Dental follicle mesenchymal stem cells highly expressing periostin, extraction and culture method thereof, and application thereof

Abstract

The present invention discloses dental follicle mesenchymal stem cells that highly express periostin, their extraction and culture methods, and applications, and belongs to the field of biomedical technology. The dental follicle mesenchymal stem cells that highly express periostin of the present invention are deposited in the China Center for Type Culture Collection with the accession number CCTCC NO: C2024384. The extraction and culture method disclosed in the present invention comprises the following steps: S1. Extraction: The dental follicle tissue is cleaned, minced, digested, centrifuged, and the centrifugal precipitate is resuspended in primary culture medium and then cultured until the cells fuse, followed by digestion and a second centrifugation. The secondary centrifugal precipitate is the primary dental follicle mesenchymal stem cell; S2. Subculture: The dental follicle mesenchymal stem cells from step S1 are resuspended in primary culture medium and supplemented with subculture medium before subculture. The present invention also discloses the dental follicle mesenchymal stem cells that highly express periostin and their application in the preparation of a method for preventing or / and treating periodontal disease. The method of the present invention has an unexpected effect of enhancing the expression of periostin by dental follicle mesenchymal stem cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to dental follicle mesenchymal stem cells that highly express periostin, and a method for extracting and culturing the same and applications thereof. Background Art

[0002] Stem cell therapy has become one of the most cutting-edge and promising research areas in disease treatment and tissue regeneration. Finding ideal seed cells and efficient in vivo cell interaction systems is crucial for achieving these goals. Dental stem cells, particularly those derived from the dental follicle, have attracted considerable attention due to their robust self-renewal and multipotential differentiation capabilities. Dental stem cells primarily 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 (SHEDs). Numerous studies have demonstrated the enormous potential of these stem cells in tissue repair and disease treatment. Among the numerous dental stem cells, dental follicle mesenchymal stem cells (DFSCs) are the only ones derived from tissue during tooth germ development. The dental follicle is the loose connective tissue layer that surrounds the enamel organ and the base of the dental papilla during the tooth germ stage and originates from the ectodermal mesenchyme. The cementum, periodontal ligament, and alveolar bone in the periodontium all develop from the dental follicle. Clinically, the dental follicle tissue attached to the neck of an extracted impacted tooth is often discarded along with the tooth as medical waste. Therefore, dental follicle tissue is an easily accessible and harmless source of stem cells. Therefore, compared to other dental stem cells, dental follicle mesenchymal stem cells have unique advantages, making them 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 increasingly important. However, there are few reports on how to maintain or even enhance the functions of DFSCs during extraction and culture. DFSCs are currently primarily used to treat periodontal diseases. Studies have demonstrated that DFSCs can promote periodontal regeneration in an inflammatory microenvironment, and this regenerative effect may be related to high expression of periostin. Studies have shown that in response to inflammatory stimuli, DFSCs highly express periostin, recruiting and reprogramming macrophages and improving the immune microenvironment of the defect area. However, silencing periostin diminishes the effects of DFSCs in promoting periodontal regeneration and regulating macrophages. Recombinant human periostin (rhPeriostin) not only directly promotes macrophage reprogramming through the integrinαM / phosphorylated extracellular signal-regulated kinase (p-Erk) / Erk signaling pathway, but also demonstrates potential to promote periodontal regeneration in rats when loaded into a collagen matrix. These results suggest that periostin may be a molecular target for DFSCs to promote periodontal regeneration. However, there are no reports in the prior art on enhancing the expression of periostin in DFSCs by improving extraction and culture methods alone.

[0004] Therefore, providing a method for extracting and culturing dental follicle mesenchymal stem cells that 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-forming abilities, has strong multidirectional differentiation ability, and can also highly express periostin.

[0006] A 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 applications of the dental follicle mesenchymal stem cells that highly express periostin.

[0008] To achieve the above object, 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 the preservation number CCTCCNO: C2024384, the preservation time is December 6, 2024, and the preservation address is: 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, minced, digested, and centrifuged. The pellet is resuspended in primary culture medium and cultured until the cells are confluent. The tissue is then digested and centrifuged again. The pellet obtained from this second centrifugation is the primary dental follicle mesenchymal stem cells.

[0012] S2. Subculture: resuspend the dental follicle mesenchymal stem cells from step S1 in subculture medium and 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 to 50 units / mL.

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

[0018] The preparation method of the primary culture medium comprises the following steps: uniformly mixing 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 culture medium contains nucleosides but does not contain phenol red.

