Dental sac mesenchymal stem cells with high periostin expression, and extraction and culture method and application of dental sac mesenchymal stem cells

Abstract

The invention discloses a dental sac mesenchymal stem cell with high periostin expression as well as an extraction and culture method and application thereof, and belongs to the technical field of biomedicine. The dental sac mesenchymal stem cell with high periostin expression is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2024384; the extraction and culture method disclosed by the invention comprises the following steps: S1, extraction: cleaning, cutting, digesting and centrifuging dental sac tissues, resuspending, centrifuging and precipitating by using a primary culture medium, culturing until cells are fused, digesting, and carrying out secondary centrifugation and precipitating to obtain primary dental sac mesenchymal stem cells; s2, subculture: resuspending the dental sac mesenchymal stem cells in the step S1 by using a primary culture medium, supplementing a subculture medium, and then subculturing. The invention also discloses the dental sac mesenchymal stem cell with high periostin expression and application of the dental sac mesenchymal stem cell in preparation of medicines for preventing or / and treating periodontal diseases. The method disclosed by the invention has an unexpected effect of enhancing periostin expression of the dental sac mesenchymal stem cells.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to dental follicle mesenchymal stem cells with high periostin expression, and an extraction and culture method and application thereof. Background Art

[0002] Stem cell therapy has become one of the most cutting-edge and promising directions in current disease treatment and tissue regeneration research. Finding ideal seed cells and efficient cell action systems in vivo is the key to achieving disease treatment and tissue regeneration. Among them, dental stem cells have attracted much attention due to their strong self-renewal and multidirectional differentiation abilities. Dental stem cells mainly include dental follicle mesenchymal stem cells (DFSCs), periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED). Many studies have confirmed that these stem cells have great application potential in tissue repair and disease treatment. Among many dental stem cells, dental follicle mesenchymal stem cells (DFSCs) are the only ones derived from tissues during tooth germ development. The dental follicle is a loose connective tissue layer that surrounds the enamel organ and the base of the dental papilla during the tooth germ period, originating from the ectodermal mesenchymal leaf. The cementum, periodontal ligament and alveolar bone in the periodontal tissue are all developed from the dental follicle. Clinically, the dental follicle tissue attached to the neck of the extracted impacted tooth is usually discarded as medical waste together with the tooth. Therefore, dental follicle tissue is an easily accessible source of stem cells that is harmless to the human body. It can be seen that compared with other dental stem cells, dental follicle mesenchymal stem cells have unique advantages and become an ideal source of seed cells for the treatment of various diseases.

[0003] With the widespread application of dental follicle mesenchymal stem cells (DFSCs), their extraction, culture and large-scale production have become particularly important. However, there are few reports in the existing technology on how to maintain or even enhance the relevant functions of DFSCs during the extraction and culture process. DFSCs are currently mostly used for the treatment of periodontal diseases. Studies have confirmed that DFSCs can promote periodontal regeneration in an inflammatory microenvironment, and this effect of promoting periodontal regeneration may be related to the high expression of periostin. Studies have shown that in response to inflammatory stimuli, DFSCs highly express periostin, recruit and reprogram macrophages, and improve the immune microenvironment of the defect area, while silencing periostin will weaken the role of DFSCs in promoting periodontal regeneration and regulating macrophages. Recombinant human periostin (rhPeriostin) can not only directly promote the reprogramming of macrophages through the integrinαM / phosphorylated extracellular signal-regulated kinase (p-Erk) / Erk signaling pathway, but also show the potential to promote periodontal regeneration in rats when loaded in a collagen matrix. These results suggest that periostin may be a molecular target for DFSCs to promote periodontal regeneration. However, there is no report in the prior art that only the extraction and culture methods are improved to enhance the expression of periostin in DFSCs.

[0004] Therefore, providing a method for extracting and culturing dental follicle mesenchymal stem cells, which can enhance the expression of periostin by dental follicle mesenchymal stem cells, has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] One of the purposes of the present invention is to provide a dental follicle mesenchymal stem cell that highly expresses periostin, has strong self-renewal and clone formation capabilities, has strong multidirectional differentiation ability, and can also highly express periostin.

[0006] The second object of the present invention is to provide a method for extracting and culturing dental follicle mesenchymal stem cells that highly express periostin.

[0007] The third object of the present invention is to provide the application of the dental follicle mesenchymal stem cells that highly express periostin.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention discloses a dental follicle mesenchymal stem cell that highly expresses periostin, which is a human dental follicle mesenchymal stem cell MSC, preserved in the China Center for Type Culture Collection, with a preservation number of CCTCCNO: C2024384, a preservation date of December 6, 2024, and a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0010] In a second aspect, the present invention discloses a method for extracting and culturing dental follicle mesenchymal stem cells that highly express periostin, the method comprising the following steps:

[0011] S1. Extraction: The dental follicle tissue is cleaned, cut into pieces, digested, and centrifuged. The centrifuged precipitate is resuspended in primary culture medium and cultured until the cells are fused. The precipitate is then digested and centrifuged again. The secondary centrifuged precipitate is the primary dental follicle mesenchymal stem cells.

[0012] S2. Subculture: resuspending the dental follicle mesenchymal stem cells in step S1 with a subculture medium for subculture;

[0013] The subculture medium is prepared by adding serum-free cell culture additives and vitamin C to MEMα medium; 24-26 mL of serum-free cell culture additives and 49-51 μL of vitamin C injection are added to every 100 mL of MEMα medium;

[0014] The primary culture medium is prepared by mixing gentamicin sulfate injection and subculture culture medium, wherein the content of gentamicin sulfate in the primary culture medium is 30-50 units / mL.

[0015] In some embodiments of the present invention, the preparation method of the subculture medium comprises the following steps: mixing 0.8-1.2 mL of vitamin C injection with 500 mL of serum-free cell culture additive to obtain a mixed solution; taking 20-30 mL of the mixed solution and mixing it with 500 mL of MEMα culture medium to obtain the subculture medium;

[0016] The preparation method of the primary culture medium comprises the following steps: uniformly mixing gentamicin sulfate injection and the subculture culture medium at a volume ratio of 0.8 to 1.2:1000 to obtain the primary culture medium.

