A cryopreserved dental follicle mesenchymal stem cell injection and its preparation method and application

Abstract

The present invention discloses a frozen dental follicle mesenchymal stem cell injection, a preparation method and an application thereof, and belongs to the field of biomedical technology. The frozen dental follicle mesenchymal stem cell injection comprises a cryopreservation protection solution and human dental follicle mesenchymal stem cells MSC with a preservation number of CCTCC NO: C2024384, the cryopreservation protection solution comprises 8-12 parts of human serum albumin, 35-45 parts of sodium chloride injection, and 45-55 parts of CryoStor® CS10 cell freezing solution; the density of the dental follicle mesenchymal stem cells is 0.5×10 7 ~20×10 7 The present invention also discloses a method for preparing the injection and its use in preparing a drug for preventing or / and treating periodontal diseases. The injection of the present invention can be used directly after thawing, can maintain the activity and biological properties of dental follicle mesenchymal stem cells, enhance their osteogenic and chondrogenic abilities, and has a good effect in treating periodontal diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a cryopreserved dental follicle mesenchymal stem cell injection, and a preparation method and application thereof. Background Art

[0002] At present, stem cell therapy is considered to be one of the most cutting-edge and promising research directions in the field of disease treatment and tissue regeneration. In order to achieve effective disease treatment and tissue regeneration, it is crucial to find the right type of stem cells and ensure that these cells function efficiently in vivo. Studies have shown that dental stem cells are derived from the neural crest (ectodermal mesenchymal leaves) and have strong self-renewal ability and multidirectional differentiation potential. In addition, this type of stem cell has a wide range of sources, is not subject to ethical restrictions, is easy to culture on a large scale, and has a low immune response. Compared with mesenchymal stem cells derived from the mesoderm, dental stem cells, as neural crest-derived mesenchymal stem cells, show a stronger tendency to differentiate into neurons and chondrocytes, as well as significant immunomodulatory and anti-inflammatory effects, which gives them unique advantages in disease treatment and tissue regeneration. So far, dental stem cells have been used in many preclinical studies and clinical trials to treat a variety of diseases, including inflammatory diseases, neurodegenerative diseases, organ damage, autoimmune diseases, orthopedic diseases, and diabetes, showing their great potential in tissue repair and disease treatment.

[0003] 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) according to their different tissue sources. Among them, PDLSCs, DPSCs and SHED are all derived from mature tissues, while DFSCs are derived from the tooth germ development stage. The dental follicle is a layer of loose connective tissue that surrounds the enamel organ and the base of the dental papilla during tooth germ development and originates from the ectodermal mesenchyme. The dental follicle will eventually develop into cementum, periodontal ligament and alveolar bone in the periodontal tissue. In clinical practice, when impacted teeth are extracted, the dental follicle tissue in the neck is usually discarded together with the teeth as medical waste. Therefore, dental follicle tissue is an easily accessible and patient-friendly source of stem cells.

[0004] Periodontitis is a common infectious disease. Clinical evidence and research have confirmed that periodontal infection may be an important infectious factor for cardiovascular and cerebrovascular diseases (such as atherosclerosis, myocardial infarction, stroke, etc.), diabetes, pregnancy complications, respiratory infections, rheumatoid arthritis, and Alzheimer's disease. The incidence of the above-mentioned systemic diseases in patients with severe periodontitis is significantly increased, seriously affecting overall health. The prevention and treatment of periodontal disease has become a major problem that needs to be urgently addressed in the medical field. The regeneration of periodontal tissue includes not only hard tissue regeneration, that is, new cementum is formed on the root surface of the diseased tooth and the normal height of the alveolar bone is restored, but also the new periodontal ligament fibers are embedded in the newly formed cementum and alveolar bone. However, existing periodontitis treatments are difficult to achieve ideal tissue regeneration.

[0005] At present, a large number of preclinical and clinical studies use fresh cells as the form of stem cell drugs, but fresh stem cell drugs have high production costs, delayed test results, short shelf life, difficult storage and long-distance transportation, which are not conducive to later clinical research and wide application. Therefore, it is urgent to develop frozen stem cell drugs that can be directly used in clinical practice. Summary of the invention

[0006] One of the purposes of the present invention is to provide a cryopreserved dental follicle mesenchymal stem cell injection, which can not only maintain the high activity and basic biological properties of dental follicle mesenchymal stem cells, but also has the same periodontal disease treatment effect as fresh dental follicle mesenchymal stem cells.

[0007] The second object of the present invention is to provide a method for preparing the cryopreserved dental follicle mesenchymal stem cell injection.

[0008] The third object of the present invention is to provide an application of the cryopreserved dental follicle mesenchymal stem cell injection.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] In the first aspect, the present invention discloses a cryopreserved dental follicle mesenchymal stem cell injection, comprising a cryopreservation protective solution and dental follicle mesenchymal stem cells, wherein the dental follicle mesenchymal stem cells are human dental follicle mesenchymal stem cells MSC, which are preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C2024384, a preservation date of December 6, 2024, and a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0011] The cryopreservation protection solution includes the following components by volume: 8-12 parts of human albumin, 35-45 parts of sodium chloride injection, and 45-55 parts of CryoStor® CS10 cell freezing solution.

[0012] The dental follicle mesenchymal stem cells of the present invention have good passage stability, strong self-renewal and clone formation capabilities, strong multidirectional differentiation ability, and highly express dental follicle mesenchymal stem cell periostin.

[0013] The density of the dental follicle mesenchymal stem cells in the frozen dental follicle mesenchymal stem cell injection is 0.5×10 7 ~20×10 7 cells / mL.

[0014] In some embodiments of the present invention, the cryopreservation protection solution includes the following components in volume parts: 9-11 parts of human albumin, 37-43 parts of sodium chloride injection, and 47-52 parts of CryoStor® CS10 cell freezing solution.

[0015] In some embodiments of the present invention, the specification of human albumin is 20wt%; the sodium chloride content in the sodium chloride injection is 9wt%.

[0016] In some embodiments of the present invention, the density of dental follicle mesenchymal stem cells is 1×10 7 ~7×10 7 cells / mL.

[0017] In a second aspect, the present invention discloses a method for preparing a cryopreserved dental follicle mesenchymal stem cell injection, comprising the following steps: uniformly mixing the dental follicle mesenchymal stem cells with a cryopreservation protection solution at a specified density, cryopreserving them by programmed cooling, and storing them in liquid nitrogen for a long time.

[0018] In some embodiments of the present invention, dental follicle mesenchymal stem cells are obtained, expanded and cultured, and cells of generations P3 to P6, preferably P5, are harvested for the preparation of cryopreserved dental follicle mesenchymal stem cell injections.

[0019] In some embodiments of the present invention, the dental follicle mesenchymal stem cells are cultured as follows: the dental follicle mesenchymal stem cells are resuspended in a subculture medium at a density of 5000-6500 cells / cm 2 The cells were inoculated at a density of 100 μg / cm2 and cultured at 37±0.5℃ and 5% CO2. When the cell confluence reached 70-90%, subculture was performed.

[0020] 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.

[0021] In some embodiments of the present invention, the serum-free cell culture additive is UltraGRO™-Advanced;

[0022] In some embodiments of the present invention, the MEMα medium contains nucleosides but does not contain phenol red.

[0023] The programmed cooling step in the present invention is a prior art. In some embodiments of the present invention, the programmed cooling step is as follows: waiting to cool down to 2~6°C; cooling down at 0.8~1.2°C / min until the sample probe temperature reaches -6~0°C; cooling down at 20~30°C / min until the chamber probe temperature reaches -40°C±5°C; cooling down at 8.0~12.0°C / min until the chamber probe temperature reaches -12°C±5°C; cooling down at 0.8~1.2°C / min until the chamber probe temperature reaches -40°C±5°C; cooling down at 8.0~12.0°C / min until the chamber probe temperature reaches -90°C±5°C.

[0024] In some embodiments of the present invention, the programmed cooling steps are as follows: wait for cooling to 4°C; cool down at 1.0°C / minute until the sample probe temperature reaches -4°C; cool down at 25.0°C / minute until the chamber probe temperature reaches -40°C; cool down at 10.0°C / minute until the chamber probe temperature reaches -12°C; cool down at 1.0°C / minute until the chamber probe temperature reaches -40°C; cool down at 10.0°C / minute until the chamber probe temperature reaches -90°C±5°C.

[0025] In a third aspect, the present invention discloses an application of the above-mentioned cryopreserved dental follicle mesenchymal stem cell injection, wherein the application is an application in the preparation of a drug for preventing and / or treating periodontal diseases.

[0026] In some embodiments of the present invention, the invention provides a method for preparing a drug for promoting periodontal regeneration.

[0027] The frozen dental follicle mesenchymal stem cell injection of the present invention can be used directly after thawing without washing.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The cryopreserved dental follicle mesenchymal stem cell injection of the present invention comprises a cryopreservation protection solution and dental follicle mesenchymal stem cells. The cryopreservation protection solution can not only maintain the high activity and basic biological properties of the dental follicle mesenchymal stem cells, but also enhance the osteogenic and chondrogenic abilities of the dental follicle mesenchymal stem cells, while having low tissue irritation and toxicity.

