A repair injection preparation containing dental pulp stem cells and a preparation method thereof

Abstract

The present invention belongs to the field of biopharmaceutical manufacturing, and specifically relates to a repair injection preparation containing dental pulp stem cells and a preparation method thereof. The repair injection preparation comprises a microcarrier complex and a dental pulp stem cell mixture. The microcarrier complex is composed of substance 1 synthesized from citric lactone, angelica lactone and pentaerythritol, substance 2 obtained by reacting calcium nitrate tetrahydrate with diammonium hydrogen phosphate, chitosan and shikonin. The dental pulp stem cell mixture is composed of passage-passaged dental pulp stem cells and an activity-maintaining reagent. The activity-maintaining reagent is obtained by extracting Hypericum perforatum with a green solvent, combining with high-voltage electric field treatment, and then mixing the obtained filtrate with trehalose. The microcarrier complex can effectively enhance the repair effect of the dental pulp stem cell mixture. Experimental verification shows that the repair injection preparation obtained by the present invention has good repair effects on dental pulp-dentin and cartilage, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical manufacturing, and particularly relates to a repair injection preparation containing dental pulp stem cells and a preparation method thereof. Background Art

[0002] Stem cells exist in different parts of the human body, such as hematopoietic, neural, and mesenchymal tissues, and exhibit different degrees of tissue reconstruction ability. Among them, dental pulp stem cells are pluripotent stem cells derived from dental pulp tissue. They have rich sources, relatively painless and less risky acquisition processes, the ability of self-renewal, can maintain proliferative activity for a long time in in vitro culture, have low immunogenicity, can also secrete growth factors to stimulate angiogenesis, thereby promoting tissue repair and regeneration, and can release a series of anti-inflammatory factors to reduce inflammatory responses. Dental pulp stem cells have high gene stability, are not prone to malignant transformation, and do not involve ethical issues during use. The above advantages make dental pulp stem cells have great application potential in multiple aspects such as tissue repair. Stem cell injection preparations are relatively common preparation forms, which have advantages such as simple operation and easy control of dosage, and at the same time put forward higher requirements for maintaining the purity and activity of stem cells. Summary of the Invention

[0003] Based on the above problems, the present invention provides a repair injection preparation containing dental pulp stem cells, which comprises a microcarrier complex and a dental pulp stem cell mixture. The microcarrier complex is composed of a combination of multiple active ingredients, such as chitosan, shikonin, etc. The dental pulp stem cell mixture is composed of passage dental pulp stem cells and an activity maintenance reagent. The activity maintenance reagent is obtained by extracting Hypericum perforatum with a green solvent, combining with high-voltage electric field treatment, and then mixing the obtained filtrate with trehalose. The repair injection preparation containing dental pulp stem cells obtained by the present invention has been experimentally verified to have good repair effects on dental pulp-dentin and cartilage, and the repair effect is stronger than that of a single dental pulp stem cell preparation, having good application prospects.

[0004] The present invention provides a repair injection preparation containing dental pulp stem cells, which comprises the following raw materials in parts by mass: 5 - 8 parts of microcarrier complex, 10 - 15 parts of dental pulp stem cell mixture.

[0005] The preparation method of the microcarrier complex is as follows:

[0006] L1. Weigh pentaerythritol, angelica lactone, isocitric acid lactone, and stannous isooctanoate according to the mass-volume ratio of 27.2 mg: 139.3 mg: 78.5 mg: 0.8 mL. Add stannous isooctanoate to toluene to obtain Solution 1. Heat Solution 1 to 80 - 90 °C, then put in pentaerythritol, angelica lactone, and isocitric acid lactone, and continuously stir at a speed of 100 - 150 rpm under a nitrogen atmosphere for 18 - 20 h. After completion, obtain Solution 2;

[0007] L2. Naturally cool the solution 2 obtained in step L1 to room temperature, add chloroform in an amount 8 - 10 times the volume of solution 2, stir until evenly mixed to obtain solution 3, add absolute ethanol in an amount 3 - 5 times the volume of solution 3, let it stand for 2 - 3 h, filter to obtain the precipitate, and vacuum dry the precipitate at 60 - 70 °C to obtain Substance 1;