[0017] Preferably, the preparation method of the subculture medium comprises the following steps: mixing 1.0 mL of vitamin C injection with 500 mL of serum-free cell culture additive to obtain a mixed solution; taking 25 mL of the mixed solution and mixing it with 500 mL of MEMα culture medium to obtain a subculture medium;

[0018] The preparation method of the primary culture medium comprises the following steps: uniformly mixing the gentamicin sulfate injection with the subculture culture medium to obtain the primary culture medium, wherein the content of gentamicin sulfate in the primary culture medium is 30 to 50 units / mL, preferably 40 units / mL.

[0019] In some embodiments of the present invention, the serum-free cell culture additive is UltraGRO TM -Advanced;

[0020] In some embodiments of the present invention, the MEMα medium contains nucleosides but does not contain phenol red.

[0021] In some embodiments of the present invention, the specification of vitamin C injection is 2mL:0.5g.

[0022] In some embodiments of the present invention, the specification of gentamicin sulfate injection is 2 mL: 80,000 units or 1 mL: 40,000 units.

[0023] In some embodiments of the present invention, in step S1, the cells are cultured until the cell confluence is 70% or more, preferably 70-90%.

[0024] In some embodiments of the present invention, in step S1, the minced dental follicle tissue is digested with collagenase I and dispase.

[0025] In some embodiments of the present invention, in step S1, the cells are digested after fusion using Gibco TM TrypLE TM Select enzyme (1X) without phenol red for digestion.

[0026] In some embodiments of the present invention, in step S1, the centrifugal precipitate is resuspended in the primary culture medium and then cultured, and the initial inoculation density is 0.07-0.08 g / 75 cm 2 , preferably 0.075g / 75cm 2 .

[0027] In some embodiments of the present invention, during the subculture in step S2, the seeding density is 5000-6500 / cm 2 .

[0028] In some embodiments of the present invention, the culture conditions in step S1 are 37±0.5°C, 5% CO 2 .

[0029] In some embodiments of the present invention, the culture conditions in step S2 are 37±0.5°C, 5% CO 2 .

[0030] In some embodiments of the present invention, dental follicle mesenchymal stem cells of generations P2 to P10 are collected as principal cells; preferably, dental follicle mesenchymal stem cells of generations P2 to P4 are collected as principal cells.

[0031] In one embodiment of the present invention, P3 dental follicle mesenchymal stem cells are collected as principal cells.

[0032] In a third aspect, the present invention discloses the use of dental follicle mesenchymal stem cells that highly express periostin, including use in the preparation of a method for preventing and / or treating periodontal diseases.

[0033] In a fourth aspect, the present invention discloses the use of dental follicle mesenchymal stem cells that highly express periostin, wherein the use is in the preparation of a drug that promotes macrophage migration and / or promotes the conversion of macrophages into M2 macrophages.

[0034] Dental follicle mesenchymal stem cells with high expression of periostin can promote macrophage migration and reprogram macrophages into M2 type. M2 macrophages can promote, including but not limited to, the healing of skin injuries, nerve damage repair, vascularization and osteogenesis, and anti-aging of tissues.

[0035] Preferably, the invention is used in the preparation of a drug for promoting periodontal regeneration.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The dental follicle mesenchymal stem cells extracted and cultured by the method of the present invention have good passage stability and strong self-renewal and clone formation ability, and have strong multidirectional differentiation ability. In addition, the applicant unexpectedly found that the method of the present invention enhances the expression of periostin in dental follicle mesenchymal stem cells, thereby enhancing the role of dental follicle mesenchymal stem cells in promoting periodontal regeneration and regulating macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Attached Figure 1 This is the morphology of primary P0 dental follicle mesenchymal stem cells;

[0039] Attached Figure 2 This is the result of flow cytometric identification of surface markers of primary P3 dental follicle mesenchymal stem cells;

[0040] Attached Figure 3 This is a statistical chart showing the changes in population doubling time of dental follicle mesenchymal stem cells at each generation;

[0041] Attached Figure 4 This is a diagram of the colony formation of dental follicle mesenchymal stem cells (500 cells / 60mm seeding), where the right picture is a partial enlarged picture of the left picture;

[0042] Attached Figure 5 The figures are the results of the investigation of the multidirectional differentiation ability of P5 dental follicle mesenchymal stem cells extracted and cultured by the method of the present invention, wherein Figure A is the result of Alizarin Red staining of dental follicle mesenchymal stem cells cultured under osteogenic induction culture conditions for 21 days; Figure B is the result of Oil Red O staining of dental follicle mesenchymal stem cells cultured under adipogenic induction culture conditions for 14 days; Figure C is the result of Alecian Blue staining of dental follicle mesenchymal stem cell microspheres cultured under chondrogenic induction culture conditions for 28 days;

[0043] Attached Figure 6This is a graph showing the relative expression differences of the periostin gene in dental follicle mesenchymal stem cells of the same generation under different culture conditions.

[0044] Attached Figure 7 Figures A, B and C are the results of the clinical trial of dental follicle mesenchymal stem cells of the present invention; wherein Figure A, Figure B and Figure C are the results of PD level, CAL level and GR level of each group before treatment and 3 months, 6 months and 12 months after treatment, respectively; Figure D is the result analysis chart of PD, CAL and GR level of DFSC group before treatment and 3 months, 6 months and 12 months after treatment; Figure E is the result analysis chart of PD, CAL and GR level of NS group before treatment and 3 months, 6 months and 12 months after treatment; Figure F and Figure G are the results of BDD level and BDW level of each group before treatment and 3, 6 and 12 months after treatment, Figure H is the result analysis chart of BDD and BDW level of DFSC group before treatment and 3, 6 and 12 months after treatment; Figure I is the result analysis chart of BDD and BDW level of NS group before treatment and 3, 6 and 12 months after treatment. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0046] The cell culture flask described in the embodiment of the present invention is Nunc TM EasYFlask TM Cell culture flasks;

[0047] The culture dish described in the embodiment of the present invention is Nunc TM EasYDish TM Petri dish

[0048] The cell factory described in the embodiment of the present invention is Nunc TM EasyFill TM -2Cell Factory TM system.

[0049] The human stem cell osteogenic differentiation kit (item number HUXXC-90021), human stem cell adipogenic differentiation kit (item number HUXXC-90031) and human stem cell chondrogenic differentiation kit (item number HUXXC-90041) described in the embodiments of the present invention are all provided by Saiye (Guangzhou) Biotechnology Co., Ltd. The RNA extraction kit (item number RC112-01) and reverse transcription kit (item number R323-01) described in the embodiments of the present invention are all provided by Nanjing Novezan Biotechnology Co., Ltd.