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

[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 reaches 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 and 5% CO2.

[0029] In some embodiments of the present invention, the subculture conditions in step S2 are 37±0.5°C and 5% CO2.

[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 that overexpress periostin can promote macrophage migration and reprogram macrophages into the M2 phenotype. M2 macrophages can promote, but are not limited to, skin wound healing, nerve damage repair, vascularization, osteogenesis, and tissue anti-aging.

[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] Dental follicle mesenchymal stem cells extracted and cultured using the present method exhibit excellent passage stability, strong self-renewal and clone-forming abilities, and robust multidirectional differentiation capabilities. Furthermore, the applicant unexpectedly discovered that the present method enhances periostin expression in dental follicle mesenchymal stem cells, thereby enhancing their ability to promote periodontal regeneration and regulate macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0041] Attachment Figure 4 The right image is a partial magnification of the left image.

[0042] Attachment Figure 5 Figures 1 and 2 show the results of an investigation into the multidirectional differentiation ability of P5 dental follicle mesenchymal stem cells extracted and cultured using the method of the present invention. Figure A shows the results of Alizarin Red staining of dental follicle mesenchymal stem cells cultured under osteogenic induction conditions for 21 days; Figure B shows the results of Oil Red O staining of dental follicle mesenchymal stem cells cultured under adipogenic induction conditions for 14 days; and Figure C shows the results of Alcian Blue staining of dental follicle mesenchymal stem cell microspheres cultured under chondrogenic induction conditions for 28 days.

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

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

[0045] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are 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 examples of the present invention were 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 examples of the present invention were both 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 medium to obtain the 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 UltraGRO TM -Advanced; In some embodiments of the present invention, the MEMα culture medium contains nucleosides but does not contain phenol red.

[0053] Example 1

[0054] This embodiment discloses the extraction and culture method of 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, passage number, cell batch number, seeding density, date, and operator.

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

[0058] Transfer the cleaned dental follicle tissue to a sterile EP tube. Mark the scale, then transfer the tissue to a new sterile EP tube. Fill the original EP tube with 0.9% sodium chloride injection to the marked scale line. Measure the volume (V) of the added 0.9% sodium chloride injection to estimate the dental follicle tissue weight. Dental follicle tissue weight = V x 1g / mL. Tissue weight must be ≥ 0.5g.

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

[0060] S14. Count and resuspend: according to 0.075g / 75cm 2 Calculate the number of inoculated bottles for cell culture flasks, using 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% CO2 incubator. Perform a complete medium change every 3 days and observe the tissue and cell adhesion and contamination under a microscope. The primary culture period is 16-22 days. When the cell confluence reaches more than 70% under a microscope, the culture supernatant from the final medium change is sterile.

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

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

[0064] S22. Digestion and centrifugation: Add 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 stop the digestion. 2 Transfer all the liquid and cells in the cell culture flask to a 50mL centrifuge tube and add 5mL / 75cm 2 Wash the cell culture flask with sodium chloride injection (0.9%) and add the washing solution into a 50 mL centrifuge tube. A 50 mL centrifuge tube can accommodate four 75 cm 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 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 cm 2 Cell culture flasks, each 225cm 2The 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 into a 37.0°C, 5% CO2 incubator for culture, so that the cells were evenly distributed.

[0066] S3. Dental follicle mesenchymal stem cells passaged from P1 to 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, passage 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 flask was aspirated and discarded in a biosafety cabinet at 225 cm 2 Add 10-15 mL of sodium chloride injection (0.9%) to the culture supernatant in the cell flask and gently shake the culture surface for 10 seconds, then discard the washing solution and repeat the washing operation.

[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, gently tap 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, you can choose to culture them in a 10-layer cell factory. After inoculating the 10-layer cell factory, if there are remaining cells that are not enough to inoculate a 10-layer cell factory, you can inoculate them 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 subculture medium. The amount of subculture 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 insufficient to inoculate a 10-layer cell factory, use 5000 cells / cm 2 -6500 cells / cm 2 The cells were passaged to a density of 225 cm 2 Cell flask, every 225cm 2 The volume of culture medium added to the cell flask was 45 mL, and then the mixture was shaken in a cross direction for 225 cm. 2 Each time the cells are subcultured, at least one 225cm 2 The cell flask was used as an observation flask for cell confluence. 2 The cell flask was returned to a 37.0°C, 5% CO2 incubator for culture.