[0008] L3. Weigh calcium nitrate tetrahydrate and diammonium hydrogen phosphate according to a mass ratio of 2 - 2.5:1, separately prepare them into solutions with distilled water to obtain solution 4 and solution 5, drop solution 5 into solution 4, and stir at a speed of 150 - 200 rpm. After 40 - 50 min, obtain solution 6, filter solution 6, wash the obtained precipitate with distilled water, and dry it to obtain Substance 2;

[0009] L4. Dissolve chitosan in acetic acid solution to obtain solution 7 with a concentration of 3 - 5 g / mL. Weigh Substance 1 obtained in step L2, Substance 2 obtained in step L3, and solution 7 according to a mass - volume ratio of 3 - 5 g:4 - 8 g:15 - 20 mL, mix them, and perform ultrasonic treatment at a power of 500 - 600 W for 15 - 20 min. After completion, obtain solution 8, and vacuum freeze - dry solution 8 to obtain a freeze - dried powder;

[0010] L5. Weigh the freeze - dried powder obtained in step L4 and shikonin according to a mass ratio of 30 - 35:1, dissolve shikonin with absolute ethanol, then add the freeze - dried powder, stir until evenly dispersed, and place it in a ventilated environment until the absolute ethanol completely volatilizes to obtain the microcarrier complex.

[0011] Preferably, in step L1, the CAS number of pentaerythritol is 115 - 77 - 5, the CAS number of isocitric acid lactone is 4702 - 32 - 3, the CAS number of angelica lactone is 591 - 12 - 8, the CAS number of stannous octoate is 301 - 10 - 0, and the amount of toluene used is 4 - 5 times the volume of stannous octoate.

[0012] Preferably, in step L3, the concentration of solution 4 is 0.2 - 0.3 g / mL, and the concentration of solution 5 is 0.1 - 0.2 g / mL.

[0013] Preferably, in step L4, the mass percentage concentration of the acetic acid solution is preferably 2%.

[0014] Preferably, in step L5, the CAS number of shikonin is 54952 - 43 - 1.

[0015] The dental pulp stem cell mixture is composed of dental pulp stem cells of the 3rd generation, or the 4th generation, or the 5th generation combined with an activity - maintaining reagent. The preparation method of the activity - maintaining reagent is as follows:

[0016] S1. Vacuum freeze-dry the flowers of Hypericum perforatum, grind them into powder after drying, obtain Hypericum perforatum powder after passing through a 2-mesh sieve, weigh Hypericum perforatum powder, proline, glucose and water according to the mass-volume ratio of 5-10 g: 1-2 g: 2-3 g: 60-80 mL, mix them evenly and heat to 40-50 °C, continuously stir and process at a rotation speed of 150-200 rpm for 45-60 min, after completion, process at an electric field strength of 3-5 kV / cm for 5-10 min, filter to obtain the filtrate;

[0017] S2. Weigh trehalose and the filtrate obtained in step S1 according to the mass-volume ratio of 5-8 mg: 10-15 mL, stir until evenly dispersed to obtain the activity-maintaining reagent.

[0018] Preferably, passage the primary dental pulp stem cells successively to obtain the 3rd, or 4th, or 5th generation of dental pulp stem cells, place the 3rd, or 4th, or 5th generation of dental pulp stem cells in the activity-maintaining reagent, so that the concentration of the passage dental pulp stem cells in the activity-maintaining reagent is 1×10 6 -10 9 cells / mL to obtain the dental pulp stem cell mixture.

[0019] The present invention also provides a preparation method of a repair injection preparation containing dental pulp stem cells, and the specific steps are as follows:

[0020] Obtain the microcarrier complex and the dental pulp stem cell mixture according to the corresponding mass parts, mix the microcarrier complex and the dental pulp stem cell mixture, and perform ultrasonic treatment at a power of 200-250 W for 1-2 min, and after completion, obtain the repair injection preparation containing dental pulp stem cells.

[0021] Preferably, the above-mentioned repair injection preparation containing dental pulp stem cells needs to be prepared and used immediately.