[0050] The subculture medium described in the embodiment of the present invention is prepared by the following method: 1.0 mL of vitamin C injection (2 mL: 0.5 g) is mixed evenly with 500 mL of serum-free cell culture additive to obtain a mixed solution; 25 mL of the mixed solution is mixed evenly with 500 mL of MEMα culture medium to obtain a subculture medium;

[0051] The preparation method of the primary culture medium comprises the following steps: uniformly mixing gentamicin sulfate injection (2 mL: 80,000 units) and the subculture culture medium at a volume ratio of 1.0:1000 to obtain the primary culture medium.

[0052] Serum-free cell culture supplement for UltraGRO TM -Advanced; In some embodiments of the present invention, the MEMα medium contains nucleosides but does not contain phenol red.

[0053] Example 1

[0054] This embodiment discloses a method for extracting and culturing dental follicle mesenchymal stem cells that highly express periostin of the present invention, which is specifically as follows:

[0055] S1. Extraction of primary (P0) dental follicle mesenchymal stem cells

[0056] S11. Preparation: Place 75cm 2 The cell culture flasks were labeled with the following information: unique code, generation number, cell lot number, seeding density, date, and operator.

[0057] S12. Cleaning: Collect the immature wisdom teeth that need to be extracted from healthy subjects, collect the dental follicle tissue around the crown; clean with sodium chloride injection (0.9%) containing 40 units / mL gentamicin sulfate solution.

[0058] Move the cleaned dental follicle tissue into a sterile EP tube, mark the scale, and then move the tissue into a new sterile EP tube. Inject sodium chloride injection (0.9%) into the original EP tube to the marked scale line, measure the volume (V) of sodium chloride injection (0.9%) added to estimate the weight of the dental follicle tissue, dental follicle tissue weight = V × 1g / mL, tissue weight must be ≥ 0.5g.

[0059] S13. Digestion and centrifugation: Cut the cleaned dental follicle tissue into pieces of 1-2 mm 3 Then, the cells were digested with a mixed enzyme solution (containing 0.05% collagenase I and 0.005% dispase (neutral protease)) in a thermostatic oscillator (37°C, 150 rpm) for 30 minutes, and the digestion was terminated with primary culture medium (containing 40 units / mL gentamicin sulfate), followed by centrifugation and removal of the supernatant.

[0060] S14. Count and resuspend: according to 0.075g / 75cm 2 Calculate the number of inoculated bottles for cell culture bottles, 5mL / 75cm 2 Calculate the resuspension volume of the cell culture flask and resuspend it again with primary culture medium (containing 40 units / mL gentamicin sulfate);

[0061] S15. Subculture: Inoculate the tissue and cell suspension prepared in step S14 to 75 cm 2 Place the inoculated cell culture flask in a 37.0°C, 5% CO 2 Culture in an incubator. Perform a full medium change every 3 days, and observe the tissue and cell adhesion and whether there is contamination under a microscope. The primary culture time is 16-22 days. When the cell fusion degree reaches more than 70% under a microscope, perform a sterility test on the supernatant of the last medium change.

[0062] S2. Dental follicle mesenchymal stem cells passage P0-P1

[0063] S21. Pipette and discard the supernatant: When the cell confluence reaches 70%-90%, pipette 75cm in the biosafety cabinet. 2 The culture supernatant in the cell culture bottle is used to remove the tissue fragments and discard them together with the culture supernatant; 10 mL of sodium chloride injection (0.9%) is added to gently shake and rinse the culture surface, the washing solution is discarded, and the washing operation is repeated again;

[0064] S22. Digestion and centrifugation: Add 1 mL / bottle of Gibco TM TrypLE TM Select enzyme (1X), without phenol red, digest at room temperature for 5-7 minutes, tap gently to remove cells, press 4mL / 75cm 2 Sodium chloride injection (0.9%) was added to the cell culture flask to terminate the digestion. 2 Transfer all the liquid and cells in the cell culture flask to a 50 mL centrifuge tube and add 5 mL / 75 cm 2 Wash the cell culture flask with sodium chloride injection (0.9%) and add the washing solution into a 50mL centrifuge tube. A 50mL centrifuge tube can hold four 75cm 2 The cell suspension harvested from the cell culture flask was centrifuged at 400 g for 5 minutes and the supernatant was discarded.

[0065] S23. Resuspend and subculture: Resuspend the cell pellet with an appropriate amount of subculture medium and take two samples for counting. 2 -6500 cells / cm 2 The cells were passaged to a density of 225 cm2 Cell culture flask, 225cm 2 The cell culture flask was supplemented with subculture medium (without gentamicin sulfate) to 45 mL and shaken for 225 cm 2 The culture flask was placed back to 37.0°C and 5% CO to allow the cells to be evenly distributed. 2 Culture in an incubator.

[0066] S3. Dental follicle mesenchymal stem cells passage P1-P2

[0067] S31. Preparation: Take 10 layers of cell factories and 225cm 2 Cell bottles are labeled in the biosafety cabinet with the following information: unique code, generation number, cell batch number, cell culture density, date, and operator.

[0068] S32. Aspirate and discard the supernatant: Take the 225cm in step S23 2 The cell flask was observed under an inverted microscope. When the cell confluence reached 70%-90%, the 225cm 2 The culture supernatant in the cell bottle; add 10-15mL of sodium chloride injection (0.9%) and gently shake to rinse the culture surface for 10 seconds, then discard the washing solution and repeat the washing operation again;

[0069] S33. Digestion and centrifugation: 3mL / 225cm 2 Cell flasks added to Gibco TM TrypLE TM Select enzyme (1X), without phenol red, digest at room temperature for 5-7 minutes, tap gently to remove cells, and press 12mL / 225cm in a biosafety cabinet. 2 Sodium chloride injection (0.9%) was added to the cell flask to terminate the digestion. 2 Transfer all the liquid and cells in the cell bottle to a 50mL centrifuge tube and add 15mL / 225cm 2 The cell flask was washed with sodium chloride injection (0.9%), and the washing solution was added into a 50 mL centrifuge tube. The tube was centrifuged at 400 g for 5 minutes, and the supernatant was discarded.

