Nano odontogenic mesenchymal stem cell vesicle as well as preparation method and application thereof

Abstract

The invention relates to a nano odontogenic mesenchymal stem cell vesicle as well as a preparation method and application thereof, and belongs to the technical field of regenerative medicine and drug carriers. The method comprises the following steps: obtaining odontogenic mesenchymal stem cells; adding a chemical vesicle stimulant and then putting the mixture into a CO2 constant-temperature incubator for stimulation to obtain a giant plasma membrane vesicle solution of the odontogenic mesenchymal stem cells; repeatedly extruding through a polycarbonate film by using a lipidosome extruder to obtain the nano odontogenic mesenchymal stem cell vesicles of which the particle sizes are less than or equal to 200nm; the chemical vesicle stimulant comprises a buffer solution, a dithiothreitol aqueous solution and a paraformaldehyde solution, wherein the buffer solution comprises 8.5-9 g / L of sodium chloride, 0.2-0.24 g / L of calcium dichloride, 2.3-2.4 g / L of 4-hydroxyethyl piperazine ethanesulfonic acid and water. The obtained vesicles are high in yield and purity, have efficient bone tissue targeting property, and can prolong the in-vivo circulation time of the medicine and enhance the bone regeneration capacity.

Description

Technical Field

[0001] The invention belongs to the technical field of regenerative medicine and drug carriers, and in particular relates to a nano-sized dental mesenchymal stem cell vesicle and a preparation method thereof. Background Art

[0002] Osteoporosis is a systemic bone disease characterized by low bone mass, damaged bone microstructure, increased bone brittleness, and susceptibility to fractures. The medical treatment and care of osteoporosis and fractures require a lot of manpower, material and financial resources, causing a heavy burden on families and society. Therefore, it is necessary to explore and develop effective treatments to alleviate bone loss, including osteoporosis.

[0003] In the past few decades, mesenchymal stem cells (MSCs) have become the main seed cells for regenerative medicine due to their strong self-renewal, multidirectional differentiation, immunomodulatory ability, and lack of ethical controversy. Among them, dental MSCs have attracted much attention because they are easy to obtain, and medical discarded tissues such as impacted wisdom teeth, orthodontically extracted premolars, and lost deciduous teeth are important sources of them. They have the advantage of no iatrogenic trauma. However, the local and systemic application of MSCs has limitations and side effects. For example, the efficiency of MSCs transport to target tissues in vivo is limited, which can lead to an increased risk of pulmonary embolism and tumor formation, which hinders their clinical transformation. Therefore, there is an urgent need to develop alternative products to MSCs that retain the beneficial properties of MSCs while reducing their adverse effects.

[0004] Extracellular vesicles (EVs) secreted by MSCs are part of the paracrine pathway of MSCs. They can inherit the characteristics of parental cells while avoiding the defects of stem cell therapy, and can efficiently deliver endogenous proteins, nucleic acids and exogenous drugs to target tissues or cells to exert therapeutic effects. However, traditional methods for preparing extracellular vesicles have one or more technical problems, such as cumbersome preparation process, limited yield, and low purity of prepared extracellular vesicles.

[0005] Therefore, it is very necessary to provide a nano-sized dental mesenchymal stem cell vesicle and a preparation method and application thereof. Summary of the invention

[0006] In order to solve one or more technical problems existing in the prior art, the present invention provides a nano-sized dental mesenchymal stem cell vesicle and a preparation method and application thereof. The vesicle obtained by the present invention has high yield and purity, has high bone tissue targeting, can prolong the in vivo circulation time of the drug, and enhance bone regeneration ability.

[0007] In a first aspect, the present invention provides a method for preparing nano-sized dental mesenchymal stem cell vesicles, the preparation method comprising the following steps: (1) Obtaining dental mesenchymal stem cells; (2) adding a chemical vesicle stimulator to the dental mesenchymal stem cells and placing the cells in a CO2 constant temperature incubator for stimulation to obtain a giant plasma membrane vesicle solution of the dental mesenchymal stem cells; the chemical vesicle stimulator comprises a buffer solution, a dithiothreitol aqueous solution and a paraformaldehyde solution; the buffer solution comprises 8.5-9 g / L sodium chloride, 0.2-0.24 g / L calcium chloride, 2.3-2.4 g / L 4-hydroxyethylpiperazineethanesulfonic acid and deionized water; (3) The dental mesenchymal stem cell giant plasma membrane vesicle solution is repeatedly extruded through a polycarbonate membrane using a liposome extruder to obtain nano-sized dental mesenchymal stem cell vesicles with a particle size of no more than 200 nm.

[0008] Preferably, in step (1), the method for obtaining the dental mesenchymal stem cells is as follows: digesting and centrifuging the periodontal ligament tissue or dental pulp tissue, discarding the supernatant, then adding α-MEM culture medium containing fetal bovine serum and penicillin-streptomycin and mixing evenly, then culturing in a CO2 constant temperature incubator until the primary stem cells reach 80% to 90% fusion, digesting with trypsin-EDTA digestion solution and culturing and amplifying to P4 to P6 generations at a ratio of 1:2 or 1:3 to obtain dental mesenchymal stem cells.

[0009] Preferably, the volume fraction of fetal bovine serum contained in the α-MEM culture medium is 10~20%, the volume fraction of penicillin-streptomycin contained in the α-MEM culture medium is 0.8~1.2%; the volume fraction of CO2 contained in the CO2 constant temperature incubator is 5%, the temperature of the CO2 constant temperature incubator is 37°C, and the humidity is 100%; and / or the trypsin-EDTA digestion solution is a digestion solution containing 0.25wt% trypsin and 0.1wt% EDTA.

[0010] Preferably, in step (2): the concentration of the dithiothreitol aqueous solution is 150-160 mg / mL; the paraformaldehyde solution is a 4% paraformaldehyde fixative; and / or the volume ratio of the buffer solution, the dithiothreitol aqueous solution and the paraformaldehyde solution is 1000:(1.5-2.5):(16-20).

[0011] Preferably, in step (2): the concentration of the dithiothreitol aqueous solution is 154 mg / mL; the volume ratio of the buffer solution, the dithiothreitol aqueous solution and the paraformaldehyde solution is 1000:2:18; and / or the buffer solution comprises 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid and deionized water.