[0071] S4. Dental follicle mesenchymal stem cells passaged from P2 to P3

[0072] S41. Preparation: Take 10 layers of cell factories and 225cm 2 Cell culture flasks should be labeled in the biosafety cabinet with the following information: unique code, passage number, cell batch number, cell culture density, date, and operator.

[0073] S42. Aspirate and discard the supernatant, digest, and centrifuge: Take the 225cm in step S34 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 layer of the culture surface. After gently shaking 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, gently tap to remove cells, and place in a biosafety cabinet at 350mL-400mL / 225cm 2 Sodium chloride injection (0.9%) was added to the cell culture flask to stop 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 2The 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 Cell culture flask, discard the culture supernatant in the biosafety cabinet; add 10-15mL sodium chloride injection (0.9%) and gently shake the culture surface for 10 seconds, discard the washing solution, and repeat the washing operation; press 3mL / 225cm 2 Gibco cell culture flasks TM TrypLE TM Select enzyme (1X), without phenol red, digest at room temperature for 5-7 minutes, gently tap 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 stop the digestion. 2 Transfer all the liquid and cells in the cell culture flask to a 225mL centrifuge tube and add 15mL / 225cm 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 count three samples. 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, you can choose to culture them in a 10-layer cell factory. After inoculating the 10-layer cell factory, if there are remaining cells that are not enough to inoculate a 10-layer cell factory, you can inoculate them 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 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, use 5000 cells / cm 2 ~6500 cells / cm 2 The cells were passaged to a density of 225 cm 2 Cell culture flasks, each 225cm 2 The volume 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 225cm2 Cell culture flasks were used as observation flasks for cell confluence. 2 The cell culture flask was returned to the carbon dioxide incubator with a CO2 concentration of 5.0% and a temperature of 37.0°C for culture.

[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 a 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 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, gently tap to remove cells, and place in a biosafety cabinet at 350mL-400mL / 225cm 2 Sodium chloride injection (0.9%) was added to the cell culture flask to stop 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 flasks, collect part of the culture supernatant of each 10-layer cell factory in the biosafety cabinet and mix them for testing, and aspirate and discard 225cm 2 The remaining culture supernatant in the cell culture flask was added with 10-15 mL of sodium chloride injection (0.9%) and gently shaken to wash the culture surface for 10 seconds, then the washing solution was discarded and the washing operation was repeated; the washing was continued at 3 mL / 225 cm 2 Gibco cell culture flasks TM TrypLE TM Select enzyme (1X), without phenol red, digest at room temperature for 5-7 minutes, gently tap to remove cells, and press 12mL / 225cm in a biosafety cabinet. 2Sodium chloride injection (0.9%) was added to the cell culture flask to stop the digestion. 2 Transfer all the liquid and cells in the cell culture flask to a 225mL centrifuge tube and add 15mL / 225cm 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%). Three samples were taken for counting. The resulting pellet suspension was the harvested human dental follicle mesenchymal stem cell principal 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 minutes, the supernatant was discarded, and the tubes were 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 20 minutes; centrifuged at 400g for 5 minutes, 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 on 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 spindle-shaped 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 percentages in each panel represent the expression levels 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 passage medium and cultured at 5000 cells / cm 2 The cells were inoculated into culture flasks and cultured for 72-96 hours. The supernatant was discarded and the cells were washed with physiological saline. The dental follicle mesenchymal stem cells were harvested and counted using recombinant trypsin digestion solution. The number of cells harvested at the P1 generation was counted. The above process was repeated to passage to the P7 generation.

[0090] The population doubling time (PDT) was calculated using the formula PDT = [log2 / (logNt - lgN0)] × t, where t is the culture time from cell seeding to harvest, Nt is the number of cells harvested at time t, and N0 is the initial seeding number. After determining the population doubling times for P0-P1, P1-P2, ..., P6-P7, a population doubling time curve was plotted.

[0091] 2. Colony formation assay:

[0092] The P5 dental follicle mesenchymal stem cells of this experimental example were collected and seeded at 500 cells / 60 mm culture dish. 3 mL of subculture medium was added and cultured at 37°C and 5% CO2. After colony formation (about 10-12 days, the number of cells in each colony was >50), the culture was terminated, and 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 is 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 abilities.

[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. Assessment of osteogenic differentiation ability

[0097] Prepare osteogenic differentiation medium according to the instructions of the human stem cell osteogenic differentiation kit, store at 4°C and use within one month. 4 cells / cm 2 Cells were seeded at a density of 100 μg / mL in a 12-well plate. 1 mL of cell culture medium (SLCPCA complete medium) was added to each well and cultured in a 37°C, 5% CO2, saturated humidity incubator. When the cell confluence reached 70%, 1 mL of osteogenic differentiation induction medium was replaced. Fresh osteogenic differentiation induction medium was used every three days. After three weeks of culture, the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with Alizarin Red to observe calcium nodules.