[0030] The cryopreserved dental follicle mesenchymal stem cell injection of the present invention mainly resides in the local area after injection and hardly spreads and metastasizes throughout the body, has no obvious systemic toxicity, and has the same periodontal disease treatment efficacy as fresh dental follicle mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Attached Figure 1 The figure is the result of investigating the number of living cells and the cell survival rate of Experimental Example 1;

[0032] Attached Figure 2 This is a graph showing the results of investigating the effective cell resuscitation rate in Experimental Example 1;

[0033] Attached Figure 3 The figure is the result of investigating the number of living cells and cell survival rate in Experimental Example 2;

[0034] Attached Figure 4 This is a graph showing the results of investigating the proliferation of DFSCs in Experimental Example 4;

[0035] Attached Figure 5 The results of the investigation of the scratch healing of DFSCs in Experimental Example 4 are shown in Figure A, where Figure A is a micrograph of the scratch healing of each group, and Figure B is a statistical result of the scratch healing of each group;

[0036] Attached Figure 6 The β-galactosidase staining result diagram of DFSCs in Experimental Example 4, wherein Figure A is the staining result diagram of each group, and Figure B is the statistical result diagram of the staining situation of each group;

[0037] Attached Figure 7 The results of the cloning of DFSCs in Experimental Example 4 are shown in Figure A, where Figure A is a result of cloning in each group, the lower figure in Figure A is a partial enlarged view of the upper figure, and Figure B is a statistical result of the number of cell clones in each group;

[0038] Attached Figure 8 This is a graph showing the results of investigating the expression levels of Oct3 / 4, P16 and P21 of the effective surviving cells in Experimental Example 5;

[0039] Attached Fig. 9 The results of alizarin red staining for osteogenic differentiation potential investigation in Experimental Example 6 are shown, wherein Figure A is a macroscopic and microscopic image of alizarin red staining after 21 days of osteogenic differentiation induction, and Figure B is a quantitative result of alizarin red staining after 21 days of osteogenic differentiation induction;

[0040] Attached Fig.10 The results of RT-PCR analysis of osteogenic differentiation potential in Experimental Example 6 are shown, wherein Figure A shows the expression of early osteogenic differentiation-related genes by RT-PCR analysis of cells collected from each group after 7 days of osteogenic differentiation induction, Figure B shows the expression of mid- and late-stage osteogenic differentiation-related genes by RT-PCR analysis of cells collected from each group after 14 days of osteogenic differentiation induction, and Figure C shows the expression of mid- and late-stage osteogenic differentiation-related genes by RT-PCR analysis of cells collected from each group after 21 days of osteogenic differentiation induction;

[0041] Attached Fig.11The ALP color development results of the osteogenic differentiation potential investigation of Experimental Example 6, wherein Figure A is a macroscopic and microscopic ALP color development image after 3 days of osteogenic differentiation induction, Figure B is a semi-quantitative analysis image of ALP color development after 3 days of osteogenic differentiation induction, Figure C is a macroscopic and microscopic ALP color development image after 7 days of osteogenic differentiation induction, and Figure D is a semi-quantitative analysis image of ALP color development after 7 days of osteogenic differentiation induction;

[0042] Attached Fig.12 Figures are the results of the adipogenic differentiation potential investigation of Experimental Example 6, wherein Figure A is a micrograph of Oil Red O staining of each group after 21 days of adipogenic differentiation induction culture, Figure B is a graph of the expression of adipogenic differentiation-related genes analyzed by RT-PCR by collecting cells from each group after 7 days of adipogenic induction, and Figure C is a graph of the expression of adipogenic differentiation-related genes analyzed by RT-PCR by collecting cells from each group after 21 days of adipogenic induction;

[0043] Attached Fig.13 The results of the investigation of chondrogenic differentiation potential of Experimental Example 6 are shown in Figure A, wherein Figure A shows the general appearance of chondrogenic spheres formed after 28 days of chondrogenic differentiation induction and micrographs of sections stained with Alcian blue, Figure B shows the expression of chondrogenic related genes in cells collected from each group after 7 days of chondrogenic induction and analyzed by RT-PCR, and Figure C shows the expression of chondrogenic related genes in cells collected from each group after 21 days of chondrogenic induction and analyzed by RT-PCR;

[0044] Attached Fig.14 This is a MicroCT image of the periodontal bone defect of SD rats treated in Experimental Example 8;

[0045] Attached Fig.15 This is a statistical graph of the results of treating periodontal bone defects in SD rats in Experimental Example 8;

[0046] Attached Fig.16 This is a graph showing the results of Experimental Example 9 on the improvement of gum bleeding in beagle dogs with periodontitis bone defects; Fig.16 A is a graph showing the gingival bleeding index scores at each observation time point within 12 weeks of medication. Fig.16 B is a graph showing the improvement of the bleeding index scores of each group after 12 weeks of medication relative to the baseline level before medication;

[0047] Attached Fig.17 This is a graph showing the results of probing depth investigation of periodontitis bone defects in beagle dogs in Experimental Example 9; Fig.17 A is the periodontal probing depth at each observation time point within 12 weeks of medication. Fig.17 B is a graph showing the improvement of periodontal probing depth in each group after 12 weeks of administration compared with the baseline level before administration;

[0048] Attached Fig.18 This is a graph showing the results of Experimental Example 9 on the improvement of attachment level loss in periodontitis bone defects in beagle dogs; Fig.18A is a graph showing the loss of periodontal clinical attachment level at each observation time point within 12 weeks of administration. Fig.18 B is a graph showing the improvement of the periodontal clinical attachment level in each group after 12 weeks of administration compared with the baseline level before administration;

[0049] Attached Fig.19 The Micro-CT analysis and statistical results of the periodontitis bone defect repair in beagle dogs in each group of Experimental Example 9 are shown in Figure 1. Fig.19 A Horizontal cross-sectional view of Micro-ct analysis (the arrow indicates the periodontal bone defect modeling area); Fig.19 B~E are the statistical results of Micro-CT analysis of BV / TV (new bone volume fraction), Tb.Th (new bone trabecular thickness), Tb.N (new bone trabecular number), and Tb.Sp (new bone trabecular separation) of the modeling defect site;

[0050] Attached Fig. 20 The hematological test results of Test Example 10 are Figure 1 ;

[0051] Attached Fig.21 The hematological test results of Test Example 10 are Figure 2 ;

[0052] Attached Fig. 22 This is a graph showing the blood biochemical test results of Test Example 10;

[0053] Attached Fig.23 This is a diagram of the lymphocyte subset detection results of Test Example 10;

[0054] Attached Fig.24 The figure is the complement, circulating immune complex and immunoglobulin detection result of Experimental Example 10;

[0055] Attached Fig.25 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

[0056] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0057] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. The reagents and other instruments used without specifying the manufacturer are all conventional reagent products that can be purchased on the market.

[0058] The dental follicle mesenchymal stem cells described in the embodiment of the present invention are human dental follicle mesenchymal stem cells MSC, which are deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: C2024384, the deposit time is December 6, 2024, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0059] The β-galactosidase staining kit (article number G1580) described in the embodiment of the present invention is provided by Beijing Solebow Technology Co., Ltd.

[0060] The RNA extraction kit (Cat. No. RC112-01) and reverse transcription kit (Cat. No. R323-01) used in the experimental examples of the present invention were both provided by Nanjing Novezan Biotechnology Co., Ltd.

[0061] The CryoStor® CS10 cell freezing solution described in the examples of the present invention is provided by BioLife Solutions, Inc., USA.

[0062] The dental follicle mesenchymal stem cells are cultured as follows: the dental follicle mesenchymal stem cells are resuspended in a culture medium at a density of 5000-6500 cells / cm 2 The cells were inoculated at a density of 100 μg / cm2 and cultured at 37±0.5℃ and 5% CO2. When the cell confluence reached 70-90%, the cells were subcultured. Any cell generation from P3 to P6 was harvested for the preparation of frozen dental follicle mesenchymal stem cell injection.

[0063] The subculture medium described in the embodiments 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; the serum-free cell culture additive is UltraGRO™-Advanced; in some embodiments of the present invention, the MEMα culture medium contains nucleosides but does not contain phenol red.

[0064] Example 1

[0065] This embodiment discloses the cryopreserved dental follicle mesenchymal stem cell injection of the present invention and its preparation method.

[0066] The cryopreservation protection solution in this embodiment is composed of the following components: 10v% human albumin, 40v% sodium chloride injection, and 50v% CryoStor® CS10 cell freezing solution. The cryopreservation protection solution is prepared by taking human albumin, sodium chloride injection, and CryoStor® CS10 cell freezing solution, and mixing them evenly according to a certain proportion.

[0067] P5 dental follicle mesenchymal stem cells were mixed with cryopreservation protection solution at a rate of 5×10 7 The cells were mixed evenly at a density of 10 cells / mL, cryopreserved at programmed temperature, and stored in liquid nitrogen for a long time.

[0068] The programmed cooling steps are as follows: wait for the temperature to drop to 4°C; cool down at 1.0°C / min until the sample probe temperature reaches -4°C; cool down at 25.0°C / min until the chamber probe temperature reaches -40°C; cool down at 10.0°C / min until the chamber probe temperature reaches -12°C; cool down at 1.0°C / min until the chamber probe temperature reaches -40°C; cool down at 10.0°C / min until the chamber probe temperature reaches -90°C±5°C.

[0069] Example 2

[0070] This embodiment discloses the cryopreserved dental follicle mesenchymal stem cell injection of the present invention and its preparation method.

[0071] The cryopreservation protection solution in this embodiment is composed of the following components: 9v% human albumin, 41v% sodium chloride injection, and 50v% CryoStor® CS10 cell freezing solution. The cryopreservation protection solution is prepared by taking human albumin, sodium chloride injection, and CryoStor® CS10 cell freezing solution, and mixing them evenly according to a certain proportion.

[0072] P3 dental follicle mesenchymal stem cells were mixed with cryopreservation protection solution at a rate of 1×10 7 The cells were mixed evenly at a density of 10 cells / mL, cryopreserved at programmed temperature, and stored in liquid nitrogen for a long time.

[0073] The programmed cooling steps of this embodiment are as follows: wait for the temperature to drop to 2°C; cool down at 0.8°C / minute until the sample probe temperature reaches 0°C; cool down at 20°C / minute until the cabin probe temperature reaches -35°C; cool down at 8.0°C / minute until the cabin probe temperature reaches -7°C; cool down at 0.8°C / minute until the cabin probe temperature reaches -35°C; cool down at 8.0°C / minute until the cabin probe temperature reaches -90°C±5°C.

[0074] Example 3

[0075] This embodiment discloses the cryopreserved dental follicle mesenchymal stem cell injection of the present invention and its preparation method.

[0076] The cryopreservation protection solution in this embodiment is composed of the following components: 11v% human albumin, 37v% sodium chloride injection, and 52v% CryoStor® CS10 cell freezing solution. The cryopreservation protection solution is prepared by taking human albumin, sodium chloride injection, and CryoStor® CS10 cell freezing solution, and mixing them evenly according to a certain proportion.