[0012] Preferably, in step (2), the volume fraction of CO2 in the CO2 constant temperature incubator is 5%, the temperature of the CO2 constant temperature incubator is 37°C, and the humidity is 100%; the stimulation time is 18-30 hours; and / or the amount of the chemical vesicle stimulator added is 18-25 microliters of the chemical vesicle stimulator per square centimeter of dental mesenchymal stem cells; and / or the particle size of the dental mesenchymal stem cell giant plasma membrane vesicles in the dental mesenchymal stem cell giant plasma membrane vesicle solution is 5-28 μm, and the concentration of the dental mesenchymal stem cell giant plasma membrane vesicles is 6.0×10 6 ~6.7×10 6 Pieces / mL.

[0013] Preferably, in step (3): the number of repeated extrusions is 8 to 12 times; and / or the polycarbonate membrane is a polycarbonate membrane with a pore size of 200 nm.

[0014] Preferably, in step (3): before repeatedly extruding through a polycarbonate membrane using a liposome extruder, the dental mesenchymal stem cell giant plasma membrane vesicle solution is first mixed with a DPP4 inhibitor.

[0015] In a second aspect, the present invention provides nanosized dental mesenchymal stem cell vesicles prepared by the preparation method of the first aspect of the present invention.

[0016] The third aspect of the present invention provides the use of nano-sized dental mesenchymal stem cell vesicles prepared by the preparation method described in the first aspect of the present invention in bone tissue repair materials.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention generates and separates giant plasma membrane vesicles of dental mesenchymal stem cells from dental mesenchymal stem cells by using a chemical vesicle stimulator, and then repeatedly extrude the vesicles through a polycarbonate membrane to obtain nano-sized dental mesenchymal stem cell vesicles. Compared with the traditional method of extracting extracellular vesicles from cell culture supernatant, the method of the present invention can obtain vesicles with higher yield and higher purity. The polyformaldehyde solution contained in the chemical vesicle stimulator used in the present invention can induce non-specific cross-linking of proteins in the cell membrane, and the dithiothreitol contained in the chemical vesicle stimulator can act as a small molecule reducing agent to cut the thioester bond, promote cell membrane foaming to form giant plasma membrane vesicles of dental mesenchymal stem cells GPMV and fall into the supernatant; at the same time, the buffer solution contained in the chemical vesicle stimulator contains calcium ions Ca 2+ The present invention found that in Ca 2+ Under the condition of presence of Ca, cells were exposed to paraformaldehyde solution and dithiothreitol solution, which can make continuous Ca 2+ Inflow is beneficial to promote cell plasma membrane bubbling. The chemical vesicle stimulator used in the present invention can make the giant plasma membrane vesicles GPMV of dental mesenchymal stem cells produced by the present invention contain no cytoskeleton or nuclear material. Directly extracting the cell membrane by extruding the cell membrane or lysing the cell will inevitably be contaminated by organelles or nuclear material. Therefore, compared with other methods for producing cell membrane vesicles, the method of the present invention produces purer cell membrane vesicles.

[0018] (2) The nanostructured dental mesenchymal stem cell vesicles prepared by the present invention have homologous targeting similar to MSCs, targeting bone tissue, and are highly efficient in being transported to target tissues in vivo. They can also prolong the in vivo circulation time of drugs, achieve targeted delivery and sustained release of drugs in bone tissue, and thus enhance bone regeneration ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a morphological picture of the giant plasma membrane vesicle solution of dental mesenchymal stem cells obtained in Example 1 of the present invention observed by ordinary optical microscope (scale bar, 100 μm); Figure 2 The morphological diagram of the giant plasma membrane vesicle solution of dental mesenchymal stem cells obtained in Example 1 of the present invention after being labeled with the fluorescent dye DiI and thioflavin T is observed by confocal microscopy (scale bar, 10 μm); in the figure, Figure (a) corresponds to the cell membrane image labeled with the DiI dye, Figure (b) corresponds to the cytoplasm image labeled with thioflavin T, and Figure (c) corresponds to the image after being labeled with the fluorescent dye DiI and thioflavin T at the same time; Figure 3 It is a small animal fluorescence imaging image of fluorescent dye DiR and nano-sized dental mesenchymal stem cell vesicles (nMSC-DiR) labeled with fluorescent dye DiR in bone tissue; Figure 4 is a transmission electron microscopic image of the nano-sized dental mesenchymal stem cell vesicle loaded with DPP4 inhibitor obtained in Example 2 of the present invention (scale bar, 200 nm); Figure 5 The osteoporosis animal model was established by removing both ovaries of mice, and treatment was given according to different groups. The femoral bone mass results were obtained by Micro-CT detection 4 weeks after intravenous administration. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] In a first aspect, the present invention provides a method for preparing nano-sized dental mesenchymal stem cell vesicles, the preparation method comprising the following steps: (1) Obtaining dental mesenchymal stem cells; (2) adding a chemical vesicle stimulator to dental mesenchymal stem cells, placing the cells in a CO2 constant temperature incubator for stimulation, and then centrifuging the cells to obtain a dental mesenchymal stem cell giant plasma membrane vesicle solution; the chemical vesicle stimulator comprises a buffer solution (also referred to as GPMV buffer solution), a dithiothreitol aqueous solution, and a paraformaldehyde solution; the buffer solution comprises sodium chloride (NaCl) 8.5-9 g / L (e.g., 8.5, 8.6, 8.7, 8.9 or 9 g / L), calcium chloride (CaCl2) 0.2-0.24 g / L (e.g., 0.2, 0.21, 0.22, 0.23 or 0 .24g / L), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) 2.3-2.4g / L (e.g., 2.3, 2.2, 2.36, 2.38 or 2.4g / L) and the remainder deionized water; in the present invention, preferably, the buffer solution is a buffer solution with a pH of 7.4, specifically, for example, by adding a 0.5-1M NaOH aqueous solution (sodium hydroxide aqueous solution) to the buffer solution to adjust the pH of the buffer solution to 7.4; the present invention does not specifically limit the amount and concentration of the sodium hydroxide aqueous solution, as long as the pH of the buffer solution reaches the target value; (3) The dental mesenchymal stem cell giant plasma membrane vesicle solution is repeatedly extruded through a polycarbonate membrane using a liposome extruder to obtain nano-sized dental mesenchymal stem cell vesicles (also referred to as mesenchymal stem cell membrane vesicles) with a particle size of no more than 200 nm.