[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. After 21 days of osteogenic induction culture, a large number of calcium nodules can be generated, and Alizarin red staining is strongly positive.

[0099] 2. Assessment of Adipogenic Differentiation Ability

[0100] Prepare adipogenic differentiation medium A and medium B according to the instructions of the human stem cell adipogenic differentiation kit, store at 4°C and use within one month. 4 cells / cm 2 Cells were seeded at a density of 1 mL in a 12-well plate. 1 mL of cell culture medium (SLCPCA complete medium) was added to each well and cultured in a 37°C, 5% CO2, saturated humidity incubator. When the cell confluence reached 100%, 1 mL of adipogenic differentiation induction medium A was replaced. After 3 days of induction, the medium was replaced with adipogenic differentiation induction medium B. After 1 day of induction, medium B was aspirated and replaced with medium A for induction. Medium A and medium B were used alternately. After 2 weeks of induction, the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with Oil Red O to observe lipid droplet formation.

[0101] The results of 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. Assessment of Chondrogenic Differentiation Ability

[0103] Prepare the chondrogenic differentiation premix and complete culture medium according to the instructions of the human stem cell chondrogenic differentiation kit, store at 4°C and use within one month. 5 Transfer P5 dental follicle mesenchymal stem cells to be induced into a 15 mL centrifuge tube and centrifuge at 250 g for 4 minutes at 20°C. Aspirate the supernatant and resuspend in 0.5 mL of chondrogenic differentiation premix. Centrifuge at 150 g for 5 minutes at 20°C. Repeat this step to wash the cells again. Resuspend in 0.5 mL of complete chondrogenic differentiation medium and centrifuge at 150 g for 5 minutes at 20°C. Loosen the cap of the centrifuge tube to facilitate gas exchange and incubate the tube upright in a 37°C, 5% CO2, saturated humidity incubator. After 24 hours, if cells begin to aggregate, gently tap the bottom of the centrifuge tube to suspend the chondrocytes in the liquid. Replace the tube with 0.5 mL of fresh complete chondrogenic differentiation medium every 2-3 days, gently tapping the bottom of the tube after each medium change. Continue induction for 28 days to form chondrocytes approximately 1 mm in diameter. Fix with 4% paraformaldehyde for 30 minutes, dehydrate with graded alcohols, embed in paraffin, and serially section at 4 μm for observation using Alcian blue staining.

[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 Alcian blue, showing the internal 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 cultured under standard culture conditions. Standard culture conditions refer to the substitution of α-MEM medium (containing nucleosides but not phenol red) for the primary and secondary culture medium, while remaining the same as in Example 1.

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

[0109] The steps for real-time PCR periostin-related gene detection are as follows:

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

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

[0112] (3) The extracted RNA concentration and 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 extracted RNA was of high purity and could be used for subsequent experiments.

[0113] (4) RNA was reverse transcribed using a reverse transcription kit containing the following components: RNase-free ddH2O, 4× gDNA wiper mix, 5× HiScript III qRT Super Mixa, and 5× No RT Control Mixb. Reverse transcription was performed according to the following steps:

[0114] ① Genomic DNA removal

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

[0116] Table 1

[0117] <![CDATA[RNase-free ddH2O]]> to 16 μL 4×gDNA wiper mix 4 μL Template RNA Total RNA: 1pg-1μg

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

[0119] ② Prepare the reverse transcription reaction system: Add 4 μL of 5× HiScript III qRT SuperMix directly to the reaction tube from step 1. Gently pipette to mix thoroughly and briefly centrifuge. Transfer to a reverse transcription instrument and perform the reverse transcription reaction: 37°C for 15 minutes → 85°C for 5 seconds to obtain cDNA.