[0077] P6 dental follicle mesenchymal stem cells were mixed with cryopreservation protection solution at a rate of 0.5×10 7 The cells were mixed evenly at a density of 10 cells / mL, cryopreserved at programmed temperature, and stored in liquid nitrogen for a long time.

[0078] The programmed cooling steps of this embodiment are as follows: wait for the temperature to drop to 6°C; cool down at 1.2°C / minute until the sample probe temperature reaches -6°C; cool down at 30°C / minute until the chamber probe temperature reaches -45°C; cool down at 12.0°C / minute until the chamber probe temperature reaches -17°C; cool down at 1.2°C / minute until the chamber probe temperature reaches -45°C; cool down at 12.0°C / minute until the chamber probe temperature reaches -90°C±5°C.

[0079] Example 4

[0080] This embodiment discloses the cryopreserved dental follicle mesenchymal stem cell injection of the present invention and its preparation method.

[0081] The cryopreservation protection solution in this embodiment is composed of the following components: 12v% human albumin, 43v% sodium chloride injection, and 45v% CryoStor® CS10 cell freezing solution. The cryopreservation protection solution is prepared by taking human albumin, sodium chloride injection, and CryoStor® CS10 cell freezing solution, and mixing them evenly according to a certain proportion.

[0082] P5 dental follicle mesenchymal stem cells were mixed with cryopreservation protection solution at 20×10 7 The cells were mixed evenly at a density of 10 cells / mL, cryopreserved at programmed temperature, and stored in liquid nitrogen for a long time.

[0083] The programmed cooling steps are as follows: wait for the temperature to drop to 4°C; cool down at 1.0°C / min until the sample probe temperature reaches -4°C; cool down at 25.0°C / min until the chamber probe temperature reaches -40°C; cool down at 10.0°C / min until the chamber probe temperature reaches -12°C; cool down at 1.0°C / min until the chamber probe temperature reaches -40°C; cool down at 10.0°C / min until the chamber probe temperature reaches -90°C±5°C.

[0084] Example 5

[0085] This embodiment discloses the cryopreserved dental follicle mesenchymal stem cell injection of the present invention and its preparation method.

[0086] The cryopreservation protection solution in this embodiment is composed of the following components: 8% by volume of human albumin and 45% by volume of sodium chloride injection and 47% by volume of CryoStor® CS10 cell cryopreservation solution. The cryopreservation protection solution is prepared by mixing human albumin, sodium chloride injection and CryoStor® CS10 cell cryopreservation solution in a certain proportion.

[0087] P4 dental follicle mesenchymal stem cells were mixed with cryopreservation protection solution at a rate of 7×10 7 The cells were mixed evenly at a density of 10 cells / mL, cryopreserved at programmed temperature, and stored in liquid nitrogen for a long time.

[0088] The programmed cooling steps are as follows: wait for the temperature to drop to 4°C; cool down at 1.0°C / min until the sample probe temperature reaches -4°C; cool down at 25.0°C / min until the chamber probe temperature reaches -40°C; cool down at 10.0°C / min until the chamber probe temperature reaches -12°C; cool down at 1.0°C / min until the chamber probe temperature reaches -40°C; cool down at 10.0°C / min until the chamber probe temperature reaches -90°C±5°C.

[0089] Test Example 1

[0090] In this experimental example, different cryopreservation protection solutions were used to prepare cryopreserved dental follicle mesenchymal stem cell injection solutions.

[0091] The cell cryopreservation protection solutions used in each group of this test example are shown in Table 1. The preparation methods of each cryopreservation protection solution are: respectively taking the formula raw materials and mixing them evenly according to the proportion.

[0092] Table 1 Cryopreservation solution formulas of Experimental Example 1

[0093]

[0094] 1. Prepare cryopreserved dental follicle mesenchymal stem cell injection solution using different cell cryopreservation protection solutions in Table 1, and cryopreserve in liquid nitrogen for one month, then thaw the dental follicle mesenchymal stem cells, and calculate the number of living cells and cell viability. Specifically: After digesting and counting P5 dental follicle mesenchymal stem cells, use different cryopreservation protection solutions in Table 1 at 5.00×10 6 After being cryopreserved in liquid nitrogen (-196°C) for one month, dental follicle mesenchymal stem cells were revived (placed in a preheated water bath at 37°C and incubated for 1 minute 40 seconds to 2 minutes) and the cell viability was calculated. The results are shown in Table 2 and Figure 1 shown.

[0095] Table 2 Results of viable cell count and cell survival rate in Experimental Example 1

[0096]

[0097] Table 2 and Figure 1 The * in the figure indicates that there is a significant difference compared with the experimental group at p≤0.05.

[0098] The results showed that there was a significant difference in the number of resuscitated cells between the experimental group and control group 3; there was a significant difference in cell viability between the experimental group and control groups 3 and 4.

[0099] 2. Resuspend the revived dental follicle mesenchymal stem cells in the culture medium at 2.00×10 6 pcs / 75cm 2 In the cell culture bottle, 5% CO2, 37℃ culture for 20 hours, digest and count, record the number of effective recovered cells, and calculate the effective cell recovery rate. The calculation formula for the effective cell recovery rate is:

[0100] Effective recovery rate (%) = number of effectively recovered cells / number of inoculated cells × 100%.

[0101] The results are shown in Table 3 and Appendix Figure 2 shown.

[0102] Table 3 Results of investigation on effective cell recovery rate of Experimental Example 1

[0103]

[0104] Table 3 and Appendix Figure 2 * indicates p≤0.05 compared with the experimental group, ** indicates p≤0.01 compared with the experimental group, and *** indicates p≤0.001 compared with the experimental group.

[0105] The above results show that the number of effectively revived cells and the effective cell recovery rate in the cryopreserved dental follicle mesenchymal stem cell injection of the experimental group of the present invention are better than those of the control groups, and the difference is statistically significant.

[0106] Test Example 2

[0107] This test example compares the cryopreservation effects of the cryopreserved dental follicle mesenchymal stem cell injection solution of the present invention and the cryopreservation solution made of a cryopreservation solution containing 5% DMSO.

[0108] The frozen dental follicle mesenchymal stem cell injection solution in this test example was prepared according to the method of test example 1. Another frozen solution containing 5% DMSO was taken and mixed evenly with dental follicle mesenchymal stem cells, and a control frozen dental follicle mesenchymal stem cell injection solution was prepared in the same way. The preparation method of the frozen solution containing 5% DMSO in this test example is as follows: 1.0mL of vitamin C injection solution (2mL: 0.5g) was mixed evenly with 500mL of serum-free cell culture additive (UltraGRO™-Advanced) to obtain a mixed solution; 25mL of the mixed solution was mixed evenly with 500mL of MEMα culture medium (containing nucleosides but not phenol red), and then DMSO was added and mixed evenly to make its volume content 5%, thereby obtaining a frozen solution containing 5% DMSO.

[0109] The frozen dental follicle mesenchymal stem cell injection of the present invention and the frozen dental follicle mesenchymal stem cell injection of the control were frozen for one month according to the method of Experimental Example 1, and then the dental follicle mesenchymal stem cells were revived, and the number of living cells and the cell viability were calculated. The results are shown in Table 4 and Attached Figure 3 shown.

[0110] Table 4 Results of viable cell count and cell survival rate in Experimental Example 2

[0111]

[0112] Table 4 and Appendix Figure 3 In the table, * indicates p≤0.05% compared with the experimental group, which is statistically different.

[0113] The above results show that the cryopreserved dental follicle mesenchymal stem cell injection solution of the experimental group of the present invention is superior to the cryopreserved dental follicle mesenchymal stem cell injection solution made with the cryopreservation solution containing 5% DMSO in terms of the viability and number of cells revived after cryopreservation, and the difference is statistically significant.

[0114] Test Example 3

[0115] This test example discloses the cryopreservation effects of different cell densities in the cryopreserved dental follicle mesenchymal stem cell injection of the present invention. Compared with Example 1, this test example differs in that the cell density is different, and the other conditions are the same.

[0116] After freezing the frozen dental follicle mesenchymal stem cell injection solutions with different cell densities in liquid nitrogen for one month, the cells were taken out for revival (same revival conditions as in Experiment 1) and counted, and the cell viability and viable cell density were counted to calculate the cell recovery rate. The calculation formula for the cell recovery rate is: number of viable cells after revival / number of cells before freezing × 100%

[0117] Table 5 Cryopreservation effects of different cell densities

[0118]

[0119] The above results show that the cell density of the frozen dental follicle mesenchymal stem cell injection solution of the present invention is 1×10 7 ~7×10 7 When the cell density was 100 cells / mL, the viability after recovery was higher than 90%, and the cell recovery rate was higher than 85%, and there was no significant difference in cell viability and cell recovery rate between different densities.

[0120] Test Example 4

[0121] This test example investigated the basic biological characteristics of dental follicle mesenchymal stem cells in the cryopreserved dental follicle mesenchymal stem cell injection of the present invention.

[0122] The cryopreserved dental follicle mesenchymal stem cell injection in this test example was prepared according to the method of Example 1. Another cryopreserved dental follicle mesenchymal stem cell injection was prepared by the same method using a commercial cryopreservation solution as a comparative example. The commercial cryopreservation solution described in this test example is CryoStor® CS10 cell cryopreservation solution provided by BioLife Solutions, USA. After each group of cryopreserved dental follicle mesenchymal stem cell injection solutions were cryopreserved in liquid nitrogen for one month, the basic biological characteristics were investigated. The details are as follows:

[0123] 1. Comparison of proliferation of P5 DFSCs in each group

[0124] 1.1 Experimental Grouping:

[0125] Fresh dental follicle mesenchymal stem cell group (DFSCs-F): P4 generation DFSCs were taken and cryopreserved dental follicle mesenchymal stem cell injection was prepared according to the method in Example 1; then cryopreserved for 1 month, taken out and revived. The revivification operation was: placed in a preheated water bath at 37°C and incubated for 1 minute 40 seconds to 2 minutes. Then centrifuged, resuspended in the subculture medium, and 5000 cells / cm 2 The cells were inoculated into T75 cells at a density of 1.00 × 10 and cultured in an incubator at 37°C with 5% CO2 for 96 h. The cells were collected and digested, centrifuged and resuspended in the subculture medium. 6 pcs / 75cm 2 Inoculate new T75 at a density of 100 μg / mL and place in a 37°C 5% CO2 incubator. Allow cells to adhere to the wall within 12-24 hours and then harvest the cells after digestion and centrifugation.