[0022] The invention generates and separates giant plasma membrane vesicles of dental mesenchymal stem cells from dental mesenchymal stem cells by using a chemical vesicle stimulator, and then repeatedly extrude the vesicles through a polycarbonate membrane to obtain nano-sized dental mesenchymal stem cell vesicles. Compared with the traditional method of extracting extracellular vesicles from cell culture supernatant, the method of the invention can obtain vesicles with higher yield and higher purity. The paraformaldehyde solution contained in the chemical vesicle stimulator used in the invention can induce non-specific cross-linking of proteins in cell membranes, and the contained dithiothreitol can be used as a small molecule reducing agent to cut thioester bonds, thereby promoting cell membrane foaming to form giant plasma membrane vesicles of dental mesenchymal stem cells GPMV and fall off into the supernatant. At the same time, the buffer solution contained in the chemical vesicle stimulator has calcium ions Ca 2+ The present invention found that in Ca 2+ Under the condition of presence of Ca, cells were exposed to paraformaldehyde solution and dithiothreitol solution, which can make continuous Ca 2+ Inflow is conducive to promoting cell plasma membrane blistering. The chemical vesicle stimulator used in the present invention can make the giant plasma membrane vesicle GPMV of dental mesenchymal stem cells produced by the present invention contain no cytoskeleton or nuclear material. Directly extruding the cell membrane or extracting the cell membrane after lysing the cell will inevitably be contaminated by organelles or nuclear material. Therefore, compared with other cell membrane vesicle production methods, the present invention method produces purer cell membrane vesicles. The nano-sized dental mesenchymal stem cell vesicles prepared by the present invention have homologous targeting similar to MSCs, targeting bone tissue, and high efficiency of transport to target tissue in vivo, and can prolong the in vivo circulation time of the drug, realize targeted delivery and sustained release of the drug in bone tissue, thereby enhancing bone regeneration ability.

[0023] According to some preferred embodiments, in step (1), the method for obtaining the dental mesenchymal stem cells is: The periodontal ligament tissue or dental pulp tissue was digested and centrifuged, the supernatant was discarded, and then α-MEM culture medium containing fetal bovine serum and penicillin-streptomycin was added and mixed evenly. Then, the tissue was cultured in a CO2 constant temperature incubator until the primary stem cells reached 80%~90% fusion, and then digested with trypsin-EDTA digestion solution and cultured and expanded (in vitro culture expansion) at a ratio of 1:2 or 1:3 to P4~P6 generations to obtain dental mesenchymal stem cells.

[0024] According to some preferred embodiments, the volume fraction of fetal bovine serum contained in the α-MEM culture medium is 10-20%, preferably 15%, and the concentration (volume fraction) of penicillin-streptomycin contained in the α-MEM culture medium is 0.8-1.2% (for example, 0.8%, 0.9%, 1%, 1.1% or 1.2%); in the present invention, the penicillin-streptomycin is, for example, commercially available penicillin-streptomycin dual antibody (10000U / mL); the volume fraction of CO2 contained in the CO2 constant temperature incubator is 5%, the temperature of the CO2 constant temperature incubator is 37°C, and the humidity is 100%; and / or the trypsin-EDTA digestion solution is a digestion solution containing 0.25wt% trypsin and 0.1wt% EDTA (ethylenediaminetetraacetic acid); in the present invention, the digestion solution containing 0.25wt% trypsin and 0.1wt% EDTA is recorded as 0.25% trypsin / 0.1%EDTA.

[0025] According to some preferred embodiments, the dental mesenchymal stem cells are periodontal ligament mesenchymal stem cells or dental pulp mesenchymal stem cells.

[0026] According to some specific embodiments, obtaining periodontal ligament mesenchymal stem cells (PDLSCs) from periodontal ligament tissue as raw material comprises the following steps: S1. After the tooth is removed from the body, it is placed in a PBS buffer containing 2 vol% penicillin-streptomycin, transferred to a clean bench, and repeatedly rinsed with a PBS buffer containing 2 vol% penicillin-streptomycin in the clean bench. Then, 1 / 3 of the periodontal ligament tissue in the tooth root is scraped with a scalpel while moistening with a serum-free cell culture medium (i.e., serum-free cell culture medium); in the present invention, unless otherwise specified, the PBS buffer used is a 1×PBS buffer with a pH of 7.4; S2. Cut the scraped periodontal ligament tissue into small pieces of 1 mm (length) × 1 mm (width) × 1 mm (height), and place them in a mixed enzyme solution containing collagenase I and neutral protease II for digestion at 37°C for 45 to 60 minutes; in the present invention, the mixed enzyme solution containing collagenase I and neutral protease II is formed by mixing a collagenase I solution and a neutral protease II solution, the collagenase I solution uses α-MEM culture medium (i.e., α-MEM liquid culture medium) as a solvent, the concentration of collagenase I in the collagenase I solution is 8 mg / mL, the neutral protease II solution uses PBS buffer as a solvent, the concentration of neutral protease II in the neutral protease II solution is 6 mg / mL, and the volume ratio of the collagenase I solution to the neutral protease II solution is 1:1; S3, terminating the digestion in step S2 with a serum-containing α-MEM medium, and then centrifuging at 300g for 3 min; in the present invention, the serum-containing α-MEM medium can be, for example, an α-MEM medium containing 10-20 vol% fetal bovine serum; in some specific embodiments, the serum-containing α-MEM medium is an α-MEM medium containing 15 vol% fetal bovine serum; S4. After centrifugation in step S3, discard the supernatant, add an appropriate amount (e.g., 3-4 mL) of α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin by volume, mix well by pipetting, and place in a culture dish with a diameter of 6 cm, and culture in a CO2 constant temperature incubator (containing 5% CO2 by volume, 37° C., 100% humidity); in some specific embodiments of the present invention, the primary cells with the supernatant discarded are placed in a cell culture dish with a diameter of 6 cm, and each cell culture dish finally contains 3 mL of α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin by volume; S5. Replace the medium every 2-3 days (i.e., replace the α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin). When the cells reach 80-90% confluence, digest them with 0.25% trypsin / 0.1% EDTA at 37°C for 60-80s, then passage them at a ratio of 1:2 or 1:3, and culture and expand them in vitro to P4-P6 to obtain uniform periodontal ligament mesenchymal stem cells (PDLSCs).