[0022] The beneficial effects of the present invention are as follows:

[0023] The repair injection preparation containing dental pulp stem cells of the present invention contains a microcarrier complex and a dental pulp stem cell mixture, wherein the microcarrier complex is composed of a plurality of active ingredients. Using pentaerythritol as an initiator, in the presence of stannous octoate, the ring-opening and further connection of citric acid lactone and angelica lactone are carried out to obtain as Figure 1The shown Substance 1 has good biodegradability and low toxicity, which is superior to common complexes such as PGCL. React calcium nitrate tetrahydrate with diammonium hydrogen phosphate to obtain Substance 2 with certain mechanical support. It can not only provide a three-dimensional culture environment for subsequent dental pulp stem cells but also support the attachment of subsequent substances such as chitosan. The presence of chitosan provides adhesiveness, promotes the uniform mixing of various materials, and helps subsequent dental pulp stem cells maintain biological activity. As the final coating, shikonin can enhance the induction differentiation and repair potential of dental pulp stem cells. In addition, the dental pulp stem cell mixture is composed of passaged dental pulp stem cells and an activity-maintaining reagent. The presence of the activity-maintaining reagent can maintain the in vitro activity of dental pulp stem cells to the greatest extent. The activity-maintaining reagent is obtained by extracting Hypericum perforatum with a green solvent, combining with high-voltage electric field treatment, and then mixing the obtained filtrate with trehalose.

[0024] The obtained repair injection preparation containing dental pulp stem cells in the present invention has been experimentally verified to have good repair effects on dental pulp-dentin and cartilage, and the repair effect is stronger than that of a single dental pulp stem cell preparation. Among them, the microcarrier complex can effectively enhance the repair effect of the dental pulp stem cell mixture. Brief Description of the Drawings

[0025] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation to the present application. In the drawings:

[0026] Figure 1 It is a schematic structural diagram of Substance 1;

[0027] Figure 2 It is a live-dead staining fluorescence microscope image (40×), and (A), (B), (C), and (D) respectively correspond to Examples 4-7;

[0028] Figure 3 It is a graph of cell survival rate results;

[0029] Figure 4 It is a graph of the detection results of the thickness of the newly formed periodontal ligament-like tissue;

[0030] Figure 5 It is a graph of the detection results of blood biochemical indexes ALT and AST;

[0031] Figure 6 It is a graph of the detection results of blood biochemical indexes BUN and CRE;

[0032] Figure 7 It is a graph of the ICRS gross evaluation score results. Detailed Embodiments

[0033] To more clearly illustrate the overall concept of this application, the following provides a detailed description by way of examples in conjunction with the accompanying drawings of the specification. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0034] Example 1: This example provides a method for preparing a microcarrier complex, and the specific steps are as follows:

[0035] L1. Weigh pentaerythritol, angelica lactone, isocitric acid lactone, and stannous octoate according to the mass-volume ratio of 27.2 mg: 139.3 mg: 78.5 mg: 0.8 mL. Add stannous octoate to toluene, and the amount of toluene used is 4 times the volume of stannous octoate to obtain Solution 1. Heat Solution 1 to 80 °C, then add pentaerythritol, angelica lactone, and isocitric acid lactone. Under a nitrogen atmosphere, continuously stir at a speed of 100 rpm for 18 h, and after completion, obtain Solution 2;

[0036] L2. Naturally cool the Solution 2 obtained in step L1 to room temperature, add chloroform 8 times the volume of Solution 2, stir until evenly mixed to obtain Solution 3, add anhydrous ethanol 3 times the volume of Solution 3, let it stand for 2 h, filter to obtain the precipitate, and vacuum dry the precipitate at 60 °C to obtain Substance 1;

[0037] L3. Weigh calcium nitrate tetrahydrate and diammonium hydrogen phosphate according to the mass ratio of 2:1. Prepare solutions of calcium nitrate tetrahydrate and diammonium hydrogen phosphate respectively using distilled water to obtain Solution 4 and Solution 5. The concentration of Solution 4 is 0.2 g / mL, and the concentration of Solution 5 is 0.1 g / mL. Drop Solution 5 into Solution 4 and stir at a speed of 150 rpm. After 40 min, obtain Solution 6. Filter Solution 6, wash the obtained precipitate with distilled water, and dry it to obtain Substance 2;