[0070] S34. Resuspend and subculture: Resuspend the cell pellet with an appropriate amount of subculture medium and take two samples for counting. According to the counting results, the total number of viable cells is ≥3.16×10 7 If there are more cells than enough to inoculate a 10-layer cell factory, then a 10-layer cell factory can be selected for culture. After inoculating a 10-layer cell factory, if there are remaining cells that are not enough to inoculate a 10-layer cell factory, then inoculate to 225cm 2 Cell culture flask. Press 3.16×10 4 cells / mL~4.11×10 4cells / mL (equivalent to 5000 cells / cm 2 ~6500 cells / cm 2 ) and resuspend the cells in subculture medium. The amount of subculture medium added to each 10-layer cell factory is 1000mL~1050mL, and then shake the 10-layer cell factory in a cross direction to make the cells evenly distributed. If the amount of cells is not enough to inoculate a 10-layer cell factory, 5000 cells / cm 2 -6500 cells / cm 2 The cells were passaged to a density of 225 cm 2 Cell bottles, every 225cm 2 Add 45 mL of culture medium to the cell bottle and shake it crosswise for 225 cm 2 Each subculture should be carried out by inoculating at least one 225cm 2 The cell flask was used as an observation flask for the cell confluence. 2 Return the cell flask to 37.0°C and 5% CO 2 Culture in an incubator.

[0071] S4. Dental follicle mesenchymal stem cells passage P2-P3

[0072] S41. Preparation: Take 10 layers of cell factories and 225cm 2 Cell culture bottles are marked in the biosafety cabinet with the following information: unique code, generation number, cell batch number, subculture density, date, and operator.

[0073] S42. Aspirate and discard the supernatant, digest, and centrifuge: Take the 225 cm 2 The cell culture flask was observed under an inverted microscope. When the cell confluence reached 70%-90%, the cell factory and 225cm 2 The culture supernatant in the cell culture flask is then digested and centrifuged. Specifically:

[0074] (1) For a 10-layer cell factory, discard the culture supernatant in a biosafety cabinet; add 500 mL of sodium chloride injection (0.9%) and evenly distribute it to each culture surface, gently shake the culture surface for 10 seconds, discard the washing solution, and repeat the above washing operation; add Gibco TM TrypLE TM Select enzyme (1X), without phenol red, digest at room temperature for 5-7 minutes, tap gently to remove cells, and place at 350mL-400mL / 225cm in a biosafety cabinet. 2 Sodium chloride injection (0.9%) was added to the cell culture flask to terminate the digestion. 2Transfer all the liquid and cells in the cell culture flask to a 225 mL centrifuge tube and add 500 mL / 225 cm 2 The cell culture flask was washed with sodium chloride injection (0.9%), and the washing solution was added into a 225 mL centrifuge tube; centrifuged at 800 g for 5 minutes.

[0075] (2) For 225cm 2 For cell culture flasks, discard the culture supernatant in a biosafety cabinet; add 10-15mL of sodium chloride injection (0.9%) and gently shake the culture surface for 10 seconds, then discard the washing solution and repeat the washing operation; press 3mL / 225cm 2 Gibco cell culture flask TM TrypLE TM Select enzyme (1X), without phenol red, digest at room temperature for 5-7 minutes, tap gently to remove cells, and press 12mL / 225cm in a biosafety cabinet. 2 Sodium chloride injection (0.9%) was added to the cell culture flask to terminate the digestion. 2 Transfer all the liquid and cells in the cell culture flask to a 225 mL centrifuge tube and add 15 mL / 225 cm 2 The cell culture flask was washed with sodium chloride injection (0.9%), and the washing solution was added into a 225 mL centrifuge tube and centrifuged at 800 g for 5 minutes.

[0076] S43. Resuspend and subculture: After centrifugation, discard the supernatant and resuspend the cell pellet with subculture medium. Combine and take 3 samples for counting. According to the counting results, the total number of viable cells is ≥3.16×10 7 If there are more cells than enough to inoculate a 10-layer cell factory, then a 10-layer cell factory can be selected for culture. After inoculating a 10-layer cell factory, if there are remaining cells that are not enough to inoculate a 10-layer cell factory, then inoculate to 225cm 2 Cell culture flask. Press 3.16×10 4 cells / mL~4.11×10 4 cells / mL (equivalent to 5000 cells / cm 2 ~6500 cells / cm 2 ) and resuspend the cells in the subculture medium. The amount of medium added to each 10-layer cell factory is 1000mL-1050mL. Then shake the 10-layer cell factory in a cross direction to make the cells evenly distributed. If the amount of cells is not enough to inoculate a 10-layer cell factory, add 5000 cells / cm 2 ~6500 cells / cm 2 The cells were passaged to a density of 225 cm 2 Cell culture flask, 225cm 2The amount of culture medium added to the cell culture flask was 45 mL, and then the flask was shaken in a cross direction for 225 cm. 2 Cell culture flasks should be used to distribute cells evenly. At least one 225cm 2 Cell culture flasks were used as observation flasks for cell confluence. 2 Return the cell culture flask to CO 2 The cells were cultured in a carbon dioxide incubator at a concentration of 5.0% and a temperature of 37.0°C.

[0077] S5. Harvesting of Human Dental Follicle Mesenchymal Stem Cells

[0078] Take 225cm in step S43 2 The cell culture flask was observed under an inverted microscope. When the cell confluence reached 70%-90%, the cell factory and 225cm 2 The culture supernatant in the cell culture flask is then digested and centrifuged. Specifically:

[0079] (1) For a 10-layer cell factory, collect part of the culture supernatant of each 10-layer cell factory in the biosafety cabinet and mix them for detection, and discard the remaining culture supernatant in the 10-layer cell factory; add 500 mL of sodium chloride injection (0.9%) and evenly distribute it to each layer of the culture surface, gently shake and rinse the culture surface for 10 seconds, discard the washing solution, and repeat the above washing operation again; add Gibco TM TrypLE TM Select enzyme (1X), without phenol red, digest at room temperature for 5-7 minutes, tap gently to remove cells, and place at 350mL-400mL / 225cm in a biosafety cabinet. 2 Sodium chloride injection (0.9%) was added to the cell culture flask to terminate the digestion. 2 Transfer all the liquid and cells in the cell culture flask to a 225 mL centrifuge tube and add 500 mL / 225 cm 2 The cell culture flask was washed with sodium chloride injection (0.9%), and the washing solution was added into a 225 mL centrifuge tube and centrifuged at 800 g for 5 minutes.