[0027] According to some specific embodiments, obtaining dental pulp mesenchymal stem cells (DPSCs) using dental pulp tissue as raw material comprises the following steps: S1. After the tooth is removed from the body, the tooth tissue is split with a dental turbine handpiece, and then placed in a PBS buffer containing 2 vol% penicillin-streptomycin, and then transferred to an ultra-clean bench. After repeated rinsing with a PBS buffer containing 2 vol% penicillin-streptomycin in the ultra-clean bench, the pulp tissue is separated from the pulp cavity with a small fine forceps or a probe while moistening with a serum-free cell culture medium (i.e., serum-free cell culture medium); S2. Cut the separated pulp tissue into small pieces of 1 mm (length) × 1 mm (width) × 1 mm (height), and place them in a mixed enzyme solution containing collagenase I and neutral protease II for digestion at 37°C for 45 to 60 minutes; in the present invention, the mixed enzyme solution containing collagenase I and neutral protease II is formed by mixing a collagenase I solution and a neutral protease II solution, the collagenase I solution uses α-MEM culture medium (i.e., α-MEM liquid culture medium) as a solvent, the concentration of collagenase I in the collagenase I solution is 8 mg / mL, the neutral protease II solution uses PBS buffer as a solvent, the concentration of neutral protease II in the neutral protease II solution is 6 mg / mL, and the volume ratio of the collagenase I solution to the neutral protease II solution is 1:1; S3, terminating the digestion in step S2 with a serum-containing α-MEM medium, and then centrifuging at 300g for 3 min; in the present invention, the serum-containing α-MEM medium can be, for example, an α-MEM medium containing 10-20 vol% fetal bovine serum; in some specific embodiments, the serum-containing α-MEM medium is an α-MEM medium containing 15 vol% fetal bovine serum; S4. After centrifugation in step S3, discard the supernatant, add an appropriate amount (e.g., 3-4 mL) of α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin by volume, mix well by pipetting, and place in a culture dish with a diameter of 6 cm, and culture in a CO2 constant temperature incubator (containing 5% CO2 by volume, 37° C., 100% humidity); in some specific embodiments of the present invention, the primary cells with the supernatant discarded are placed in a cell culture dish with a diameter of 6 cm, and each cell culture dish finally contains 3 mL of α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin by volume; S5. Replace the medium every 2-3 days (i.e., replace the α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin). When the cells reach 80-90% confluence, digest them with 0.25% trypsin / 0.1% EDTA at 37°C for 60-80s, then passage them at a ratio of 1:2 or 1:3, and culture and expand them in vitro to P4-P6 to obtain uniform dental pulp mesenchymal stem cells (DPSCs).

[0028] According to some preferred embodiments, in step (2): the concentration of the dithiothreitol aqueous solution is 150-160 mg / mL (for example, 150, 152, 154, 156, 158 or 160 mg / mL); the paraformaldehyde solution is 4% paraformaldehyde fixative (4% PFA); and / or the volume ratio of the buffer, the dithiothreitol aqueous solution and the paraformaldehyde solution is 1000:(1.5-2.5):(16-20) (for example, 1000:1.5:16, 1000:1.5:18, 1000:1.5:20, 1000:2:16, 1000:2:18, 1000:2:20, 1000:2.5:16, 1000:2.5:18 or 1000:2.5:20).

[0029] In the present invention, the rational selection of the components of the chemical vesicle stimulator and the optimization of their concentrations and ratios are helpful to obtain high-concentration, high-purity, high-quality giant plasma membrane vesicles of dental mesenchymal stem cells, thereby improving the efficiency of the nano-sized dental mesenchymal stem cell vesicles finally obtained in targeting bone tissue, prolonging the in vivo circulation time of the drug, and enhancing the bone regeneration ability; wherein the NaCl at a concentration of 8.5-9 g / L contained in the buffer is beneficial to maintaining the extracellular osmotic pressure and preventing cell stress caused by osmotic pressure changes, and the CaCl2 at a concentration of 0.2-0.24 g / L is beneficial to promoting the bone regeneration by providing calcium ions. The cell membrane blebs and promotes the formation and release of giant plasma membrane vesicles. If the concentration of CaCl2 is too low, the cell's ability to respond to chemical stimulation will be weakened, thereby reducing the formation of giant plasma membrane vesicles. If the concentration of CaCl2 is too high, it will lead to excessive activation of intracellular calcium-dependent enzymes and induce cell apoptosis. HEPES with a concentration of 2.3-2.4 g / L can avoid plasma membrane damage caused by pH fluctuations during vesicle formation. In addition, the chemical vesicle stimulator also contains a suitable concentration of dithiothreitol, which is conducive to cutting thioester bonds and promoting cell membrane blebbing, which is sufficient to induce the formation of a large number of giant plasma membrane vesicles in a short time, but It will not cause excessive cell lysis or non-specific reaction, thus helping to maintain the purity and structural integrity of the vesicles. The chemical vesicle stimulator also contains a suitable concentration of paraformaldehyde, which can effectively fix the structure of the vesicles while inducing cell vesicle formation relative to a low-concentration aldehyde solution, making the structure of the vesicles more stable. The high-purity and high-integrity mesenchymal stem cell membrane vesicles of the present invention can more effectively interact with target tissues (such as bone tissue). The natural mesenchymal stem cell membrane proteins and lipid molecules retained on the vesicle membrane help the mesenchymal stem cell membrane vesicles to exert the characteristics of homing to bone tissue similar to those of the parent cells, thereby interacting with the characteristics of bone tissue. Heterotropic receptor binding improves targeting efficiency, and the present invention uses high-purity and structurally intact vesicles as drug carriers to avoid rapid drug failure due to nonspecific degradation or rapid clearance by the immune system, and can remain in the body for a long time to avoid early release or degradation, thereby extending the in vivo half-life of the drug. The present invention can accurately deliver more drugs or therapeutic factors to bone tissue through the high targeting of vesicles and the extended drug circulation time, which is beneficial to enhancing the bone regeneration effect. By extending the release time and concentration of the drug in bone tissue, it can provide a sustained therapeutic effect, which is also beneficial to the repair and regeneration of bone tissue.

[0030] According to some preferred embodiments, in step (2): the concentration of the dithiothreitol aqueous solution is 154 mg / mL; the volume ratio of the buffer solution, the dithiothreitol aqueous solution and the paraformaldehyde solution is 1000:2:18; and / or the buffer solution comprises 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid and the remainder deionized water.