[0038] L4. Dissolve chitosan in an acetic acid solution with a mass percentage concentration of 2% to obtain Solution 7 with a concentration of 3 g / mL. Weigh Substance 1 obtained in step L2, Substance 2 obtained in step L3, and Solution 7 according to the mass-volume ratio of 3 g: 4 g: 15 mL, mix them, and perform ultrasonic treatment at a power of 500 W for 15 min. After completion, obtain Solution 8, and vacuum freeze-dry Solution 8 to obtain a freeze-dried powder;

[0039] L5. Weigh the freeze-dried powder obtained in step L4 and shikonin according to the mass ratio of 30:1. Dissolve shikonin in anhydrous ethanol, then add the freeze-dried powder, stir until evenly dispersed, and place it in a ventilated environment until the anhydrous ethanol completely evaporates to obtain the microcarrier complex.

[0040] Example 2: This example provides a method for preparing a microcarrier complex, and the specific steps are as follows:

[0041] L1. Weigh pentaerythritol, angelica lactone, isocitric lactone, and stannous octoate according to the mass-volume ratio of 27.2 mg: 139.3 mg: 78.5 mg: 0.8 mL. Add stannous octoate to toluene, and the amount of toluene used is 4.5 times the volume of stannous octoate to obtain Solution 1. Heat Solution 1 to 85 °C, then add pentaerythritol, angelica lactone, and isocitric lactone. Under a nitrogen atmosphere, continuously stir at a speed of 120 rpm for 19 h, and after completion, obtain Solution 2;

[0042] L2. Naturally cool the Solution 2 obtained in step L1 to room temperature, add chloroform 9 times the volume of Solution 2, stir until evenly mixed to obtain Solution 3, add anhydrous ethanol 4 times the volume of Solution 3, let it stand for 2.5 h, filter to obtain the precipitate, and vacuum dry the precipitate at 65 °C to obtain Substance 1;

[0043] L3. Weigh calcium nitrate tetrahydrate and diammonium hydrogen phosphate according to the mass ratio of 2.2:1. Dissolve calcium nitrate tetrahydrate and diammonium hydrogen phosphate in distilled water respectively to obtain Solution 4 and Solution 5. The concentration of Solution 4 is 0.25 g / mL, and the concentration of Solution 5 is 0.15 g / mL. Drop Solution 5 into Solution 4 and stir at a speed of 180 rpm for 45 min. After completion, obtain Solution 6. Filter Solution 6, wash the obtained precipitate with distilled water, and dry it to obtain Substance 2;

[0044] L4. Dissolve chitosan in an acetic acid solution with a mass percentage concentration of 2% to obtain Solution 7 with a concentration of 4 g / mL. Weigh Substance 1 obtained in step L2, Substance 2 obtained in step L3, and Solution 7 according to the mass-volume ratio of 4 g: 6 g: 18 mL. After mixing, perform ultrasonic treatment at a power of 550 W for 18 min. After completion, obtain Solution 8. Vacuum freeze-dry Solution 8 to obtain a freeze-dried powder;

[0045] L5. Weigh the freeze-dried powder obtained in step L4 and shikonin according to the mass ratio of 32:1. Dissolve shikonin in anhydrous ethanol, then add the freeze-dried powder, stir until evenly dispersed, and place it in a ventilated environment until the anhydrous ethanol completely volatilizes to obtain the microcarrier complex.

[0046] Example 3: This example provides a method for preparing a microcarrier complex, and the specific steps are as follows:

[0047] L1. Weigh pentaerythritol, angelica lactone, isocitric acid lactone, and stannous octoate according to the mass-to-volume ratio of 27.2 mg : 139.3 mg : 78.5 mg : 0.8 mL. Add stannous octoate to toluene, and the amount of toluene used is 5 times the volume of stannous octoate to obtain Solution 1. Heat Solution 1 to 90 °C, then add pentaerythritol, angelica lactone, and isocitric acid lactone. Under a nitrogen atmosphere, continuously stir at a speed of 150 rpm for 20 h, and after completion, obtain Solution 2;

[0048] L2. Naturally cool the Solution 2 obtained in step L1 to room temperature, add chloroform with a volume 10 times that of Solution 2, stir until evenly mixed to obtain Solution 3, add anhydrous ethanol with a volume 5 times that of Solution 3, let it stand for 3 h, filter to obtain the precipitate, and vacuum dry the precipitate at 70 °C to obtain Substance 1;