[0126] The frozen dental follicle mesenchymal stem cell injection group (DFSCs-C1) of the present invention: P5 generation DFSCs were taken and frozen dental follicle mesenchymal stem cell injection was prepared according to the method of Example 1; then frozen for 1 month, taken out, revived (same as DFSCs-F), washed and centrifuged, resuspended in the passage medium, and heated at 2.00×10 6 pcs / 75cm 2The cells were inoculated into T75 at a density of 100 μg / mL and allowed to adhere to the wall for 12-24 hours. The cells were then harvested after digestion and centrifugation.

[0127] Commercially cryopreserved dental follicle mesenchymal stem cell group (DFSCs-C2): Compared with the DFSCs-C1 group, the cryopreservation medium was replaced with commercially cryopreserved medium, and the other conditions were the same.

[0128] 1.2 Add 100 μL of each group of cell suspension to each well of a 96-well plate, 2000 cells per well, and culture in an incubator at 37°C with 5% CO2. Replace the culture medium every 2 days. Use CCK8 kit to detect proliferation, detect absorbance at 450nm, and use GraphPad Prism statistical software for statistical analysis. The results are shown in the attached figure. Figure 4 The results showed that the CCK8 proliferation experiment showed that the OD value of the frozen dental follicle mesenchymal stem cell injection group (DFSCs-C1) of the present invention was better than that of the other two groups on the 5th and 6th days, and the difference was statistically significant. There was no significant statistical difference among the three groups at other time points.

[0129] 2. Comparison of scratch healing of P5 DFSCs in each group

[0130] 2.1 Experimental grouping: the same as the experimental grouping under the item “Comparison of proliferation of P5 generation DFSCs in each group” in this experimental example.

[0131] 2.2 Cells in each group were plated at a density of 5x10 5 Cells / well were inoculated in a 12-well plate. When the cells were full, a 1ml pipette tip was used to draw a line perpendicular to the well plate, and the old culture medium was discarded. The cells were gently rinsed with PBS two to three times until the cells were washed clean, and fresh culture medium was added. A photo was taken at 0h under a microscope as a control. After culturing in a 37℃, 5% CO2 incubator for 13h, the cells were taken out, the width of the scratch at the same position was observed under a microscope, and photos were taken. ImageJ software was used to analyze the migration area of ​​the scratch experiment.

[0132] The results are attached Figure 5 The results showed that 13 hours after scratching, there was no significant difference in the migration area among the fresh dental follicle mesenchymal stem cell group (DFSCs-F), the frozen dental follicle mesenchymal stem cell injection group (DFSCs-C1) of the present invention, and the commercial frozen dental follicle mesenchymal stem cell injection group (DFSCs-C2), and the scratch test results showed no significant statistical difference. Figure 5 ns means no statistical significance.

[0133] 3. β-galactosidase staining results of P5 DFSCs in each group

[0134] 3.1 Experimental grouping: the same as the experimental grouping under the item “Comparison of proliferation of P5 generation DFSCs in each group” in this experimental example.

[0135] 3.2 Cells in each group were plated at 5000 cells / cm 2 Inoculate into a 6-well plate and add subculture medium to culture until the cell confluence reaches 70%. Aspirate the medium, gently rinse the cells 2-3 times with PBS buffer, then add an appropriate amount of fixative 4% paraformaldehyde and fix at room temperature for 15-30 minutes. After fixation, rinse with PBS 3 times, 5 minutes each time. Perform β-galactosidase staining according to the instructions of the β-galactosidase staining kit, and observe the cell staining under a microscope. β-galactosidase-positive cells (senescent cells) will appear blue. According to the number and distribution of blue cells, GraphPadPrism statistical software can be used for analysis and statistics.

[0136] The results are attached Figure 6 The results showed that there was no significant statistical difference in the β-galactosidase staining results among the fresh dental follicle mesenchymal stem cell group (DFSCs-F), the frozen dental follicle mesenchymal stem cell injection group (DFSCs-C1) and the commercial frozen dental follicle mesenchymal stem cell injection group (DFSCs-C2). Figure 6 ns means no statistical significance.

[0137] 4. Comparison of clone formation of P5 DFSCs in each group

[0138] 4.1 Experimental grouping: the same as the experimental grouping under the item “Comparison of proliferation of P5 generation DFSCs in each group” in this experimental example.

[0139] 4.2 Inoculate cells at 500 cells / 60mm culture dish, add 3ml of subculture medium, and culture at 37℃, 5% CO2. After clone colonies are formed (about 12 days, the number of cells in each colony is greater than 50), terminate the culture, fix with 4% paraformaldehyde for 15 minutes, and observe the colony formation by crystal violet staining. The results are shown in the attached figure. Figure 7 As shown. The statistical results of clone formation showed that the clone formation ability of the frozen dental follicle mesenchymal stem cell injection group (DFSCs-C1) of the present invention was better than that of the other two groups, and the difference was statistically significant. The above results show that the frozen dental follicle mesenchymal stem cell injection of the present invention can maintain the basic biological characteristics of dental follicle mesenchymal stem cells, such as proliferation, migration, clone formation, cell senescence state, etc.

[0140] Figure 7 In the table, * indicates statistically significant difference, and ns indicates no statistical significance.

[0141] Test Example 5

[0142] This test example investigated the expression of Oct3 / 4, P16 and P21 in the effectively surviving dental follicle mesenchymal stem cells after the cryopreserved dental follicle mesenchymal stem cell injection solution of the present invention was cryopreserved for one month and then taken out and revived.

[0143] The cryopreserved dental follicle mesenchymal stem cell injection in this test example was prepared according to the method of Example 1. Another cryopreserved dental follicle mesenchymal stem cell injection was prepared by the same method using a commercial cryopreservation solution (same as in Test Example 4) as a control.

[0144] 1. Experimental Grouping

[0145] The frozen dental follicle mesenchymal stem cell injection group (DFSCs-C1) of the present invention was as follows: P5 DFSCs were taken and frozen dental follicle mesenchymal stem cell injection was prepared according to the method of Example 1; then frozen for 1 month, taken out, revived (placed in a water bath preheated at 37°C, incubated for 1 minute 40 seconds to 2 minutes), washed and centrifuged, resuspended in the passage medium, and heated at 2.00×10 6 cells / 75cm 2 The cells were inoculated into T75 at a density of 100 μg / mL and allowed to adhere to the wall for 12-24 h. After digestion and centrifugation, the cells were harvested for RT-PCR.

[0146] Commercially cryopreserved dental follicle mesenchymal stem cell group (DFSCs-C2): Compared with DFSCs-C1, the cryopreservation medium was replaced with commercial cryopreservation medium, and the other conditions were the same.

[0147] Fresh dental follicle mesenchymal stem cell group (DFSCs-F): Fresh P5 dental follicle mesenchymal stem cells P5 were directly subcultured, and the cells were digested and collected within 24 hours for RT-PCR, which was recorded as DFSCs-F.

[0148] 2. RT-PCR detection

[0149] The cells collected from each group were subjected to real-time polymerase chain reaction (RT-PCR) to detect the expression of Oct3 / 4, P16 and P21.

[0150] The detection methods of Oct3 / 4, P16 and P21 are based on the existing technology. The total RNA was extracted using an RNA extraction kit, and the RNA was reverse transcribed using a reverse transcription kit. The specific operations were performed according to the instructions of the kit.

[0151] The results are attached Figure 8The results showed that there were some differences in the expression of cell cycle-related genes P16 and P21 and multidirectional differentiation potential-related transcription factors Oct3 / 4 among the three groups. In particular, the expression of Oct3 / 4 was significantly upregulated in the cells that survived effectively in the two groups of frozen dental follicle mesenchymal stem cell injections, while the expression of P16 and P21 was reduced, indicating that these effectively surviving cells may be a group of stem cells with weaker aging and stronger multidirectional differentiation potential.

[0152] Figure 8 In the table, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference.

[0153] Test Example 6

[0154] This test example investigated the multidirectional differentiation potential of the effective surviving cells after the cryopreserved dental follicle mesenchymal stem cell injection of the present invention was taken out after one month of cryopreservation. The cryopreserved dental follicle mesenchymal stem cell injection in this test example was prepared according to the method of Example 1. In addition, a commercial cryopreserved solution (same as in Test Example 4) was used to prepare a cryopreserved dental follicle mesenchymal stem cell injection as a control. The human-related stem cell osteogenic differentiation kit (item number HUXXC-90021), human-related stem cell adipogenic differentiation kit (item number HUXXC-90031) and human-related stem cell chondrogenic differentiation kit (item number HUXXC-90041) described in this test were all provided by Saiye (Guangzhou) Biotechnology Co., Ltd. The alkaline phosphatase colorimetric kit (item number C3250S) was provided by Shanghai Biyuntian Biotechnology Co., Ltd.

[0155] 1. Experimental Grouping

[0156] Cryopreserved dental follicle mesenchymal stem cell group (DFSCs-C1): P5 DFSCs were taken and cryopreserved dental follicle mesenchymal stem cell injection was prepared according to the method of Example 1; then frozen for 1 month, taken out, revived (placed in a water bath preheated at 37°C, incubated for 1 minute 40 seconds to 2 minutes), washed and centrifuged, resuspended in the subculture medium, and heated at 2.00×10 6 cells / 75cm 2 The cells were inoculated at a density of 100 μg / mL into T75 and adhered to the wall for 12-24 h. After digestion and centrifugation, the cells were harvested for investigation of multidirectional differentiation potential.

[0157] Commercially cryopreserved dental follicle mesenchymal stem cell group (DFSCs-C2): Compared with DFSCs-C1, the cryopreservation medium was replaced with commercial cryopreservation medium, and the other conditions were the same.

[0158] Fresh dental follicle mesenchymal stem cell group (DFSCs-F): Fresh P5 dental follicle mesenchymal stem cells were obtained and directly subcultured. The cells were digested and collected within 24 hours for investigation of multidirectional differentiation potential, which was recorded as DFSCs-F.