[0031] According to some preferred embodiments, in step (2): the volume fraction of CO2 contained in the CO2 constant temperature incubator is 5%, the temperature of the CO2 constant temperature incubator is 37°C, and the humidity is 100%; the stimulation time is 18-30 hours (for example, 18, 20, 22, 24, 26, 28 or 30 hours); and / or the amount of the chemical vesicle stimulator added is 18-25 microliters of the chemical vesicle stimulator per square centimeter of dental mesenchymal stem cells; and / or the particle size of the dental mesenchymal stem cell giant plasma membrane vesicles in the dental mesenchymal stem cell giant plasma membrane vesicle solution is 5-28 μm, and the concentration of the dental mesenchymal stem cell giant plasma membrane vesicles is 6.0×10 6 ~6.7×10 6 The purity is not less than 95%, preferably not less than 99%.

[0032] According to some specific embodiments, step (2) is: select PDLSCs and / or DPSCs cultured in vitro to P4-P6, when the cells reach 90% fusion, discard the culture medium, rinse with PBS buffer for 3 times, and then use 20uL / cm 2 A chemical vesicle stimulator was added, and the cells were stimulated in a CO2 constant temperature incubator (CO2 volume fraction of 5%, 37°C, 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution of dental mesenchymal stem cells; wherein the chemical vesicle stimulator was prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:18, the buffer solution used deionized water as a solvent, contained 8.75 g / L sodium chloride, 0.22 g / L calcium dichloride, and 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution was a buffer solution with a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution used deionized water as a solvent, contained a dithiothreitol concentration of 154 mg / mL, and the paraformaldehyde solution was 4% PFA.

[0033] According to some specific embodiments, micron-sized dental mesenchymal stem cell giant plasma membrane vesicles GPMV with a particle size distribution range of 5 to 28 μm and an average particle size of 12.25 μm were obtained, and the vesicle concentration was 6.0×106 ~6.7×10 6 / mL, and the protein concentration determined by A280 (nm) ultraviolet absorption method is 1-1.5 mg / mL; the present invention obtains high-concentration GPMV, which is conducive to obtaining high-yield vesicles after nano-sizing.

[0034] According to some preferred embodiments, in step (3): the number of repeated extrusions is 8 to 12 times (e.g., 8, 9, 10, 11 or 12 times), preferably 10 times; and / or the polycarbonate membrane is a polycarbonate membrane with a pore size of 200 nm.

[0035] According to some specific embodiments, step (3) is: using a liposome extruder, repeatedly extruding the dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution) through a 200nm polycarbonate membrane 10 times to obtain nanosized dental mesenchymal stem cell vesicles with a particle size of less than 200nm, recorded as nMSC.

[0036] According to some preferred embodiments, in step (3): before repeatedly extruding through a polycarbonate membrane using a liposome extruder, the dental mesenchymal stem cell giant plasma membrane vesicle solution is first mixed with a DPP4 inhibitor (i.e., a dipeptidyl peptidase 4 inhibitor); in the present invention, the dental mesenchymal stem cell giant plasma membrane vesicle solution contains a protein concentration of, for example, 1 to 1.5 mg / mL; the dental mesenchymal stem cell giant plasma membrane vesicle solution and the DPP4 inhibitor (DPP-4 inhibitor) are mixed, for example, by first mixing with deionized water; A DPP4 inhibitor solution with a concentration of 2 mg / mL is prepared, and then 100 μL of the DPP4 inhibitor solution is added to every 1000 μL of the dental mesenchymal stem cell giant plasma membrane vesicle solution with a protein concentration of 1 mg / mL, and the DPP4 inhibitor solution is added to the dental mesenchymal stem cell giant plasma membrane vesicle solution and mixed evenly; in the present invention, the protein concentration of the dental mesenchymal stem cell giant plasma membrane vesicle solution is measured by A280 (nm) ultraviolet absorption method (i.e., 280 nanometer ultraviolet absorption method).

[0037] According to some specific embodiments, step (3) is: firstly, a DPP4 inhibitor Sitagliptin solution is mixed with a dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution), and then the mixture is repeatedly co-extruded 10 times through a liposome extruder through a 200 nm polycarbonate membrane to obtain nano-sized dental mesenchymal stem cell vesicles loaded with a DPP4 inhibitor, which are recorded as nMSC-DPP4i.

[0038] In a second aspect, the present invention provides nanosized dental mesenchymal stem cell vesicles prepared by the preparation method described in the first aspect of the present invention.

[0039] The third aspect of the present invention provides the use of nano-sized dental mesenchymal stem cell vesicles prepared by the preparation method described in the first aspect of the present invention in bone tissue repair materials.

[0040] The present invention will be further described below by way of examples, but the protection scope of the present invention is not limited to these embodiments. The present invention may also have other various embodiments, and those skilled in the art may make various corresponding changes and deformations according to the present invention without departing from the spirit and essence of the present invention, but these corresponding changes and deformations shall all belong to the protection scope of the claims attached to the present invention.

[0041] Example 1 ① Preparation of periodontal ligament mesenchymal stem cells PDLSCs: After the tooth is removed from the body, it is placed in a PBS buffer containing 2 vol% penicillin-streptomycin, and then transferred to a clean bench. After repeated rinsing with a PBS buffer containing 2 vol% penicillin-streptomycin in the clean bench, 1 / 3 of the periodontal ligament tissue in the tooth root is scraped with a scalpel while moistening with a serum-free cell culture medium (i.e., serum-free cell culture medium); the scraped periodontal ligament tissue is cut into small pieces of 1 mm (length) × 1 mm (width) × 1 mm (height), and placed in a mixed enzyme solution containing collagenase I and neutral protease II for digestion at 37°C for 60 min; the digestion in step S2 is terminated with a serum-containing α-MEM medium, and then centrifuged at 300g for 3 min. The serum-containing α-MEM medium is a medium containing 15 vol% fetal bovine blood. α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin was added after centrifugation, and the mixture was mixed by pipetting and placed in a 6 cm culture dish, and cultured in a CO2 constant temperature incubator (containing 5% CO2, 37°C, 100% humidity); α-MEM medium containing 15% fetal bovine serum and 1% penicillin-streptomycin was replaced every 2 days. When the cells reached 80% fusion, they were digested with 0.25% trypsin / 0.1% EDTA at 37°C for 1 min, and then passaged at a ratio of 1:3. The cells were cultured and expanded in vitro to the P6 generation to obtain uniform periodontal ligament mesenchymal stem cells (PDLSCs).

