeded in 96-well plates (5000 cells per well) and cultured with medium containing different concentrations of F127DA-DPSC-EXO (0, 1, 5, 10 and 20 μg / mL, respectively). After 1, 3, 5 and 7 days, 10% CCK-8 solution was added to each well. After incubating the plate at 37°C for 1 hour, the optical density (OD) value at 450nm was appropriately measured using a microplate reader (Tecan, Infinite f200 PRO, Switzerland). To further confirm the proliferation effect of F127DA-DPSC-EXO, an EdU proliferation kit (Beyotime, China) was used. Periodontal ligament fibroblasts and dental pulp stem cells were seeded in a well plate at a density of 104 cells per well and then cultured with 10μg / mL and 20μg / mL F127DA-DPSC-EXO for 7 days. After staining the nuclei with Adize488 and DAPI, six random fields of each group were photographed using a laser confocal microscope (Zeiss LSM 980, Germany). The proportion of EdU-positive nuclei in each group was calculated using Image J (National Institutes of Health, Bethesda, MD). In addition, the cell cycle profile after treatment with 20μg / mL F127DA-DPSC-EXO was established by flow cytometry.

[0088] 2. Assessment of migration capability

[0089] Transwell chambers with 12 μm pores (Corning, USA) were used to evaluate the migration ability of periodontal ligament fibroblasts and dental pulp stem cells. 5 100 μL suspension of DPSCs (in α-MEM without FBS) was seeded in the upper chamber. In the lower chamber, 20 μg / mL F127DA-DPSC-EXO was added. After 24 hours, the migrating DPSCs on the lower surface of the membrane were properly fixed with formaldehyde, stained with 0.1% crystal violet, and recorded using an inverted microscope.

[0090] 3. Evaluation of in vitro dentinogenic potential

[0091] To evaluate the effect of F127DA-DPSC-EXO on calcium nodule formation, DPSCs were plated at 1 × 10 per well. 5The cells were seeded at a density of 1.57 × 10 cells / mL in a 12-well plate and treated with odontoblast induction medium containing 20 μg / mL F127DA-DPSC-EXO. After 7 and 14 days, the cells were fixed with 4% paraformaldehyde and stained with Alizarin red solution, and then the calcium nodules were observed under an inverted microscope. Then, the calcium nodules were measured by incubating with cetylpyridinium chloride and measuring the OD value at a wavelength of 562 nm. In addition, after 7 days of odontoblast induction, ALP activity detection and staining were performed using an ALP kit (Nanjing Jiancheng Bioengineering Institute, China) and an NBT / BCIP staining kit (Beyotime, China) according to the manufacturer's instructions.

[0092] Example 6 Preliminary proof of concept and safety evaluation of F127DA-DPSC-EXO for periodontal tissue repair after transplantation in rat periodontitis model

[0093] 1. Animal model and transplantation grouping: All animal experiments were approved by the Animal Care and Use Committee of Beijing Jishuitan Hospital, Capital Medical University, and were conducted in strict accordance with the "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T 35,892-2018). This study used rat periodontitis inflammation and alveolar bone defect models to evaluate the function of F127DA-DPSC-EXO.

[0094] For the experimental model, a total of 24 male Wistar rats (6 weeks old) were randomly divided into four groups (n=6 in each group) as follows: 1) blank control group (blank); 2) negative control group (periodontitis); 3) experimental group I (F127DA); 4) experimental group II (F127DA-DPSC-EXO).