[0159] 2. Multidirectional differentiation potential assessment

[0160] 2.1 Investigation of osteogenic differentiation potential

[0161] According to the instructions of the human stem cell osteogenic differentiation kit, the osteogenic differentiation medium was prepared and stored at 4°C for use within one month. 4 viable cells / cm 2 The cells were seeded at a density of 100 μg / mL in a 12-well plate, 1 ml of culture medium was added to each well, and the plates were cultured in an incubator at 37°C, 5% CO2, and saturated humidity. When the cell confluence reached 70%, 1 ml of osteogenic differentiation induction medium was replaced, and fresh osteogenic differentiation induction medium was used every 3 days. After 7, 14, and 21 days of induction, the cells in each group were collected for real-time polymerase chain reaction (RT-PCR) to detect the expression of osteogenic-related genes (ALP, RUNX2, OCN, BSP). In addition, the samples collected after 3, 7, and 21 days of induction were fixed with 4% paraformaldehyde for 15 minutes, and the 3- and 7-day samples were stained with an alkaline phosphatase colorimetric kit to evaluate the alkaline phosphatase activity, or the 21-day samples were stained with Alizarin Red to observe calcium nodules. The results are shown in the attached figure. Fig. 9 ~Attached Fig.11 The results showed that the frozen dental follicle mesenchymal stem cells (DFSCs-C1) prepared by the freezing solution of the present invention had excellent osteogenic differentiation ability, and a large number of calcium nodules were produced after 21 days of osteogenic induction culture, and Alizarin red staining was strongly positive ( Fig. 9 A, B). RT-PCR results showed that at 7 days of osteogenic induction, the expression of early osteogenic-related genes ALP and RUNX2 in the frozen dental follicle mesenchymal stem cells (DFSCs-C1) prepared by the freezing solution of the present invention was better than that in the other two groups; at 14 days and 21 days of osteogenic induction, the expression of mid- and late-stage osteogenic-related genes OCN and BSP in the frozen dental follicle mesenchymal stem cells (DFSCs-C1) prepared by the freezing solution of the present invention was better than that in the other two groups, and the differences were statistically significant ( Fig.10 A~C). In addition, the ALP staining results showed that the ALP activity of the frozen dental follicle mesenchymal stem cells (DFSCs-C1) prepared by the cryopreservation solution of the present invention was superior to that of the other two groups at 3 days and 7 days of osteogenic induction ( Fig.11A~D), and the difference is statistically significant. This shows that the cryopreservation solution of the present invention can enhance the osteogenic differentiation ability of dental follicle mesenchymal stem cells, and is superior to the cryopreserved dental follicle mesenchymal stem cells prepared by the same method using the commercial cryopreservation solution.

[0162] Figure 9~Figure 11 In the table, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference.

[0163] 2.2 Investigation of adipogenic differentiation potential

[0164] According to the instructions of the human stem cell adipogenic differentiation kit, the adipogenic differentiation medium A and B were prepared and stored at 4°C. They were used within one month. 4 viable cells / cm 2 The cells were inoculated in a 12-well plate at a density of 1 ml of culture medium per well, and the plates were cultured in an incubator at 37°C, 5% CO2, and saturated humidity. 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, the medium B was removed and replaced with medium A for induction. The medium A and medium B were used alternately. After 7 and 21 days of induction, the cells in each group were collected for real-time polymerase chain reaction (RT-PCR) to detect the expression of adipogenic-related genes (Adiponectin, PPARγ). In addition, the samples collected 21 days after induction were fixed with 4% paraformaldehyde for 15 minutes, and the formation of lipid droplets was observed by oil red O staining.

[0165] The results are attached Fig.12 The results showed that the expression of adipogenic-related genes Adiponectin and PPARγ in the frozen dental follicle mesenchymal stem cells of the injection of the present invention after 7 days and 21 days of adipogenic induction was lower than that of the other two groups ( Fig.12 B, C), indicating that the frozen dental follicle mesenchymal stem cells of the injection of the present invention have lower adipogenic differentiation ability than the other two groups, but they still retain good adipogenic differentiation ability. After 21 days of adipogenic induction, obvious lipid droplets can be seen in the cytoplasm, and Oil Red O staining is positive ( Fig.12 A).

[0166] Fig.12 **p<0.01, ***p<0.001, ns indicates no significant difference.

[0167] 2.3 Investigation of chondrogenic differentiation potential

[0168] According to the instructions of the human stem cell chondrogenic differentiation kit, prepare the chondrogenic differentiation premix and complete medium, store at 4°C, and use within one month. 5 Transfer the effectively surviving dental follicle mesenchymal stem cells of each group to be induced into a 15 ml centrifuge tube, centrifuge at 20°C, 250g for 4 minutes, and remove the supernatant; add 0.5 ml of chondrogenic differentiation premix solution to resuspend, centrifuge at 20°C, 150g for 5 minutes; repeat this step to wash the cells again; add 0.5 ml of chondrogenic differentiation complete medium to resuspend, centrifuge at 20°C, 150g for 5 minutes; loosen the cap of the centrifuge tube to facilitate gas exchange, and place it upright in an incubator at 37°C, 5% CO2, and saturated humidity for culture; after 24 hours, the cells will aggregate, and the bottom of the centrifuge tube will be flicked to suspend the chondrocytes in the liquid. Replace with 0.5 ml of fresh chondrogenic differentiation complete medium every 2 to 3 days, and flick the bottom of the tube every time the medium is changed. After 7 and 21 days of induction, the cells of each group are collected for real-time polymerase chain reaction (RT-PCR) ) to detect the expression of cartilage-related genes (YWHAZ, ACAN, MIA, COL10A1). In addition, continuous induction for 28 days can form chondrocytes with a diameter of about 1 mm, which were fixed with 4% paraformaldehyde for 30 min, dehydrated with gradient alcohol, embedded in paraffin, and sliced ​​continuously at 4 μm, and observed with Alecian blue staining.

[0169] The results are attached Fig.13 As shown. The results showed that at 7 days of induction, the expression of chondrogenesis-related genes YWHAZ, ACAN, MIA, and COL10A1 in the frozen dental follicle mesenchymal stem cells (DFSCs-C1) of the injection of the present invention was significantly higher than that in the fresh cell group (DFSCs-F), among which the expression of the MIA gene was significantly better than that in the commercial preparation group (DFSCs-C2); there was no significant difference among the three at 21 days of induction, indicating that the frozen dental follicle mesenchymal stem cells of the injection of the present invention had stronger chondrogenic differentiation ability than fresh dental follicle mesenchymal stem cells in the early stage. After 28 days of chondrogenic induction culture, all three could form smooth chondrocytes, which were blue in color with Alecin blue staining, indicating the internal acidic mucopolysaccharide in the cartilage tissue.

[0170] Fig.13 In the table, *p<0.05, ***p<0.001, ns indicates no significant difference.

[0171] Test Example 7

[0172] This test example discloses an investigation on the systemic distribution of the cryopreserved dental follicle mesenchymal stem cell injection solution of the present invention after injection.

[0173] 1. Biodistribution study of cryopreserved DFSCs injection solution after single periodontal injection in F344RG rats (immunodeficient rats)

[0174] A mandibular defect (3×2×1 mm) was made in the buccal apical region of 70 F344RG rats. After modeling, 60 F344RG rats with consistent bone defect depth, small weight difference and good general condition were selected for inclusion in the group, half of which were male and half were female. 6 The invention provides a cryopreserved dental follicle mesenchymal stem cell injection solution of 1 cell / mouse. On days 1, 3, 7, 14, 28 and 42 after administration, samples of the heart, liver, spleen, lung, kidney, brain, salivary gland, reproductive system (female: ovary; male: testis), bone marrow (femur on both sides), the contralateral side of the administration site, oral site (pharynx, bilateral), the surrounding tissue on the contralateral side of the administration site (gingiva), the surrounding tissue (gingiva) of the administration site and the administration site are collected. The concentration of the target gene in the whole blood and each tissue sample is detected by qPCR, and the concentration range of the standard curve is 0.150-60000 pg / μL.

[0175] qPCR detection method:

[0176] Whole blood and tissue homogenate and DNA extraction: Whole blood and tissue homogenate and DNA extraction: All solid tissues were thawed in an ice bath, minced, and appropriate amount of tissue samples were weighed. The spleen was homogenized at a ratio of 1:40, and other tissues were homogenized at a ratio of 1:20. All administration sites were uniformly homogenized with 1500 μL of lysis buffer, and 400 μL of tissue homogenate, 200 μL of whole blood and bone marrow were taken for DNA extraction. The extracted DNA samples were frozen for testing.

[0177] Sample detection: After extraction, the total DNA concentration of the sample was determined by a multifunctional microplate reader, and the concentration of the target gene in the blood and tissue samples at each time point was detected by a validated qPCR method, with a standard curve range of 0.150-60000 pg / μL, a lower limit of quantification (LLOQ) of 0.150 pg / μL, and a detection limit of 0.0375 pg / μL. The results are shown in the following table.

[0178] Table 6 Single periodontal injection of 1.5×10 in F344RG rats 6 The concentration of target gene in whole blood and tissue after injection of cryopreserved dental follicle mesenchymal stem cells

[0179]

[0180] In the above table, the total number of animals tested at each time point was 10 (half male and half female), the units of the administration site and the contralateral side of the administration site were pg / μL homogenate, the units of other tissues were pg / mg tissue, and the units of whole blood / bone marrow were pg / μL sample; BLD means below the detection limit, BLQ means below the quantitative limit, and "-" means no relevant calculation 0.