[0042] ② Preparation of dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution): Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2A chemical vesicle stimulator was added, and the cells were stimulated in a CO2 constant temperature incubator (CO2 volume fraction of 5%, 37°C, 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes, and the supernatant was taken to obtain a giant plasma membrane vesicle solution of dental mesenchymal stem cells; wherein the chemical vesicle stimulator was prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:18, the buffer solution used deionized water as a solvent, and contained 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, and 4-hydroxyethyl The invention relates to a method for preparing a tooth-derived mesenchymal stem cell giant plasma membrane vesicle (DMS) containing 2.38 g / L of 1,2-dithiothreitol piperazine ethanesulfonic acid, and the buffer solution is a buffer solution with a pH value of 7.4 after being adjusted with a NaOH aqueous solution; the dithiothreitol aqueous solution uses deionized water as a solvent, and contains 154 mg / mL of dithiothreitol; the paraformaldehyde solution is 4% PFA; in this step, giant plasma membrane vesicles of dental mesenchymal stem cells with a particle size distribution range of 5 to 28 μm and an average particle size of 12.25 μm are obtained, and the concentration of the giant plasma membrane vesicles of dental mesenchymal stem cells in the giant plasma membrane vesicle solution is 6.7×10 6 / mL, and the protein concentration was 1.5mg / mL determined by A280 (nm) UV absorption method.

[0043] ③ Preparation of Nano-sized Dental Mesenchymal Stem Cell Vesicles: The dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution) obtained in step ② was repeatedly extruded through a 200 nm polycarbonate membrane 10 times using a liposome extruder to obtain nanosized dental mesenchymal stem cell vesicles with a particle size less than 200 nm, which were recorded as nMSC.

[0044] The present invention observed the morphology of the giant plasma membrane vesicle solution of dental mesenchymal stem cells obtained in this embodiment through an ordinary optical microscope, as shown in FIG. Figure 1 As shown in FIG. 1 , the morphology of the giant plasma membrane vesicle solution of dental mesenchymal stem cells obtained in this example after being labeled with fluorescent dye DiI and thioflavin T was observed by confocal microscopy, as shown in FIG. Figure 2 shown.

[0045] The present invention also uses fluorescent dye to label the nano-sized dental mesenchymal stem cell vesicles nMSC prepared in this embodiment, and performs small animal fluorescence imaging 24 hours after mouse tail vein injection to observe the enrichment of nMSC in bone tissue. The results are as follows: Figure 3 The average fluorescence intensity is shown in Table 1. Figure 3 It can be seen that compared with the free fluorescent dye DiR, the nanostructured dental mesenchymal stem cell vesicles (nMSC-DiR) labeled with the fluorescent dye DiR showed stronger fluorescence intensity in bone tissue, indicating that nMSC can efficiently target bone tissue and be enriched in bone tissue.

[0046] Table 1: Fluorescence intensity of nMSC-DiR labeled with fluorescent dye DiR in bone tissue.

[0047]

[0048] Example 2 Embodiment 2 is substantially the same as Embodiment 1, except that: ③ Preparation of Nano-sized Dental Mesenchymal Stem Cell Vesicles Loaded with DPP4 Inhibitor: A DPP4 inhibitor (sitagliptin) solution with a concentration of 2 mg / mL was prepared with deionized water, and then 150 μL of the DPP4 inhibitor solution was added to every 1000 μL of the dental mesenchymal stem cell giant plasma membrane vesicle solution with a protein concentration of 1.5 mg / mL. The DPP4 inhibitor solution was added to the dental mesenchymal stem cell giant plasma membrane vesicle solution obtained in step ② and mixed evenly. The solution was then repeatedly co-extruded 10 times through a liposome extruder through a 200 nm polycarbonate membrane to obtain nano-sized dental mesenchymal stem cell vesicles loaded with DPP4 inhibitor, recorded as nMSC-DPP4i.

[0049] The transmission electron microscopy image of the nano-sized dental mesenchymal stem cell vesicle loaded with DPP4 inhibitor obtained in this example (scale bar, 200 nm) is as follows Figure 4 shown.

[0050] The present invention measured the particle size, polydispersity index and Zeta potential characteristic results of the nMSC prepared in Example 1 and the nMSC-DPP4i prepared in Example 2, as shown in Table 2.

[0051] Table 2: Particle size, polydispersity index and zeta potential characteristics of nMSCs and nMSC-DPP4i.

[0052]

[0053] The present invention established an osteoporosis animal model by removing both ovaries of mice, and gave treatment (100uL / mouse) according to different groups. After 4 weeks of intravenous administration, the femoral bone mass was detected by Micro-CT. The results were as follows Figure 5 As shown; grouping: bilateral oophorectomy group (OVX), bilateral oophorectomy post-intravenous injection of nMSC group in Example 1, bilateral oophorectomy post-intravenous injection of DPP4i group, bilateral oophorectomy post-intravenous injection of nMSC-DPP4i group in Example 2; from Figure 5It can be seen that compared with OVX, nMSC group and DPP4i group, nMSC-DPP4i group can significantly increase bone mass; in the present invention, nMSC and nMSC-DPP4i are dialyzed with PBS buffer before application to animals (PBS buffer dosage during dialysis: 1000 mL / time, replaced every 30-40 minutes, a total of 4 dialysis times) to remove residual dithiothreitol and paraformaldehyde. For nMSC-DPP4i, dialysis can also remove unencapsulated (loaded) DPP4 inhibitors.

[0054] Example 3 Embodiment 3 is substantially the same as Embodiment 1, except that: ② Preparation of dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution): Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 A chemical vesicle stimulator was added, and the cells were stimulated in a CO2 constant temperature incubator (CO2 volume fraction of 5%, 37°C, 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution of dental mesenchymal stem cells; wherein the chemical vesicle stimulator was prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:4:30, the buffer solution used deionized water as a solvent, contained 8.75 g / L sodium chloride, 0.22 g / L calcium dichloride, and 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution was a buffer solution with a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution used deionized water as a solvent, contained a dithiothreitol concentration of 180 mg / mL, and the paraformaldehyde solution was 4% PFA.