[0080] (2) For 225cm 2 Cell culture flask, collect part of the culture supernatant of each 10-layer cell factory in the biosafety cabinet and mix them for detection, and discard 225cm 2 The remaining culture supernatant in the cell culture bottle; add 10-15mL sodium chloride injection (0.9%) and gently shake the culture surface for 10 seconds, then discard the washing solution and repeat the washing operation; press 3mL / 225cm 2 Gibco cell culture flask TM TrypLETM Select enzyme (1X), without phenol red, digest at room temperature for 5-7 minutes, tap gently to remove cells, and press 12mL / 225cm in a biosafety cabinet. 2 Sodium chloride injection (0.9%) was added to the cell culture flask to terminate the digestion. 2 Transfer all the liquid and cells in the cell culture flask to a 225 mL centrifuge tube and add 15 mL / 225 cm 2 The cell culture flask was washed with sodium chloride injection (0.9%), and the washing solution was added into a 225 mL centrifuge tube and centrifuged at 800 g for 5 minutes.

[0081] After centrifugation, the supernatant in the 225 mL centrifuge tube was discarded in a biosafety cabinet, and the cell pellet was resuspended with an appropriate amount of sodium chloride injection (0.9%), and three samples were taken for counting. The obtained pellet suspension was the harvested human dental follicle mesenchymal stem cell chief cell suspension.

[0082] Test Example 1

[0083] This experiment discloses the morphology of primary P0 dental follicle mesenchymal stem cells and the flow cytometric identification of P3 dental follicle mesenchymal stem cells surface markers. 6 P3 dental follicle mesenchymal stem cells to be tested were placed in a flow cytometry tube, centrifuged at 400g for 5 min, the supernatant was discarded, and the tube was resuspended in 300μL of normal saline. 5-10μL of corresponding antibodies were added according to the marker grouping, and isotype controls were set up. The tubes were incubated at room temperature and protected from light for 20min; centrifuged at 400g for 5min, the supernatant was discarded, and the tubes were washed twice with 1mL of normal saline; the tubes were resuspended in 300μL of normal saline, and tested using an Agilent NovoExpress flow cytometer.

[0084] The morphology of primary P0 dental follicle mesenchymal stem cells is shown in the attached figure. Figure 1 Shown: Typical spindle-shaped or long-fusiform fibroblast-like morphology.

[0085] The results of surface marker flow cytometric identification are shown in the attached Figure 2 As shown: positive expression of CD90, CD105, and CD73, negative expression of CD34, CD19, CD45, CD11b, and HLA-DR, consistent with the phenotypic characteristics of mesenchymal stem cells. Figure 2 9008024311003 / E1 is the cell number, APC-H, FITC-H, PE-H, and PerCP-H are the types of flow cytometry dyes; the percentage in each small figure represents the expression level of each surface marker.

[0086] Test Example 2

[0087] This test example investigated the population doubling time and colony formation of the dental follicle mesenchymal stem cells of the present invention.

[0088] 1. Population doubling time test:

[0089] P0 dental follicle mesenchymal stem cells were resuspended in the culture medium and cultured at 5000 cells / cm 2 Inoculate in a culture bottle, culture for 72-96 hours, discard the supernatant, wash with physiological saline, digest with recombinant pancreatic enzyme digestion solution, harvest dental follicle mesenchymal stem cells and count, and count the number of cells harvested in the P1 generation; repeat the above process to pass down to the P7 generation.

[0090] The population doubling time was calculated by the formula PDT = [log2 / (lgNt-lgN0)] × t, where t is the culture time from cell inoculation to harvest, Nt is the number of cells harvested at time t, and N0 is the number of cells inoculated at the beginning. After obtaining the population doubling times of P0-P1, P1-P2, ...P6-P7, a population doubling time curve was drawn.

[0091] 2. Clonal colony formation test:

[0092] The P5 dental follicle mesenchymal stem cells of this test example were collected and inoculated at 500 cells / 60 mm culture dish. 3 mL of subculture medium was added and placed at 37°C and 5% CO. 2 The cells were cultured statically and the culture was terminated after clone colonies were formed (about 10-12 days, the number of cells in each colony was >50). The cells were fixed with 4% paraformaldehyde for 15 minutes and the colony formation was observed by crystal violet staining.

[0093] The results of population doubling time are shown in the attached Figure 3 The colony formation was shown in the attached Figure 4 The results show that the dental follicle mesenchymal stem cells of the present invention have good passage stability and strong self-renewal and clone formation capabilities.

[0094] Test Example 3

[0095] This test example investigated the multidirectional differentiation ability of the dental follicle mesenchymal stem cells of the present invention.

[0096] 1. Investigation of osteogenic differentiation ability

[0097] Prepare osteoinduction differentiation medium according to the instructions of the human stem cell osteoinduction differentiation kit, store at 4°C, and use within one month. 4 cells / cm 2 The cells were inoculated into a 12-well plate at a density of 1 mL. 1 mL of cell culture medium (SLCPCA complete medium) was added to each well and placed at 37°C and 5% CO 2, saturated humidity incubator. When the cell confluence reaches 70%, replace 1 mL of osteogenic differentiation induction medium, and replace with fresh osteogenic differentiation induction medium every 3 days. After 3 weeks of culture, fix with 4% paraformaldehyde for 15 minutes, and observe calcium nodules with Alizarin red staining.

[0098] The results of osteogenic differentiation ability test are shown in the attached Figure 5 As shown in Figure A, it can be seen from the figure that the dental follicle mesenchymal stem cells of the present invention have excellent osteogenic differentiation ability, and can produce a large number of calcium nodules after 21 days of osteogenic induction culture, and Alizarin red staining is strongly positive.

[0099] 2. Investigation of adipogenic differentiation ability

[0100] According to the instructions of the human stem cell adipogenic differentiation kit, prepare adipogenic differentiation medium A and medium B, store at 4°C, and use within one month. 4 cells / cm 2 The cells were inoculated into a 12-well plate at a density of 1 mL. 1 mL of cell culture medium (SLCPCA complete medium) was added to each well and placed at 37°C and 5% CO 2 , and cultured in an incubator with saturated humidity. When the cell confluence reached 100%, 1 mL of adipogenic differentiation induction medium A was replaced. After 3 days of induction, it was replaced with adipogenic differentiation induction medium B. After 1 day, the B solution was removed and replaced with A solution for induction. A solution and B solution were used alternately. After 2 weeks of induction, 4% paraformaldehyde was used for fixation for 15 minutes, and oil red O staining was used to observe lipid droplet formation.