[0049] L3. Weigh calcium nitrate tetrahydrate and diammonium hydrogen phosphate according to the mass ratio of 2.5 : 1. Prepare solutions of calcium nitrate tetrahydrate and diammonium hydrogen phosphate respectively with distilled water to obtain Solution 4 and Solution 5. The concentration of Solution 4 is 0.3 g / mL, and the concentration of Solution 5 is 0.2 g / mL. Drop Solution 5 into Solution 4 and stir at a speed of 200 rpm. After 50 min, obtain Solution 6. Filter Solution 6, wash the obtained precipitate with distilled water, and after drying, obtain Substance 2;

[0050] L4. Dissolve chitosan in an acetic acid solution with a mass percentage concentration of 2% to obtain Solution 7 with a concentration of 5 g / mL. Weigh Substance 1 obtained in step L2, Substance 2 obtained in step L3, and Solution 7 according to the mass-to-volume ratio of 5 g : 8 g : 20 mL, mix them, and perform ultrasonic treatment at a power of 600 W for 20 min. After completion, obtain Solution 8, and vacuum freeze-dry Solution 8 to obtain a freeze-dried powder;

[0051] L5. Weigh the freeze-dried powder obtained in step L4 and shikonin according to the mass ratio of 35 : 1. Dissolve shikonin with anhydrous ethanol, then add the freeze-dried powder, stir until evenly dispersed, and place it in a ventilated environment until the anhydrous ethanol completely volatilizes to obtain the microcarrier complex.

[0052] Example 4: This example provides a method for preparing dental pulp stem cells, and the specific content is as follows:

[0053] V1. Collect intact and healthy third molars extracted from humans, place them in a sterile PBS buffer solution, wipe them 3 times with alcohol cotton balls in a biosafety cabinet. After disinfection, break the enamel and dentin to expose the dental pulp. Wash the dental pulp tissue with sterile PBS buffer solution repeatedly 3 times, and cut it into tissue fragments with a size of 0.5 mm × 0.5 mm × 0.5 mm with ophthalmic scissors;

[0054] V2. Centrifuge the tissue fragments obtained in step V1 at a centrifugal force of 4800 g for 5 min, discard the PBS buffer, then add Dispase II enzyme and type I collagenase, and place them in a carbon dioxide incubator for sufficient digestion. After completion, add an equal volume of high-glucose medium containing 10% FBS to terminate the digestion, centrifuge again at a centrifugal force of 4800 g for 5 min, and resuspend the precipitate to obtain primary dental pulp stem cells;

[0055] V3. Add the primary dental pulp stem cells obtained in step V2 to the medium for culture. When the cell density reaches 80% under the microscope, passage the cells to successively obtain the 3rd, or 4th, or 5th generation of dental pulp stem cells.

[0056] Example 5: This example provides a method for preparing a dental pulp stem cell mixture, and the specific steps are as follows:

[0057] S1. Vacuum freeze-dry the flowers of Hypericum perforatum, and after completion, grind them into powder. Obtain Hypericum perforatum powder after passing through a 2-mesh sieve. Weigh Hypericum perforatum powder, proline, glucose, and water according to the mass-volume ratio of 5 g: 1 g: 2 g: 60 mL, mix them evenly, heat to 40 °C, continuously stir and process at a speed of 150 rpm for 45 min. After completion, process at an electric field strength of 3 kV / cm for 10 min, and filter to obtain the filtrate;

[0058] S2. Weigh trehalose and the filtrate obtained in step S1 according to the mass-volume ratio of 5 mg: 10 mL, and stir until evenly dispersed to obtain the activity-maintaining reagent;

[0059] S3. Obtain the 3rd generation of dental pulp stem cells according to the method of Example 4, and place the 3rd generation of dental pulp stem cells in the activity-maintaining reagent obtained in step S2, so that the concentration of the 3rd generation of dental pulp stem cells in the activity-maintaining reagent is 1×10 6 cells / mL to obtain the dental pulp stem cell mixture.