[0055] Example 4 Embodiment 4 is substantially the same as Embodiment 1, except that: ② Preparation of dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution): Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2A chemical vesicle stimulator was added, and the cells were stimulated in a CO2 constant temperature incubator (CO2 volume fraction of 5%, 37°C, 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution of dental mesenchymal stem cells; wherein the chemical vesicle stimulator was prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:10, the buffer solution used deionized water as a solvent, contained 8.75 g / L sodium chloride, 0.22 g / L calcium dichloride, and 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution was a buffer solution with a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution used deionized water as a solvent, contained a dithiothreitol concentration of 120 mg / mL, and the paraformaldehyde solution was 4% PFA.

[0056] Comparative Example 1 Comparative Example 1 is substantially the same as Example 1, except that: ② Preparation of dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution): Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 A chemical vesicle stimulator was added, and the cells were stimulated in a CO2 constant temperature incubator (CO2 volume fraction of 5%, 37°C, 100% humidity) for 24 hours, and the supernatant was collected. The cells were centrifuged at 100g for 3 minutes to remove cell debris, and the supernatant was taken to obtain a giant plasma membrane vesicle solution of dental mesenchymal stem cells; wherein the chemical vesicle stimulator was prepared from a buffer solution and a paraformaldehyde solution in a volume ratio of 1000:18, the buffer solution used deionized water as a solvent, and contained 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, and 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution was a buffer solution with a pH value of 7.4 after adjustment with a NaOH aqueous solution; the paraformaldehyde solution was 4% PFA.

[0057] Comparative Example 2 Comparative Example 2 is substantially the same as Example 1, except that: ② Preparation of dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution): Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2A chemical vesicle stimulator was added, and the cells were stimulated in a CO2 constant temperature incubator (CO2 volume fraction of 5%, 37°C, 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution of dental mesenchymal stem cells; wherein the chemical vesicle stimulator was prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:18, the buffer solution used deionized water as a solvent, contained 8.75 g / L of sodium chloride, 2.38 g / L of 4-hydroxyethylpiperazineethanesulfonic acid, and the pH value of the buffer solution after adjustment with a NaOH aqueous solution was 7.4; the dithiothreitol aqueous solution used deionized water as a solvent, contained 154 mg / mL of dithiothreitol, and the paraformaldehyde solution was 4% PFA.

[0058] Comparative Example 3 Comparative Example 3 is substantially the same as Example 1, except that: ② Preparation of dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution): Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2 A chemical vesicle stimulator was added, and the cells were stimulated in a CO2 constant temperature incubator (CO2 volume fraction of 5%, 37°C, 100% humidity) for 24 hours, and the supernatant was collected. The cells were centrifuged at 100g for 3 minutes to remove cell debris, and the supernatant was taken to obtain a giant plasma membrane vesicle solution of dental mesenchymal stem cells; wherein the chemical vesicle stimulator was prepared from deionized water and paraformaldehyde solution in a volume ratio of 1000:18, and the paraformaldehyde solution was 4% PFA.

[0059] Comparative Example 4 Comparative Example 4 is substantially the same as Example 1, except that: ② Preparation of dental mesenchymal stem cell giant plasma membrane vesicle solution (GPMV solution): Step ① Culture and expand PDLSCs to P6 in vitro. When the cells reach 90% confluence, discard the culture medium, rinse with PBS buffer 3 times, and then rinse at a dosage of 20uL / cm 2A chemical vesicle stimulator was added, and the cells were stimulated in a CO2 constant temperature incubator (CO2 volume fraction of 5%, 37°C, 100% humidity) for 24 hours, the supernatant was collected, and the cell debris was removed by centrifugation at 100g for 3 minutes. The supernatant was taken to obtain a giant plasma membrane vesicle solution of dental mesenchymal stem cells; wherein the chemical vesicle stimulator was prepared by a buffer solution, a dithiothreitol aqueous solution, and a paraformaldehyde solution in a volume ratio of 1000:2:18, the buffer solution used deionized water as a solvent, contained 8.75 g / L sodium chloride, 13.5 g / L calcium chloride, and 2.6 g / L 4-hydroxyethylpiperazineethanesulfonic acid, and the buffer solution was a buffer solution with a pH value of 7.4 after adjustment with a NaOH aqueous solution; the dithiothreitol aqueous solution used deionized water as a solvent, contained a dithiothreitol concentration of 154 mg / mL, and the paraformaldehyde solution was 4% PFA.

[0060] Comparative Example 5 ①Same as step ① of Example 1.

[0061] ② Preparation of nanovesicles: Select the periodontal ligament mesenchymal stem cells PDLSCs that were cultured and expanded in vitro to P6 in step ①. When the cells reached 90% fusion, they were digested with a digestion solution containing 0.25wt% trypsin at 37°C for 1 min, centrifuged, and then the collected periodontal ligament mesenchymal stem cells were resuspended in PBS buffer to obtain a periodontal ligament mesenchymal stem cell PDLSCs suspension; the periodontal ligament mesenchymal stem cell PDLSCs suspension was extruded successively using a liposome extruder with 800nm, 400nm and 200nm polycarbonate membranes, and then repeatedly extruded 7 times through a 200nm polycarbonate membrane to obtain nanovesicles.

[0062] In this comparative example, it is not possible to directly use a 200nm polycarbonate membrane to repeatedly extrude the periodontal ligament mesenchymal stem cell PDLSCs suspension, as the cells are too large to be extruded smoothly; and although this comparative example is first extruded through 800nm ​​and 400nm polycarbonate membranes and then extruded through a 200nm polycarbonate membrane, which can avoid the problem of too large cell size, it is impossible to avoid the contamination of cell membrane vesicles by organelles or nuclear substances during the extrusion process, which will have undesirable side effects on subsequent therapeutic effects.

[0063] The present invention tests the concentration and purity of giant plasma membrane vesicles of dental mesenchymal stem cells in the giant plasma membrane vesicle solutions of dental mesenchymal stem cells obtained in Example 1, Examples 3-4 and Comparative Examples 1-4, and the results are shown in Table 3.

[0064] Table 3: Concentration and purity of giant plasma membrane vesicle solutions of dental mesenchymal stem cells obtained in Example 1, Examples 3-4 and Comparative Examples 1-4.