[0095] To establish the periodontitis model, rats were anesthetized and orthodontic ligatures (0.2 mm) were placed around the cervical region of the bilateral maxillary first molars. During the ligation of the orthodontic wires, we were careful not to damage the gingiva or buccal mucosa. During this period, LPS (1 mg mL^-1, 0.02 mL) was injected subgingivally at the site of the bilateral maxillary first molars every day. After 4 days, the orthodontic wires were removed, and then food debris, plaque, and calculus were thoroughly scraped off. In brief, animals in the periodontitis group were treated with 0.9% NaCl solution (20 μL), while animals in the F127DA and F127DA-DPSC-EXO groups were injected into the periodontal pocket with pregel solution (20 μL), which was then polymerized under blue light for 15 seconds. After 4 days, the gingiva was collected and fixed with 4% paraformaldehyde. Subsequently, the samples were dehydrated using a series of graded ethanol, embedded in paraffin, and cut into 3.0 μm thick sections. Immunohistochemical staining was used to detect inflammatory factors in the gingiva, using rabbit anti-TNF-α and anti-COX2 antibodies. Images of stained samples were captured by optical microscopy. Before euthanasia, the gingival margin was wiped with a sterile cotton swab and placed in 1 mL of PBS (pH = 7.4). After sufficient preparation, 20 μL of the suspension was evenly spread on a blood agar plate containing defibrinated sheep blood, hemoglobin and vitamin K. Cultured at 37 ° C in an anaerobic environment. After 2 days, the colonies were photographed using a digital camera. In order to establish an alveolar bone defect model, the rats were anesthetized and orthodontic ligatures (0.2 mm) were placed around the necks of the bilateral maxillary first molars. After 4 weeks, the orthodontic wires were removed, and then food debris, plaque and tartar were thoroughly scraped off. The treatment regimen was the same as above, except for the treatment time. The operation was performed once a week. After 4 weeks, all rats were anesthetized and perfused with 4% paraformaldehyde. Maxillary bones were collected and subjected to micro-computed tomography (micro-CT) with the following scanning parameters: voltage 80 kV; current 100 μA; voxel size 9.0 μm; exposure time 4200 ms. Three-dimensional (3D) images were reconstructed using CTVOX. Next, two-dimensional (2D) images were randomly selected from each group, and the distance from the cementoenamel junction (CEJ) between the first and second molars to the apex of the alveolar bone ridge (ABC) (CEJ-ABC) was calculated. Subsequently, the bone volume / tissue volume (BV / TV) ratio of the area between the first and second molars was further evaluated using CTAN to assess the alveolar bone repair status at the 3D level. After micro-CT scanning, the samples were decalcified in ethylenediaminetetraacetic acid (EDTA) for 8 weeks. Subsequently, the samples were dehydrated using a series of graded ethanol, embedded in paraffin, cut into 3.0 μm thick sections, and stained with hematoxylin-eosin (H&E) and Masson's trichrome. The alveolar bone between the maxillary first and second molars was observed by light microscopy.

[0096] Histological analysis: To evaluate in vivo toxicity, the heart, liver, spleen, lung and kidney of all groups of animals were collected for H&E staining. 10% formalin was used. After decalcification and dehydration, the samples were embedded in paraffin. Paraffin sections were stained with H&E and Safranine O-Fast Green (S&O) as indicated. Immunohistochemical staining was performed with anti-COL I and anti-COL II antibodies.

[0097] The results of tissue repair status after using F127DA-DPSC-EXO repair material are as follows Fig. 9 As shown, the results showed that the exosomes derived from dental pulp stem cells were mixed with the hydrogel matrix as a bone filling material, supplemented to the tissue in need of bone repair and then photocured. After a period of growth and repair, it was seen that the exosome hydrogel group had obvious bone growth, indicating that it has the ability to repair bone. The control group had low bone density and incomplete bone imaging; correspondingly, the bone density of the exosome hydrogel group increased significantly and had better integrity.

[0098] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A dental pulp stem cell-derived exosome hydrogel, the hydrogel comprising the following weight percentages: 0.1-1% of exosomes derived from dental pulp stem cells, 98-99.9% of a hydrogel matrix, wherein the hydrogel matrix is ​​selected from natural or synthetic biocompatible polymer materials; Preferably, the hydrogel matrix is ​​an acrylated polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer hydrogel.

2. The exosome hydrogel according to claim 1, further comprising a pharmaceutically acceptable excipient; Preferably, the auxiliary materials include excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH regulators and / or buffers, solvents, surfactants or emulsifiers.

3. The exosome hydrogel according to claim 1, wherein the exosome hydrogel is administered orally, by suppository, around the lesion, on the lesion surface, topically, by corneal stroma injection, by subconjunctival injection, by intravenous administration, by parenteral administration, by intraperitoneal administration, by muscle administration, by intralesional administration, by intrathecal administration, by intranasal administration, or by subcutaneous administration; Preferably, the exosome hydrogel further comprises a drug selected from anti-apoptotic agents, mitotic inhibitors, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, anti-metabolites, boron-containing agents, chemotherapeutic protectants, hormone agents, anti-hormonal agents, antiviral drugs, photosensitizing therapeutic agents, oligonucleotides, radionuclide agents, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and antibiotic drugs.