[0120] (5) Real-time PCR reaction

[0121] ① The primer sequences were designed and synthesized by Sangon Biotech (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., Cat. 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 ice 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 10 μL Front primer 1 μL rear primer 1 μL cDNA 2μL <![CDATA[ddH2O]]> 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: initial denaturation: 95°C, 30 seconds, 1 cycle; PCR cycling: 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 levels of genes were 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 higher periostin expression levels than the same generation dental follicle mesenchymal stem cells cultured under conventional conditions. The expression of periostin in the P5 generation dental follicle mesenchymal stem cells from Example 1 and conventional culture was slightly lower than that in the P3 generation, but the expression of periostin in the P5 generation dental follicle mesenchymal stem cells from Example 1 was still significantly higher than that in the P3 generation dental follicle mesenchymal stem cells cultured under conventional 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 experimental 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 serum albumin and counted, and the density of dental follicle mesenchymal stem cells was adjusted to 5×10 7 cells / mL, and 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 30 to 45 years old;

[0139] (2) Subjects were found to have a two-wall or three-wall subosseous pocket with a PD ≥ 5 mm, or grade I / II furcation lesions during 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 for at least 1 year after surgery;

[0142] (5) The subjects were able to complete good self-plaque control, including brushing teeth correctly and using dental floss correctly.

[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 radiographic examinations show untreated acute infection at the surgical site, apical lesions, root fractures, severe root deformities, difficult-to-remove cementum / enamel protrusions, untreated caries at the cementoenamel junction or root surface, metal restorations or restorations that extend subgingivally and / or poorly fitted restoration margins at or below the cementoenamel 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) Suffering from malignant tumors or having a history of malignant tumors;

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

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

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

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

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

[0157] (14) Patients received other research treatments within 30 days of surgery;

[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, Behçet'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 enrolled and divided into the fresh dental follicle mesenchymal stem cell treatment group (DFSC group): 11 cases, and the normal saline control group (NS group): 4 cases. Each patient received the following treatment:

[0164] (1) Under local anesthesia, the study tooth 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 equal volume of sterile saline into the periodontal pocket;

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

[0170] (7) One week later, the suppository will be removed after a follow-up visit.

[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 at six sites of the treated teeth using a Florida electronic periodontal probe: probing depth (PD), which is the distance from the gingival margin to the bottom of the gingival sulcus; clinical attachment level (CAL), which is the distance from the CEJ (cementoenamel junction) to the bottom of the gingival sulcus; and gingiva recession (GR), which is the distance from the CEJ to the gingival margin.

[0174] (2) Radiographic 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, 6, 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 dental radiologists who were unaware of the subjects' grouping independently measured the CBCT results.

[0175] V. Research Results

[0176] The results are as 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 months, 6 months 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 levels of decrease 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 3 months, 6 months, and 12 months after injection (Figure C).

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

[0181] (5) Figure 7As shown in Figure F, compared with the NS group, the mean bone defect depth of the DFSC group decreased 3 months, 6 months, and 12 months after dental follicle mesenchymal stem cell injection, but there was no statistical significance between the two groups. This may be limited by the individual error of the small sample and cannot show the statistical difference 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 months, 6 months, and 12 months after treatment, ( Figure 7 H), the depth of bone defect in DFSC group decreased significantly at 3 and 6 months after injection, while there was no significant change in NS group. No significant change was found in bone defect width 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 baseline before treatment; "3m," "6m," and "12m" indicate 3, 6, and 12 months after periodontal pocket treatment. Intergroup comparisons were performed using a two-way analysis of variance with repeated measures, and intragroup comparisons were performed using a one-way analysis of variance with repeated measures. "*" indicates statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001; "ns" indicates not statistically significant. Error bars represent mean ± SD.

[0185] The present invention has completed screening and clinical trials on 15 subjects, preliminarily confirming that the use of allogeneic dental follicle mesenchymal stem cells for periodontal tissue regeneration showed no significant abnormalities in immunological indicators, a good safety profile, and no significant adverse reactions. Periodontal probing examinations and imaging analysis of the subjects showed improvement in all periodontal probing parameters at 3, 6, and 12 months after dental follicle mesenchymal stem cell injection. CBCT imaging analysis showed a significant reduction in bone defect depth and volume. Therefore, DFSC injection can, to a certain extent, promote periodontal tissue regeneration at the bone defect site, 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 merely preferred embodiments of the present invention and are intended to illustrate the technical solutions of the present invention, not to limit the present invention, and certainly not to limit the scope of the present invention. Any modifications or improvements that are insignificant to the main design concept and spirit of the present invention, provided that the technical problems they solve are consistent with the present invention, shall be included within the scope of protection of the present invention. Furthermore, any direct or indirect application of the technical solutions of the present invention to other related technical fields shall also be included within the scope of protection of the present invention.

Claims

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

2. The use of dental follicle mesenchymal stem cells that highly express periostin according to claim 1, characterized in that: Application in the preparation of medicines for treating periodontitis.

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