[0065]

[0066] The present invention also tests the plasma biological half-life (T) of the drug after the nano-sized dental mesenchymal stem cell vesicles obtained in Example 1 of the present invention encapsulate the simulated free drug (fluorescent dye). 1 / 2 The test method is as follows: a fluorescent dye is used to simulate free drugs, which are encapsulated by the nano-sized dental mesenchymal stem cell vesicles (nMSC) prepared in Example 1, and blood is collected from the orbital vein at 5 minutes, 30 minutes, 4 hours, 8 hours, 24 hours, 48 ​​hours and 72 hours after administration via tail vein injection of mice, followed by small animal imaging, and the fluorescence intensity is used to calculate the plasma biological half-life of the drug (T 1 / 2 ), T 1 / 2 =0.693 / k e (k e is the elimination rate constant), and the T of the free fluorescent dye was measured. 1 / 2 The T of the fluorescent dye encapsulated by nMSCs was 20.12 hours. 1 / 2 The result shows that the nMSC prepared by the present invention can significantly prolong the in vivo circulation time of the drug, realize the targeted delivery and sustained release of the drug in bone tissue, and can be better applied to bone tissue repair and regeneration.

[0067] The present invention also makes the nano-sized dental mesenchymal stem cell vesicles prepared in Examples 3-4 and the vesicles finally prepared in Comparative Examples 1-4 loaded with DPP4 inhibitor (sitagliptin) by the same method as in Example 2, and also establishes an osteoporosis animal model by removing both ovaries of mice for drug treatment (100uL / mouse), and detects femoral bone mass by Micro-CT after 4 weeks of intravenous administration, and the results of femoral bone mass are shown in Table 4; in the present invention, after the nano-sized dental mesenchymal stem cell vesicles prepared in Examples 3-4 and the vesicles finally prepared in Comparative Examples 1-4 are loaded with DPP4 inhibitor, and before being applied to animals, they are also dialyzed with PBS buffer (PBS buffer dosage during dialysis: 1000mL / time, replaced every 30-40min, and dialyzed 4 times in total) to remove residual dithiothreitol, paraformaldehyde and remove the unencapsulated (loaded) DPP4 inhibitor.

[0068] Table 4: Femoral bone mass results of each experimental group.

[0069]

[0070] Parts of the present invention that are not described in detail are well known to those skilled in the art.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing nano-sized dental mesenchymal stem cell vesicles, characterized in that: The preparation method comprises the following steps: (1) Obtaining dental mesenchymal stem cells; (2) adding a chemical vesicle stimulator to the dental mesenchymal stem cells and placing the cells in a CO2 constant temperature incubator for stimulation to obtain a giant plasma membrane vesicle solution of the dental mesenchymal stem cells; the chemical vesicle stimulator comprises a buffer solution, a dithiothreitol aqueous solution and a paraformaldehyde solution; the buffer solution comprises 8.5-9 g / L sodium chloride, 0.2-0.24 g / L calcium chloride, 2.3-2.4 g / L 4-hydroxyethylpiperazineethanesulfonic acid and deionized water; (3) The dental mesenchymal stem cell giant plasma membrane vesicle solution is repeatedly extruded through a polycarbonate membrane using a liposome extruder to obtain nanosized dental mesenchymal stem cell vesicles with a particle size of no more than 200 nm.

2. The preparation method according to claim 1, characterized in that: In step (1), the method for obtaining the dental mesenchymal stem cells is: The periodontal ligament tissue or dental pulp tissue was digested and centrifuged, the supernatant was discarded, and then α-MEM culture medium containing fetal bovine serum and penicillin-streptomycin was added and mixed evenly. Then, it was cultured in a CO2 constant temperature incubator until the primary stem cells reached 80%~90% fusion, and then digested with trypsin-EDTA digestion solution and cultured and expanded to P4~P6 generations at a ratio of 1:2 or 1:3 to obtain dental mesenchymal stem cells.

3. The preparation method according to claim 2, characterized in that: The volume fraction of fetal bovine serum contained in the α-MEM medium is 10-20%, and the volume fraction of penicillin-streptomycin contained in the α-MEM medium is 0.8-1.2%; The volume fraction of CO2 in the CO2 constant temperature incubator is 5%, the temperature of the CO2 constant temperature incubator is 37°C, and the humidity is 100%; and / or The trypsin-EDTA digestion solution is a digestion solution containing 0.25 wt % of trypsin and 0.1 wt % of EDTA.

4. The preparation method according to claim 1, characterized in that: In step (2): The concentration of the dithiothreitol aqueous solution is 150-160 mg / mL; The paraformaldehyde solution is a 4% paraformaldehyde fixative; and / or The volume ratio of the buffer solution, the dithiothreitol aqueous solution and the paraformaldehyde solution is 1000:(1.5-2.5):(16-20).

5. The preparation method according to claim 4, characterized in that: In step (2): The concentration of the dithiothreitol aqueous solution is 154 mg / mL; The volume ratio of the buffer solution, the dithiothreitol aqueous solution and the paraformaldehyde solution is 1000:2:18; and / or The buffer solution comprises 8.75 g / L sodium chloride, 0.22 g / L calcium chloride, 2.38 g / L 4-hydroxyethylpiperazineethanesulfonic acid and deionized water.

6. The preparation method according to claim 1, characterized in that: In step (2): The volume fraction of CO2 contained in the CO2 constant temperature incubator is 5%, the temperature of the CO2 constant temperature incubator is 37°C, and the humidity is 100%; The stimulation time is 18 to 30 hours; and / or The amount of the chemical vesicle stimulator added is 18-25 microliters of the chemical vesicle stimulator per square centimeter of dental mesenchymal stem cells; and / or The particle size of the giant plasma membrane vesicles of dental mesenchymal stem cells in the giant plasma membrane vesicle solution of dental mesenchymal stem cells is 5-28 μm, and the concentration of the giant plasma membrane vesicles of dental mesenchymal stem cells is 6.0×10 6 ~6.7×10 6 Pieces / mL.

7. The preparation method according to claim 1, characterized in that: In step (3): The number of repeated extrusions is 8 to 12 times; and / or The polycarbonate membrane is a polycarbonate membrane with a pore size of 200 nm.

8. The preparation method according to claim 1, characterized in that: In step (3): The dental mesenchymal stem cell giant plasma membrane vesicle solution was mixed with the DPP4 inhibitor before repeated extrusion through a polycarbonate membrane using a liposome extruder.

9. Nanosized dental mesenchymal stem cell vesicles prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the nanosized dental mesenchymal stem cell vesicles prepared by the preparation method according to any one of claims 1 to 8 in bone tissue repair materials.

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