4. A method for preparing the exosome hydrogel according to any one of claims 1 to 3, the method comprising: Extraction of exosomes from dental pulp stem cells and preparation of hydrogels; The extraction of exosomes derived from dental pulp stem cells comprises the following steps: Isolate and culture dental pulp stem cells from the dental pulp of healthy teeth; collect exosomes secreted by dental pulp stem cells by inducing and culturing dental pulp stem cells; purify exosomes by centrifugation and filtration to obtain exosomes derived from dental pulp stem cells; The preparation of the hydrogel comprises the steps of: The exosomes derived from dental pulp stem cells are mixed with a hydrogel matrix; the pH value, ionic strength and temperature are adjusted so that the mixture forms a stable hydrogel; and the formed hydrogel is sterilized to obtain the exosome hydrogel according to any one of claims 1 to 3.

5. The method according to claim 4, wherein the healthy tooth is an impacted third molar of an adult; Preferably, the adult is a healthy adult aged 18-25 years old; Preferably, the exosomes secreted by dental pulp stem cells are collected from cells extracted from dental pulp stem cells of passage 3-5.

6. The method according to claim 4, wherein the specific step of collecting exosomes secreted by dental pulp stem cells comprises: After culturing in osteogenic induction medium for 10 days, the cells were appropriately cultured in serum-free OM for 48 hours for OM-EV isolation. Ultracentrifugation was used to eliminate cell debris and large vesicles, and then ultracentrifuged at 100,000 g for 90 minutes. To eliminate the influence of different cell culture media, the precipitated EXOs were washed twice with pre-cooled PBS and centrifuged again at 100,000 g for 90 minutes. The precipitate was resuspended with cold PBS, and the suspended precipitate was the extracted exosomes. Preferably, the osteogenic induction medium is OM, which is composed of α-MEM supplemented with 10% FBS, 1% P / S, 10 mmol / L β-glycerophosphate, 50 μmol / L ascorbic acid, and 100 nmol / L dexamethasone; Preferably, the specific steps of collecting exosomes secreted by dental pulp stem cells include: The serum-free conventional culture medium of dental pulp stem cells was collected for CM-EV isolation. Ultracentrifugation was used to eliminate cell debris and large vesicles, and then ultracentrifuged at 100,000 g for 90 minutes. To eliminate the influence of different cell culture media, the precipitated EXOs were washed twice with pre-cooled PBS and centrifuged again at 100,000 g for 90 minutes. The precipitate was resuspended with cold PBS. The suspended precipitate was the extracted exosomes. Preferably, the serum-free conventional culture medium components are α-MEM and 1% P / S; Preferably, the centrifugation conditions used to eliminate cell debris and large vesicles are centrifugation at 300 g for 10 minutes, 2000 g for 15 minutes, and 10,000 g for 30 minutes, respectively.

7. The method of claim 4, wherein the preparation of the hydrogel comprises the steps of: Take the photoinitiator powder, add 20 ml PBS to dissolve, and obtain the photoinitiator solution; take 0.2 g of F127DA, add 4 ml of the dissolved photoinitiator solution, mix evenly at 2-8°C, and place for 15 minutes to dissolve, and filter through a 0.22 μm filter membrane after dissolution; prepare exosome hydrogels at a ratio of 1:4, 1:9, and 1:19 respectively; Preferably, the exosomes are frozen exosomes with a density of 5E10 / ml or more, and the exosomes need to be heated in a 37°C water bath to dissolve; Preferably, the protein concentration of the exosomes is 0.5 mg / ml.

8. Use of the exosome hydrogel according to any one of claims 1 to 3, or the method according to any one of claims 4 to 7 in preparing a product for the treatment of periodontitis; Preferably, the periodontitis includes mild periodontitis and severe periodontitis.

9. Use of the exosome hydrogel according to any one of claims 1 to 3, or the method according to any one of claims 4 to 7, in the preparation of a product that promotes the proliferation of periodontal ligament fibroblasts, local periodontal tissue regeneration and repair, regulates the periodontal microenvironment, and provides anti-oxidation or anti-inflammatory effects at periodontitis sites.

10. An exosome hydrogel product, comprising the exosome hydrogel according to any one of claims 1 to 3, and an auxiliary laminating material; Preferably, the auxiliary laminating material comprises a patch; Preferably, the patch is made of materials including non-woven fabrics, glass fibers, polyester films, glass fiber films, hydrophilic polyethylene materials, and nylon fibers.

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