[0101] The results of the adipogenic differentiation ability test are shown in the attached Figure 5 As shown in Figure B, it can be seen from this figure that the dental follicle mesenchymal stem cells of the present invention have good adipogenic differentiation ability. After 14 days of adipogenic induction culture, obvious lipid droplets can be seen in the cytoplasm, and Oil Red O staining is positive.

[0102] 3. Study on Chondrogenic Differentiation Ability

[0103] Prepare chondrogenic differentiation premix and complete medium according to the instructions of the human stem cell chondrogenic differentiation kit, store at 4°C, and use within one month. 5 Transfer the P5 dental follicle mesenchymal stem cells to be induced into a 15 mL centrifuge tube, centrifuge at 20°C, 250 g for 4 min, and remove the supernatant; add 0.5 mL of chondrogenic differentiation premix to resuspend, centrifuge at 20°C, 150 g for 5 min; repeat this step to wash the cells again; add 0.5 mL of chondrogenic differentiation complete medium to resuspend, centrifuge at 20°C, 150 g for 5 min; loosen the cap of the centrifuge tube to facilitate gas exchange, and place it upright at 37°C, 5% CO 2, saturated humidity incubator; after 24 hours, cells began to aggregate, and the bottom of the centrifuge tube was flicked to suspend the chondrocytes in the liquid. Every 2 to 3 days, 0.5 mL of fresh complete chondrogenic differentiation medium was used, and the bottom of the tube was flicked after each change of the medium. After 28 days of continuous induction, chondrocytes with a diameter of about 1 mm were formed. 4% paraformaldehyde was used for fixation for 30 minutes, gradient alcohol dehydration was performed, paraffin was embedded, 4 μm continuous sections were cut, and Alecian blue staining was performed for observation.

[0104] The results of the cartilage differentiation ability test are shown in the attached Figure 5 As shown in Figure C, it can be seen from this figure that the dental follicle mesenchymal stem cells of the present invention have good chondrogenic differentiation ability. After 28 days of chondrogenic induction culture, the sections were sliced ​​and stained with Alecin blue to show blue, indicating the intrinsic acidic mucopolysaccharide in the cartilage tissue.

[0105] Test Example 4

[0106] This test example discloses an experiment to investigate the ability of the dental follicle mesenchymal stem cells of the present invention to express periostin.

[0107] The P3 and P5 dental follicle mesenchymal stem cells extracted and cultured in Example 1 were compared with the same generation dental follicle mesenchymal stem cells under normal culture conditions. Normal culture conditions refer to that compared with Example 1, α-MEM medium (containing nucleosides and not containing phenol red) is used to replace the primary culture medium and the subculture medium, and the other conditions are the same.

[0108] Each charge at least 1×10 6 Real-time polymerase chain reaction (RT-PCR) was used to detect the periostin gene expression in P3 and P5 dental follicle mesenchymal stem cells cultured under different conditions. Total RNA was extracted using an RNA extraction kit.

[0109] Real-time PCR periostin-related gene detection steps are as follows:

[0110] (1) Cell treatment: 1 ～ 5×10 6 Add 500 μL of Buffer RL to P3 and P5 dental follicle mesenchymal stem cells cultured under different conditions and vortex until no obvious cell clusters are found.

[0111] (2) Total RNA extraction: Follow the instructions of the RNA extraction kit.

[0112] (3) The concentration of the extracted RNA and the A260 / A280 and A230 / A280 values ​​were determined using a Nanodrop ND-1000 micro-nucleic acid analyzer. When the A260 / A280 ratio was 1.9-2.1 and the A230 / A280 ratio was >2, the purity of the extracted RNA was high and could be used for subsequent experiments.

[0113] (4) RNA was reverse transcribed using a reverse transcription kit, which contained the following components: RNase-free ddH 2 O, 4×gDNA wiper Mix, 5×HiScript III qRTSuperMixa, 5×No RT Control Mixb, and reverse transcription according to the following steps:

[0114] ① Genomic DNA removal

[0115] Prepare the following mixture in an RNase-free centrifuge tube (pre-cooled on ice):

[0116] Table 1

[0117] <![CDATA[RNase-free ddH 2 The]]> Up to 16 μL 4×gDNA wiper Mix 4μL Template RNA Total RNA: 1pg-1μg

[0118] Mix well by gently pipetting, centrifuge briefly, and place in a reverse transcription apparatus at 42°C for 2 minutes.

[0119] ②Prepare the reverse transcription reaction system: directly add 4μL 5×HiScript IIIqRTSuperMix to the reaction tube in step ①, gently pipette to mix, and centrifuge briefly. Transfer to the reverse transcription instrument for reverse transcription reaction: 37℃15 minutes→85℃5 seconds to obtain cDNA.

[0120] (5) Real-time PCR reaction

[0121] ① The primer sequences were designed and synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. The primer sequences are shown in Table 2:

[0122] Table 2

[0123]

[0124]

[0125] ② SYBR (Taq Pro Universal SYBR qPCR Master Mix, Nanjing Novezan Biotechnology Co., Ltd., Catalog No. Q712-02) was used as the substrate source to prepare the reaction system. The following reagents were added to a 96-well plate on an ice bath to a total volume of 20 μl, as shown in Table 3:

[0126] Table 3

[0127] Reagents volume 2×Taq Pro Universal SYBR qPCR Master Mix 10μL Forward Primer 1μL Post Primer 1μL cDNA 2μL <![CDATA[ddH 2 The]]> 6μL

[0128] ③ Add the mixed solution to a 96-well plate, shake, centrifuge (2,000 rpm, 3 minutes), and place in a PCR instrument for reaction. The RT-PCR reaction program is as follows: pre-denaturation: 95°C, 30 seconds, 1 cycle; PCR cycle reaction: 95°C 10 seconds → 60°C 30 seconds, 40 cycles; melting curve: 95°C 15 seconds → 60°C 60 seconds → 95°C 15 seconds;

[0129] ④After the reaction is completed, record the CT value of each group of genes according to the formula:

[0130] Relative expression level = 2 -ΔΔCt

[0131] The relative expression of genes was calculated (ΔΔCt = (CTgene–CTGAPDH) experimental group – (CTgene–CTGAPDH) control group).