[0181] As can be seen from the table above, a single periodontal injection of 1.5×10 6 After injection of cryopreserved dental follicle mesenchymal stem cells at a concentration of 1 cell / rat, the target gene could be continuously detected in the administration site of rats from 1 day to 42 days after administration; the target gene could also be detected in the tissues surrounding the administration site (gingiva) of rats from 1 day to 42 days after administration, but the detection rate was low. Except for 7 / 10 cases detected on the 1st day after administration, the detection rate at other time points did not exceed 3 / 10 cases; in addition, the target gene was occasionally detected in the lungs (3 / 60 cases), salivary glands (1 / 60 cases) and the contralateral side of the administration site (1 / 60 cases) of rats, but the level was low, while the target gene concentration was not detected in the whole blood, heart, liver, spleen, kidney, brain, genitals (testis, epididymis), bone marrow, and oral cavity (pharynx). The vast majority of the test results were BLD, and a few were BLQ. The results showed that after a single periodontal injection of cryopreserved dental follicle mesenchymal stem cells, the dental follicle mesenchymal stem cells were mainly distributed at the administration site, and their concentration gradually decreased with time. At 42 days after administration, the target gene concentration dropped to about 10% of the peak concentration; there was a small amount of distribution in the tissues around the administration site (gingiva), and no obvious distribution was found in other tissues.

[0182] In conclusion, after local application of cryopreserved dental follicle mesenchymal stem cell injection to periodontal bone defects, it was mainly distributed at the injection site.

[0183] Test Example 8

[0184] This test example discloses an investigation on the effects of fresh dental follicle mesenchymal stem cell injection and the cryopreserved dental follicle mesenchymal stem cell injection of the present invention on promoting the regeneration and repair of periodontal bone defects in SD rats.

[0185] 1. Experimental Grouping

[0186] Fresh dental follicle mesenchymal stem cell group (DFSCs-F): Fresh dental follicle mesenchymal stem cell injection was used, and the preparation method was to inject fresh P5 dental follicle mesenchymal stem cells at 5×10 7 The cells were resuspended in normal saline at a density of 10 cells / mL.

[0187] Cryopreserved dental follicle mesenchymal stem cell group (DFSCs-C1): The dental follicle mesenchymal stem cell injection solution was prepared according to the method of Example 1, and was taken out and thawed after being frozen for one month.

[0188] Commercial cryopreserved dental follicle mesenchymal stem cell group (DFSCs-C2): Compared with the cryopreserved dental follicle mesenchymal stem cell group, the difference is that the dental follicle mesenchymal stem cell injection solution is prepared using commercial cryopreservation solution, and the other conditions are the same. The commercial cryopreservation solution described in this experimental example is CryoStor® CS10 cell cryopreservation solution provided by BioLife Solutions, USA.

[0189] Cryopreservation protection solution solvent group (C1 (SLPCA)): the cryopreservation protection solution of Example 1 was used.

[0190] Normal saline group (0.9% NaCl): normal saline was used.

[0191] The specific tests are as follows:

[0192] A periodontal bone defect model (3×2×1 mm) was made in the buccal apical region of 70 8-week-old SD rats to expose the distal root of the mandibular first molar. After modeling, 60 SD rats with consistent bone defect depth, intact tooth root, small weight difference and good general condition were selected and divided into the following 5 groups (n=12): fresh dental follicle mesenchymal stem cell group, frozen dental follicle mesenchymal stem cell group of the present invention, commercial frozen dental follicle mesenchymal stem cell group, cryopreservation protection solution solvent group, and normal saline group. Drugs were administered within 24-48 hours after modeling, and 0.03 mL / rat of dental follicle mesenchymal stem cell injection solution and corresponding solvent of each group were injected into the periodontal modeling defect site. Jaw bone samples were collected 5 weeks after drug administration for Micro-CT analysis. Evaluation of bone tissue regeneration: Micro-CT was used to reconstruct and analyze the sampled mandibles, and parameters such as BV / TV (new bone volume fraction), Tb.Th (new trabecular bone thickness), Tb.N (new trabecular bone number), and Tb.Sp (new trabecular bone separation) were used to evaluate the regeneration of bone tissue in periodontal defects.

[0193] The results are attached Fig.14 and attached Fig.15 shown. Fig.14 In the middle, the arrow points to the periodontal bone defect modeling area; Fig.15 In the data, BV / TV represents the volume fraction of new bone, Tb.Th represents the thickness of new trabecular bone, Tb.N represents the number of new trabecular bone, and Tb.Sp represents the separation degree of new trabecular bone; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns represents no significant difference.

[0194] From the above results, it can be seen that: within the observation time point, the bone surface repair of the defect area in the frozen dental follicle mesenchymal stem cell group of the present invention is similar to that in the fresh dental follicle mesenchymal stem cell group, the surface bone repair is relatively smooth, and no root exposure is seen. The Micro-CT data analysis results show that within the observation time point, the new bone volume fraction, new trabecular thickness, and new trabecular number of the frozen dental follicle mesenchymal stem cell group of the present invention are better than those of the fresh dental follicle mesenchymal stem cell group, but there is no statistical difference between the two; in addition, the results of the frozen dental follicle mesenchymal stem cell group and the fresh dental follicle mesenchymal stem cell group in the above parameters are better than the commercial frozen dental follicle mesenchymal stem cell group and the cryopreservation protective liquid solvent group. It shows that the frozen dental follicle mesenchymal stem cell preparation of the present invention and the fresh dental follicle mesenchymal stem cell preparation have equivalent efficacy in treating periodontal bone defects in SD rats, and the treatment effect is significantly better than that of the commercial preparation.

[0195] Test Example 9

[0196] This test example discloses an investigation on the effects of fresh dental follicle mesenchymal stem cell injection and the cryopreserved dental follicle mesenchymal stem cell injection of the present invention on promoting the regeneration and repair of periodontitis bone defects in beagle dogs.

[0197] The preparation methods of the fresh dental follicle mesenchymal stem cell injection and the frozen dental follicle mesenchymal stem cell injection in this test example are the same as those in Test Example 8. The specific test is as follows:

[0198] 1. Construction of the Beagle Dog Periodontal Inflammation Model

[0199] An incision was made in the gingiva of the 2nd, 3rd, and 4th premolars of the left and right mandibles of beagle dogs at about 12 months of age to expose the alveolar ridge. A three-wall bone bag of 2.5×3×7 mm (length×width×depth, the defect size and depth can be adjusted according to actual conditions) was constructed in the mesial of the 3rd and 4th premolars using a high-speed turbine. Photos of the modeling site were taken, and the length, width, and depth of the modeling site were recorded. The enamel-cementum interface was ligated with 3-0 sutures. The defect site of the model animals was injected with LPS (lipopolysaccharide of Porphyromonas gingivalis) for 1 month, with a frequency of 1 injection every 2 days, and each injection dose of 20 μg / μL / tooth. The normal control group was injected with the same amount of sterile injection solution. The ligature was removed 4 weeks after the operation, and drug treatment was started (5 weeks after the operation). One month after modeling, the beagle dogs had obvious gingival swelling, bleeding on probing, and attachment loss on both sides of the mandible. X-rays showed horizontal absorption of the buccal and lingual alveolar bones, proving that the periodontal inflammation + defect model was successfully established.

[0200] 2. Stem cell injection therapy

[0201] After the inflammation + defect model was successfully established, the drugs were administered according to the following groups: model control group, fresh cell group, and frozen dental follicle mesenchymal stem cell group of the present invention. The dosage was 5×10 6cells / tooth, once every 2 weeks, for 2 consecutive weeks (2 times in total). Observation and recording period after medication: 3 months. During the observation period, clinical indicators were observed once every 2 weeks, and the probing depth, gingival bleeding index, attachment loss and other indicators were recorded. After 3 months, the beagle dogs were killed to collect jaw bone samples, and Micro-ct analysis was performed on the modeling area to analyze the BV / TV (new bone volume fraction), Tb.Th (new trabecular thickness), Tb.N (new trabecular number), and Tb.Sp (new trabecular separation) indicators of the modeling defect site. The results are shown in the attached Fig.16 , Attachment Fig.17 , Attachment Fig.18 , Attachment Fig.19 shown.

[0202] in Fig.16 A is the gingival bleeding index score at each observation time point within 12 weeks of medication. Fig.16 A shows that at all observation time points after administration, the gingival bleeding index score of the frozen dental follicle mesenchymal stem cell group of the present invention was significantly lower than that of the model control group; at the observation time points of 2, 10, and 12 weeks after administration, the frozen dental follicle mesenchymal stem cell group of the present invention was even better than the fresh cell group. Fig.16 B is the improvement of the bleeding index score in each group after 12 weeks of medication compared with the baseline level before medication. Fig.16 B shows that compared with the model control group, the gingival bleeding index of the fresh cell group and the frozen cell group of the present invention was significantly improved, and the difference was statistically significant, and there was no significant statistical difference between the latter two. The above results show that both the frozen dental follicle mesenchymal stem cells and fresh cells of the present invention can significantly improve gingival inflammation. As of the 12-week observation period, the effects of the two on treating gingival inflammation are comparable. Fig.16 The cryopreserved cell group is the cryopreserved dental follicle mesenchymal stem cell group of the present invention. Fig.16 In the table, * represents the statistical difference between the model control group and the frozen dental follicle mesenchymal stem cell group of the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001); # represents the statistical difference between the model control group and the fresh cell group (#p<0.05, ##p<0.01, ###p<0.001), & represents the statistical difference between the fresh cell group and the frozen dental follicle mesenchymal stem cell group of the present invention (&p<0.05), if not specified, it means there is no statistical difference between the two, and ns means no significant difference.

[0203] Fig.17 A is the periodontal probing depth at each observation time point within 12 weeks of medication. Fig.17 A shows that there was a baseline difference between the model control group and the frozen dental follicle mesenchymal stem cell group of the present invention before administration. At 12 weeks after administration, the periodontal probing depth of the frozen dental follicle mesenchymal stem cell group of the present invention was significantly smaller than that of the model control group. Fig.17 B shows the improvement of periodontal probing depth in each group after 12 weeks of medication compared with the baseline level before medication. Fig.17 B shows that compared with the model control group, the periodontal probing depth of the fresh cell group and the frozen dental follicle mesenchymal stem cell group of the present invention has been significantly improved, and the difference is statistically significant. The frozen dental follicle mesenchymal stem cell group of the present invention is numerically superior to the fresh cell group, but there is no significant statistical difference between the two. The above results show that both the frozen dental follicle mesenchymal stem cells and fresh cells of the present invention can promote the repair of periodontal tissue and significantly improve the probing depth of the periodontal pocket of periodontitis. As of the 12-week observation period, the effects of the two on promoting the shallowing of the periodontal pocket depth are comparable. Fig.17 The cryopreserved cell group is the cryopreserved dental follicle mesenchymal stem cell group of the present invention. Fig.17 In the table, * represents the statistical difference between the model control group and the frozen dental follicle mesenchymal stem cell group of the present invention (*p<0.05, **p<0.01, ***p<0.001), if not specified, it means there is no statistical difference between the two groups, and ns means no significant difference.