[0132] The results showed that the dental follicle mesenchymal stem cells extracted and cultured in Example 1 had a higher expression level of periostin than the dental follicle mesenchymal stem cells of the same generation under ordinary culture conditions. The expression of periostin in the P5 generation dental follicle mesenchymal stem cells in Example 1 and ordinary culture was slightly lower than that in the P3 generation, but the expression of periostin in the P5 generation dental follicle mesenchymal stem cells in Example 1 was still significantly higher than that in the P3 generation dental follicle mesenchymal stem cells under ordinary culture conditions.

[0133] Test Example 5

[0134] This test example discloses a clinical trial of the dental follicle mesenchymal stem cells of the present invention.

[0135] The preparation method of the fresh dental follicle mesenchymal stem cell preparation described in this test example is as follows:

[0136] P5 dental follicle mesenchymal stem cells were cultured and harvested according to the above-mentioned subculture method, resuspended in physiological saline containing 6‰ human albumin and counted, and the density of dental follicle mesenchymal stem cells was adjusted to 5×10 7 Cells / mL, fresh dental follicle mesenchymal stem cell preparation was dispensed into 300 μL / bottle (a total of 1.5×10 7 Fresh dental follicle mesenchymal stem cell preparations were transported in a low-temperature transport box and used within 6 hours.

[0137] 1. Patient inclusion criteria

[0138] (1) Adults aged between 30 and 45 years old;

[0139] (2) Subjects were found to have two-wall or three-wall subosseous pockets with a PD ≥ 5 mm, or grade I / II furcation lesions by clinical exploration or imaging examination;

[0140] (3) The mobility of the affected teeth in the surgical area of ​​the subject is ≤Ⅱ°, and the width of the attached gingiva is sufficient to completely cover the bone defect and implant;

[0141] (4) The subjects gave informed consent, understood the purpose of the study, and were able to cooperate with the study and complete routine follow-up after surgery for at least 1 year;

[0142] (5) The subjects were able to achieve good self-plaque control, including correct brushing and flossing.

[0143] II. Exclusion criteria

[0144] (1) Failure to maintain good oral hygiene during the introduction period;

[0145] (2) Pregnant or lactating women or women planning to become pregnant during this study;

[0146] (3) history of oral cancer or HIV;

[0147] (4) the study teeth had a history of periodontal surgery;

[0148] (5) Clinical or radiological examinations showed untreated acute infection at the surgical site, apical lesions, root fractures, severe root deformities, difficult-to-remove cementum / enamel protrusions, untreated caries at the enamel-cementum junction or root surface, metal restorations or restorations that extend below the gingiva and / or poorly fitted restoration margins at or below the enamel-cementum junction;

[0149] (6) chewing smokeless tobacco or smoking ≥1 time per week or smoking ≥10 cigarettes per day in the past 6 months;

[0150] (7) Suffering from mental cognitive disorders;

[0151] (8) Those who have malignant tumors or have a history of malignant tumors;

[0152] (9) having diabetes or glycated hemoglobin ≥ 6.5%;

[0153] (10) Taking calcium antagonists within four weeks before surgery;

[0154] (11) Use of adrenal corticosteroids within four weeks before surgery;

[0155] (12) Taking medications other than those specified for observation during the subject's observation period;

[0156] (13) Allergy to biologically active products or drugs;

[0157] (14) Within 30 days of surgery, the patient received other research treatments;

[0158] (15) Research team members and their families;

[0159] (16) Patients with immune system abnormalities (such as autoimmune diseases);

[0160] (17) Patients with systemic diseases (Sjögren's syndrome, Behcet's disease, etc.) that may cause oral problems;

[0161] (18) Any other factors that may cause trial bias.

[0162] 3. Test operation

[0163] According to the above criteria, a total of 15 patients were included and divided into: fresh dental follicle mesenchymal stem cell treatment group (DFSC group): 11 cases, normal saline control group (NS group): 4 cases. Each patient received the following treatment:

[0164] (1) Under local anesthesia, the tooth under study was scraped clean of subgingival calculus and granulation tissue with the assistance of periodontal endoscope using ultrasonic instruments;

[0165] (2) Rinse repeatedly with hydrogen peroxide + chlorhexidine three times each;

[0166] (3) Rinse three times with sterile saline;

[0167] (4) Take the fresh dental follicle mesenchymal stem cell preparation out of the low-temperature transport box, remove the outer packaging, shake it back and forth 15-20 times to mix the cells, and install a special injection needle;

[0168] (5) In the fresh dental follicle mesenchymal stem cell treatment group, 100 μL (5×10 6 cells), and the control group was injected with an equal volume of sterile saline into the periodontal pocket;

[0169] (6) Study on the use of dental plugging agent (Kang Youning, a mixture of periodontal hemostatic powder and a small amount of cloves) to protect the surgical area;

[0170] (7) Follow up after one week and remove the suppository.

[0171] IV. Efficacy Evaluation

[0172] The content includes imaging indicators and clinical treatment indicators.

[0173] (1) Clinical treatment indicators: Before treatment and 3, 6, and 12 months after treatment, the following indicators were measured using a Florida electronic periodontal probe at six sites of the treated teeth: mesio-buccal, buccal, disto-buccal, mesio-lingual, lingual, and disto-lingual. Probing depth (PD), i.e., the distance from the gingival margin to the bottom of the gingival sulcus; clinical attachment level (CAL), i.e., the distance from the CEJ (cementoenamel junction) to the bottom of the gingival sulcus; gingival recession (GR), i.e., the distance from the CEJ to the gingival margin.

[0174] (2) Imaging treatment indicators: Cone beam computed tomography (CBCT) was used to measure bone defect depth (BDD) and bone defect width (BDW); CBCT examinations were performed before treatment and 3 months, 6 months, and 12 months after treatment. The distance from the lowest point of the defect to the cementoenamel junction (CEJ) and the maximum width of the bone defect were measured. Two oral radiologists who were unaware of the subjects' grouping independently measured the CBCT results of the subjects.

[0175] V. Research Results

[0176] The results are attached Figure 7 shown.

[0177] (1) Figure 7 As shown in A, compared with the NS (normal saline) group, the mean PD value of the DFSC group decreased compared with the NS group at 3, 6, and 12 months after dental follicle mesenchymal stem cell injection, but there was no statistical significance between the two.

[0178] (2) Figure 7 As shown in B, the level of clinical attachment loss in the DFSC group continued to decrease at 3, 6, and 12 months after injection, and the decrease levels at 6 and 12 months were significantly lower than those in the NS group.

[0179] (3) Figure 7 As shown in Figure C, the mean GR value in the DFSC group did not increase significantly compared with the NS group, but showed statistical significance at 3, 6, and 12 months after injection (Figure C).

[0180] (4) Figure 7 As shown in Figures D and 7E, when the results of the DFSC and NS groups were analyzed separately at 3, 6, and 12 months after treatment, the average PD and CAL levels in the DFSC group decreased significantly at 3, 6, and 12 months after injection, while there was no significant change in the NS group. No significant changes were found in GR at each time point within each group.

[0181] (5) Figure 7As shown in Figure F, compared with the NS group, the mean bone defect depth of the DFSC group decreased at 3, 6, and 12 months after dental follicle mesenchymal stem cell injection, but there was no statistical significance between the two. This may be limited by the individual errors of the small sample and cannot show statistical differences between the groups.

[0182] (6) Figure 7 As shown in G, 7H, and 7I, when the results of the DFSC group and the NS group were analyzed separately at 3, 6, and 12 months after treatment, ( Figure 7 H), the depth of bone defect in the DFSC group decreased significantly at 3 and 6 months after injection, while there was no significant change in the NS group. No significant changes were found in the width of bone defect between or within the groups ( Figure 7 G, 7I).

[0183] Figure 7 In the table, the Chinese meanings of the English abbreviations are as follows: PD, probing depth; CAL, clinical attachment loss; GR, gingival recession; BDD, bone defect depth; BDW, bone defect width.

[0184] "pre" indicates the baseline level before treatment; "3m", "6m", and "12m" indicate 3 months, 6 months, and 12 months after periodontal pocket treatment. The comparison between the groups was performed by repeated measures two-way analysis of variance, and the comparison within the group was performed by repeated measures one-way analysis of variance. "*" indicates statistical significance. *p<0.05; **p<0.01; ***p<0.001; "ns" indicates no statistical significance. Error bars indicate mean ± SD.

[0185] The present invention has completed the screening and clinical trials of 15 subjects, and preliminarily confirmed that the immunological indicators of allogeneic dental follicle mesenchymal stem cells used in periodontal tissue regeneration showed no obvious abnormalities, good safety, and no obvious adverse reactions. The periodontal probing examination and imaging analysis of the subjects showed that all periodontal probing indicators improved at 3 months, 6 months, and 12 months after the injection of dental follicle mesenchymal stem cells. The imaging CBCT analysis showed that the depth and volume of bone defects were significantly reduced. Therefore, DFSC injection can promote the regeneration of periodontal tissue at the bone defect to a certain extent, promote periodontal new attachment and alveolar bone regeneration, promote the recovery of periodontal soft tissue in patients with periodontitis, and inhibit gingival recession.

[0186] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial meaning, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields is also included in the patent protection scope of the present invention.

Claims

1. A dental follicle mesenchymal stem cell that highly expresses periostin, characterized in that: It is deposited in the China Center for Type Culture Collection, with the accession number being CCTCC NO:C2024384 and the preservation date being December 6, 2024.

2. The method for extracting and culturing dental follicle mesenchymal stem cells that highly express periostin according to claim 1, characterized in that: The steps include: S1. Extraction: The dental follicle tissue is cleaned, cut into pieces, digested, and centrifuged. The centrifuged precipitate is resuspended in primary culture medium and cultured until the cells are fused. The precipitate is then digested and centrifuged again. The secondary centrifuged precipitate is the primary dental follicle mesenchymal stem cells. S2. Subculture: resuspending the dental follicle mesenchymal stem cells in step S1 with a subculture medium and then subcultured; The subculture medium is prepared by adding serum-free cell culture additives and vitamin C to MEMα medium; 24-26 mL of serum-free cell culture additives and 49-51 μL of vitamin C injection are added to every 100 mL of MEMα medium; The primary culture medium is prepared by mixing gentamicin sulfate injection and subculture culture medium, wherein the content of gentamicin sulfate in the primary culture medium is 30-50 units / mL.

3. The method for extracting and culturing dental follicle mesenchymal stem cells that highly express periostin according to claim 1, characterized in that: The preparation method of the subculture medium comprises the following steps: mixing 0.8-1.2 mL of vitamin C injection with 500 mL of serum-free cell culture additive to obtain a mixed solution; taking 20-30 mL of the mixed solution and mixing it with 500 mL of MEMα culture medium to obtain a subculture medium; The preparation method of the primary culture medium comprises the following steps: uniformly mixing gentamicin sulfate injection and the subculture culture medium at a volume ratio of 0.8 to 1.2:1000 to obtain the primary culture medium.

4. The method for extracting and culturing dental follicle mesenchymal stem cells that highly express periostin according to claim 2 or 3, characterized in that: In step S1, the cells are cultured until the cell confluence reaches 70% or more.

5. The method for extracting and culturing dental follicle mesenchymal stem cells that highly express periostin according to claim 2 or 3, characterized in that: The culture conditions in step S1 were 37 ± 0.5 °C, 5% CO2; The culture conditions in step S2 were 37±0.5°C and 5% CO2.

6. The method for extracting and culturing dental follicle mesenchymal stem cells that highly express periostin according to claim 2 or 3, characterized in that: P2 to P10 dental follicle mesenchymal stem cells are collected, preferably P2 to P4 dental follicle mesenchymal stem cells are used as main cells.

7. The use of dental follicle mesenchymal stem cells highly expressing periostin according to claim 6, characterized in that: Application in the preparation of the method for preventing and / or treating periodontal diseases.

8. The use of dental follicle mesenchymal stem cells highly expressing periostin according to claim 1, characterized in that: Application in the preparation of drugs for promoting periodontal regeneration.

9. The use of dental follicle mesenchymal stem cells highly expressing periostin according to claim 7, characterized in that: The application is application in the preparation of drugs for promoting macrophage migration and / or promoting macrophage conversion into M2 macrophages.

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