[0204] Fig.18 A is the loss of periodontal clinical attachment level at each observation time point within 12 weeks of medication. Fig.18 A shows that there was a baseline difference between the model control group and the frozen dental follicle mesenchymal stem cell group of the present invention before administration. At 12 weeks after administration, the loss of periodontal clinical attachment level in the frozen dental follicle mesenchymal stem cell group was significantly less than that in the model control group. Fig.18 B is the improvement of periodontal clinical attachment level in each group after 12 weeks of medication compared with the baseline level before medication. Fig.18 B shows that the frozen dental follicle mesenchymal stem cells of the present invention have the best effect in improving the clinical attachment level of periodontitis, followed by the fresh cell group, and the model control group has the worst effect, and there are statistical differences among the three. The above results show that the frozen dental follicle mesenchymal stem cells of the present invention can significantly improve the clinical attachment level of periodontitis and can significantly promote the repair of periodontal tissue; as of the 12-week observation period, the frozen dental follicle mesenchymal stem cells of the present invention are better than the fresh cell group in promoting the improvement of the clinical attachment level of periodontitis. Fig.18 The cryopreserved cell group is the cryopreserved dental follicle mesenchymal stem cell group of the present invention. Fig.18 In the table, * represents the statistical difference between the model control group and the frozen dental follicle mesenchymal stem cell group of the present invention (*p<0.05, **p<0.01, ****p<0.0001), if not specified, it means there is no statistical difference between the two groups, and ns means no significant difference.

[0205] Attached Fig.19A is a horizontal cross-sectional view of the three groups of jaw bone samples analyzed by Micro-CT (the arrows indicate the periodontal bone defect modeling area). The results show that the modeling defect area of ​​the fresh cell group and the frozen dental follicle mesenchymal stem cell group of the present invention have good bone regeneration effects, while the model control group is relatively poor; Fig.19 B~E are the statistical results of Micro-CT analysis of BV / TV (new bone volume fraction), Tb.Th (new trabecular thickness), Tb.N (new trabecular number), and Tb.Sp (new trabecular separation) of the modeling defect. The results show that: (1) BV / TV: There is no significant statistical difference between the fresh cell group and the frozen dental follicle mesenchymal stem cell group of the present invention, but both are better than the model control group, and the difference is statistically significant; (2) Tb.N: The frozen dental follicle mesenchymal stem cell group of the present invention is the best, and there is a statistical difference between it and the model control group, followed by the fresh cell group, which is numerically better than the model control group, but there is no statistical difference; (3) There is no statistical difference in Tb.Th and Tb.Sp between the three groups. The above results show that the frozen dental follicle mesenchymal stem cells of the present invention can significantly promote bone regeneration in periodontitis with periodontal bone defects, and its overall effect is equivalent to that of the fresh cell group, and can replace fresh cells to play a corresponding therapeutic role. Fig.19 The cryopreserved cell group is the cryopreserved dental follicle mesenchymal stem cell group of the present invention. Fig.19 In the table, * represents the statistical difference between the model control group and the frozen dental follicle mesenchymal stem cell group of the present invention (*p<0.05), if not specified, it means there is no statistical difference between the two, and ns means no significant difference.

[0206] Test Example 10

[0207] This test example conducted a toxicity test on the cryopreserved dental follicle mesenchymal stem cell injection of the present invention.

[0208] This study set up three groups: a vehicle control group and a human dental follicle mesenchymal stem cell injection solution of 0.4×10 8 , 1×10 8 Cell / dog group, 10 beagles per group, half male and half female. Each group of dogs received a subalveolar mucosal injection of a solvent control substance or a corresponding concentration of cryopreserved human dental follicle mesenchymal stem cell injection at a volume of 1 mL / dog, once every 2 weeks for 4 consecutive weeks (3 times in total), followed by 4 weeks of observation after drug withdrawal. The day of the first administration was defined as the first day of the administration period / the first day of the test, and the day after the last administration was defined as the first day of the recovery period.

[0209] The cryopreserved dental follicle mesenchymal stem cell injection solution of this test example was prepared according to the method of Example 1. The cryopreservation protection solution of Example 1 was also taken as a solvent reference.

[0210] The basic vital signs such as general condition, body weight and food intake, body temperature, II-lead electrocardiogram and blood pressure examination, ophthalmological examination, as well as hematology, blood biochemistry, urine examination, immune indexes, bone marrow smear, organ weight and coefficient after autopsy, gross anatomical observation, and histopathological examination during the test period were evaluated. No abnormal changes related to the test article were found.

[0211] 1. Hematological test indicators and results

[0212] The hematological test indicators are shown in Table 7, and the hematological test results are shown in Appendix Fig. 20 and attached Fig.21 The results showed that compared with the vehicle control group: on the 17th day of the administration period, 0.4×10 8 Cells / dog group female dogs and 1×10 8 The EOS of male dogs in the cell / dog group increased; on the 29th day of the administration period, 0.4×10 8 The MONO% of female dogs in the cell / dog group decreased, 1×10 8 The APTT of male dogs in the cell / dog group was shortened; on the second day of the recovery period, 0.4×10 8 The MONO% of male dogs in the cell / dog group was increased. The above differences were statistically significant (P≤0.05).

[0213] In addition, on the 17th and 29th days of the administration period and the second day of the recovery period, there were no significant differences in WBC, NEUT, NEUT%, LYMPH, LYMPH%, MONO, BASO, BASO%, EOS%, RBC, HCT, HGB, MCV, MCH, MCHC, PLT, RET, RET%, PT between the female and male dogs in the cryopreserved dental follicle mesenchymal stem cell injection groups and those in the vehicle control group during the same period (P>0.05). At the end of the recovery period, no abnormal changes were observed in the hematological indicators of the female and male dogs in the cryopreserved dental follicle mesenchymal stem cell injection groups. Combined with the specific data, on the 17th day of the administration period, 1×10 8 In the cell / dog group, a male dog (animal number 3M005) showed a transient decrease in PLT, but no changes in related indicators were observed, and histopathological examinations of all organs / tissues examined did not reveal any lesions related to the cryopreserved dental follicle mesenchymal stem cell injection, which had no toxicological significance.

[0214] Among the above changes, no obvious abnormal changes were found in EOS compared with the baseline value before administration, the change in MONO% was not dose-related and there was no corresponding change in absolute counts, and the shortening of APTT was small (6.52%); the above changes had no toxicological significance.

[0215] Table 7 Hematological examination items

[0216]

[0217] 2. Blood biochemical test items and results

[0218] The blood biochemical test items are shown in Table 8, and the test results are shown in Attachment Fig. 22 The results showed that compared with the solvent control group: on the 17th day of the administration period, 1×10 8 In the male dogs of the cell / dog group, GLU increased and K+ decreased; on the 29th day of administration, 1×10 8 GLU increased in male dogs in the cell / dog group; on the second day of the recovery period, 1×10 8 The AST level of female dogs in the cell / dog group was increased. The above differences were statistically significant (P≤0.05).

[0219] In addition, on the 17th and 29th days of the administration period and the 2nd day of the recovery period, there were no significant differences in ALT, CK, LDH, ALP, GGT, TP, ALB, GLB, A / G, TG, CHOL, TBIL, CREA, UREA, Na+, and Cl- between the female and male dogs in the cryopreserved dental follicle mesenchymal stem cell injection groups and those in the vehicle control group during the same period (P>0.05). At the end of the recovery period, no abnormal changes were found in the blood biochemical indicators of the female and male dogs in the cryopreserved dental follicle mesenchymal stem cell injection groups.

[0220] Among the above changes, no obvious abnormal changes were observed in GLU, K+, and AST compared with their baseline values ​​before administration, so they had no toxicological significance.

[0221] Table 8 Blood biochemical test items

[0222]

[0223] 3. Immune index examination items and results

[0224] Examination items: Canine T lymphocytes (CD3 + 、CD3 + CD4 + 、CD3 + CD8 + 、CD3 + CD4 + / CD3 + CD8 + ); Complement 3 (C3), complement 4 (C4), circulating immune complex (CIC); canine immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM); canine IL-2, IL-6, IL-10, TNF-α, IFN-γ cytokines;

[0225] 3.1 Lymphocyte subset detection results

[0226] Lymphocyte subset detection results Fig.23As shown, compared with the vehicle control group, on the 17th day of the administration period, 0.4×10 8 , 1×10 8 Cell / dog group male dog CD3 + Elevated, recovery day 2, 0.4×10 8 Cell / dog group male dog CD3 + The differences were statistically significant ( P ≤0.05). However, there was no dose correlation, and no significant abnormal changes were observed compared with the baseline values ​​before administration. The above changes had no toxicological significance. In addition, on the 17th day of the administration period and the 2nd day of the recovery period, CD3 + CD4 + / CD3 + CD8 + 、CD3 + CD4 + 、CD3 + CD8 + There was no significant difference compared with the vehicle control group (P>0.05). At the end of the recovery period, no obvious abnormal changes were found in lymphocyte subsets of male and female dogs injected with frozen dental follicle mesenchymal stem cell injection.

[0227] 3.2 Complement, circulating immune complexes, and immunoglobulin test results

[0228] The results of complement, circulating immune complexes and immunoglobulins are shown in the attached Fig.24 The results showed that compared with the solvent control group, on the 17th day of the administration period, 0.4×10 8 In the cell / dog group, C4 of female dogs increased, and IgM of male dogs increased, and the differences were statistically significant (P≤0.05); but there was no dose correlation, and no abnormal changes were observed compared with the baseline values ​​before administration, and the above changes had no toxicological significance. In addition, on the 17th day of the administration period, there were no statistically significant differences in C3, circulating immune complexes, IgA, and IgG in female and male dogs injected with frozen dental follicle mesenchymal stem cell injection in each group, and on the second day of the recovery period, there were no statistically significant differences in complement, circulating immune complexes, and immunoglobulins in female and male dogs injected with frozen dental follicle mesenchymal stem cell injection in each group compared with the solvent control group ( P> 0.05). At the end of the recovery period, no obvious abnormal changes were found in complement, circulating immune complexes and immunoglobulins in the female and male dogs injected with frozen dental follicle mesenchymal stem cell injection.

[0229] 3.3 Cytokine detection results

[0230] The cytokine test results showed that 2 hours after the third administration, compared with the baseline value before the first administration, 0.4×10 8 , 1×108 In some or most of the female and male dogs in the cell / dog group, IL-2, IL-6, IL-10, and TNF-α were elevated. At the end of the recovery period, the elevated cytokines in the female and male dogs in the frozen dental follicle mesenchymal stem cell injection groups decreased to a certain extent. In addition, 2 and 24 hours after the first administration, there were no obvious abnormal changes in IL-2, IL-6, IL-10, TNF-α, and IFN-γ in the female and male dogs in the frozen dental follicle mesenchymal stem cell injection groups compared with the solvent control group. During the experiment, 0.4×10 8 , 1×10 8 The animals in the cell / dog group with elevated cytokines were in good condition and had no elevated body temperature. Except for the injection site, no abnormal changes were found in the histopathological examination of all other organs examined. In addition, the basal values ​​of IL-2, IL-6, IL-10, and TNF-α cytokines of some animals in the solvent control group before the first dose were high, indicating that cytokines vary greatly among individuals in dogs and that the body will not be abnormal even at higher levels. There was a trend of recovery at the end of the recovery period. Therefore, although the above cytokines are related to the test article, they have no obvious toxicological significance.

[0231] Test Example 11

[0232] This test example discloses a clinical trial of the dental follicle mesenchymal stem cells of the present invention.

[0233] P5 dental follicle mesenchymal stem cells were obtained, resuspended in physiological saline containing 6‰ human albumin, and counted. 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 cells), fresh dental follicle mesenchymal stem cell preparations were transported in a low-temperature transport box and used within 6 hours. The details are as follows:

[0234] 1. Patient inclusion criteria

[0235] (1) Adults aged between 30 and 45 years old;

[0236] (2) The subject was found to have a two-wall or three-wall subosseous pocket with a PD ≥ 5 mm, or grade I / II furcation lesions by clinical exploration or imaging examination;

[0237] (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;

[0238] (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;

[0239] (5) The subjects are able to achieve good self-plaque control, including correct brushing and flossing.

[0240] II. Exclusion criteria

[0241] (1) Failure to maintain good oral hygiene during the introduction period;

[0242] (2) Pregnant or lactating women, or women who plan to become pregnant during this study;

[0243] (3) History of oral cancer or HIV;

[0244] (4) the studied teeth had a history of periodontal surgery;

[0245] (5) Clinical or radiological 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 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;

[0246] (6) chewing smokeless tobacco or smoking ≥1 time per week or smoking ≥10 cigarettes per day in the past 6 months;

[0247] (7) Suffering from mental cognitive disorders;

[0248] (8) Those who suffer from malignant tumors or have a history of malignant tumors;

[0249] (9) Having diabetes or glycated hemoglobin ≥ 6.5%;

[0250] (10) Taking calcium antagonists within four weeks before surgery;

[0251] (11) Use of adrenal corticosteroids within four weeks before surgery;

[0252] (12) Taking medications other than those specified for observation during the observation period;

[0253] (13) Allergy to biologically active products or drugs;

[0254] (14) The patient received other research treatments within 30 days of surgery;

[0255] (15) Research team members and their families;

[0256] (16) Patients with immune system abnormalities (such as autoimmune diseases);

[0257] (17) Patients with systemic diseases (Sjögren's syndrome, Behcet's disease, etc.) that may cause oral problems;

[0258] (18) Any other factors that may cause trial bias.

[0259] 3. Test operation

[0260] 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:

[0261] (1) Under local anesthesia, the tooth under study was scraped clean with ultrasonic instruments with the assistance of periodontal endoscope.

[0262] (2) Repeatedly rinse with hydrogen peroxide + chlorhexidine three times;

[0263] (3) Rinse three times with sterile saline;

[0264] (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 the special injection needle;

[0265] (5) In the fresh dental follicle mesenchymal stem cell treatment group, 100 μL (5×10 6 cells), the normal saline control group was injected with an equal volume of sterile saline into the periodontal pocket;

[0266] (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;

[0267] (7) Return for follow-up visit one week later to remove the suppository.

[0268] IV. Efficacy Evaluation

[0269] The content includes imaging indicators and clinical treatment indicators.

[0270] (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; gingiva recession (GR), i.e., the distance from the CEJ to the gingival margin.

[0271] (2) Imaging treatment indicators: Cone beam CT (CBCT) was used to measure the 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 area to the cementoenamel junction (CEJ) and the maximum width of the bone defect area were measured. Two oral radiologists who were unaware of the subjects' grouping independently measured the subjects' CBCT results.

[0272] V. Research Results

[0273] The results are attached Fig.25 shown.

[0274] (1) If Fig.25 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.

[0275] (2) If Fig.25 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.

[0276] (3) If Fig.25 As shown in 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.

[0277] (4) If Fig.25 D. Fig.25 As shown in Figure E, when the results of the DFSC group and the NS group were analyzed separately at 3 months, 6 months, and 12 months after treatment, the average PD and CAL levels in the DFSC group decreased significantly at 3 months, 6 months, 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.

[0278] (5) Fig.25 As 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.

[0279] (6) Fig.25 As shown in G, 25H, and 25I, when the results of the DFSC group and the NS group were analyzed separately at 3, 6, and 12 months after treatment, ( Fig.25H), 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 ( Fig.25 G, 25I).

[0280] Fig.25 In the table, the Chinese meanings of the English abbreviations are as follows: DFSC, fresh dental follicle mesenchymal stem cell treatment group; NS, normal saline control group; PD, probing depth; CAL, clinical attachment loss; GR: gingival recession; BDD, bone defect depth; BDW, bone defect width;

[0281] "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 two-way analysis of variance with repeated measures, and the comparison within the group was performed by one-way analysis of variance with repeated measures. * indicates statistical significance. * p<0.05; ** p<0.01; *** p<0.001; ns indicates no statistical significance. Error bars indicate mean ± SD.

[0282] 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.

[0283] The CCK8 proliferation experiment showed that the OD value of the frozen dental follicle mesenchymal stem cell injection of the present invention was better than that of fresh dental follicle mesenchymal stem cells on the 5th and 6th days, and the difference was statistically significant. There was no significant statistical difference in the scratch test results and β-galactosidase staining aging staining results between the frozen dental follicle mesenchymal stem cell injection of the present invention and fresh dental follicle mesenchymal stem cells; the clone formation ability of the frozen dental follicle mesenchymal stem cell injection group (DFSCs-C1) of the present invention was better than that of fresh dental follicle mesenchymal stem cells, and the difference was statistically significant.

[0284] Compared with fresh dental follicle mesenchymal stem cells, the frozen dental follicle mesenchymal stem cell injection of the present invention significantly downregulated the expressions of cell cycle-related genes P16 and P21, and significantly upregulated the expressions of multidirectional differentiation potential-related transcription factors Oct3 / 4; at 3, 7, 14, and 21 days of induction, respectively, the expressions of osteogenesis-related genes ALP, RUNX2, OCN, and BSP were significantly upregulated; at 7 days of induction, the expressions of chondrogenesis-related genes YWHAZ, ACAN, MIA, and COL10A1 were significantly upregulated.

[0285] Animal experiments show that the frozen dental follicle mesenchymal stem cells of the present invention and the fresh dental follicle mesenchymal stem cell preparation have equivalent efficacy in treating periodontal bone defects in SD rats and periodontitis bone defects in beagle dogs.

[0286] The cryopreserved dental follicle mesenchymal stem cells of the present invention can replace fresh dental follicle mesenchymal stem cells to improve gingival inflammation, prevent further gingival recession, and promote the regeneration of periodontal tissue in patients with periodontitis.

[0287] 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 cryopreserved dental follicle mesenchymal stem cell injection, characterized in that: It includes a cryopreservation protection solution and dental follicle mesenchymal stem cells, wherein the dental follicle mesenchymal stem cells are human dental follicle mesenchymal stem cells MSC, which are preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C2024384 and a preservation time of December 6, 2024; the cryopreservation protection solution is composed of the following components in volume parts: 8-12 parts of human serum albumin, 35-45 parts of sodium chloride injection, and 45-55 parts of CryoStor® CS10 cell freezing solution; The density of the dental follicle mesenchymal stem cells in the frozen dental follicle mesenchymal stem cell injection is 1×10 7 ~7×10 7 cells / mL; The specification of human albumin is 20wt%; the sodium chloride content in sodium chloride injection is 9wt%.

2. The cryopreserved dental follicle mesenchymal stem cell injection according to claim 1, characterized in that: The cryopreservation protection solution is composed of the following components by volume: 9-11 parts of human albumin, 37-43 parts of sodium chloride injection, and 47-52 parts of CryoStor® CS10 cell freezing solution.

3. The method for preparing a cryopreserved dental follicle mesenchymal stem cell injection according to claim 1 or 2, characterized in that: The steps include: The dental follicle mesenchymal stem cells were mixed evenly with the cryopreservation protection solution at a specified density, cryopreserved by programmed cooling, and stored in liquid nitrogen for a long time.

4. The method for preparing a cryopreserved dental follicle mesenchymal stem cell injection according to claim 3, characterized in that: Dental follicle mesenchymal stem cells were obtained, expanded and cultured, and P3-P6 cells were harvested for the preparation of cryopreserved dental follicle mesenchymal stem cell injection.

5. The use of a cryopreserved dental follicle mesenchymal stem cell injection according to claim 1 or 2, characterized in that: Application in the preparation of medicines for preventing and / or treating periodontal diseases.

6. The use of a cryopreserved dental follicle mesenchymal stem cell injection according to claim 1 or 2, characterized in that: Application in the preparation of drugs for promoting periodontal regeneration.

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