[0060] Example 6: This example provides a method for preparing a dental pulp stem cell mixture, and the specific steps are as follows:

[0061] S1. Vacuum freeze-dry the flowers of Hypericum perforatum, and after completion, grind them into powder. Obtain Hypericum perforatum powder after passing through a 2-mesh sieve. Weigh Hypericum perforatum powder, proline, glucose, and water according to the mass-volume ratio of 8 g: 1.5 g: 2.5 g: 70 mL, mix them evenly, heat to 45 °C, continuously stir and process at a speed of 180 rpm for 52 min. After completion, process at an electric field strength of 4 kV / cm for 8 min, and filter to obtain the filtrate;

[0062] S2. Weigh trehalose and the filtrate obtained in step S1 according to the mass-volume ratio of 7 mg: 12 mL, and stir until evenly dispersed to obtain the activity-maintaining reagent;

[0063] S3. Obtain the fourth-generation dental pulp stem cells according to the method of Example 4, and place the fourth-generation dental pulp stem cells in the activity-maintaining reagent obtained in step S2, so that the concentration of the fourth-generation dental pulp stem cells in the activity-maintaining reagent is 1×10 7 cells / mL, thus obtaining the dental pulp stem cell mixture.

[0064] Example 7: This example provides a method for preparing a dental pulp stem cell mixture, and the specific steps are as follows:

[0065] S1. Vacuum freeze-dry the flowers of Hypericum perforatum, grind them into powder after drying, pass through a 2-mesh sieve to obtain Hypericum perforatum powder, weigh Hypericum perforatum powder, proline, glucose and water according to the mass-volume ratio of 10 g: 2 g: 3 g: 80 mL, mix them evenly and heat to 50°C, continuously stir and process at a speed of 200 rpm for 60 min, after completion, process at an electric field strength of 5 kV / cm for 5 min, and filter to obtain the filtrate;

[0066] S2. Weigh trehalose and the filtrate obtained in step S1 according to the mass-volume ratio of 8 mg: 15 mL, and stir until evenly dispersed to obtain the activity-maintaining reagent;

[0067] S3. Obtain the fifth-generation dental pulp stem cells according to the method of Example 4, and place the fifth-generation dental pulp stem cells in the activity-maintaining reagent obtained in step S2, so that the concentration of the fifth-generation dental pulp stem cells in the activity-maintaining reagent is 1×10 9 cells / mL, thus obtaining the dental pulp stem cell mixture.

[0068] Example 8: This example provides a method for preparing a repair injection preparation containing dental pulp stem cells, and the specific steps are as follows:

[0069] Weigh the raw materials according to the following mass parts: 5 parts of microcarrier complex, 10 parts of dental pulp stem cell mixture. The microcarrier complex is obtained by the preparation method of Example 1, and the dental pulp stem cell mixture is obtained by the preparation method of Example 5. After mixing the microcarrier complex and the dental pulp stem cell mixture, perform ultrasonic treatment at a power of 200 W for 1 min, and after completion, obtain the repair injection preparation containing dental pulp stem cells of this example.

[0070] Example 9: This example provides a method for preparing a repair injection preparation containing dental pulp stem cells, and the specific steps are as follows:

[0071] Weigh the raw materials according to the following mass parts: 7 parts of microcarrier complex, 13 parts of dental pulp stem cell mixture. The microcarrier complex is obtained by the preparation method of Example 2, and the dental pulp stem cell mixture is obtained by the preparation method of Example 6. After mixing the microcarrier complex and the dental pulp stem cell mixture, perform ultrasonic treatment at a power of 220 W for 1.5 min, and after completion, obtain the repair injection preparation containing dental pulp stem cells of this example.

[0072] Example 10: This example provides a method for preparing a repair injection preparation containing dental pulp stem cells. The specific steps are as follows:

[0073] Weigh the raw materials according to the following mass fractions: 8 parts of microcarrier complex and 15 parts of dental pulp stem cell mixture. The microcarrier complex is obtained by the preparation method of Example 3, and the dental pulp stem cell mixture is obtained by the preparation method of Example 7. After mixing the microcarrier complex and the dental pulp stem cell mixture, ultrasonic treatment is carried out at a power of 250 W for 2 minutes, and then the repair injection preparation containing dental pulp stem cells of this example is obtained.

[0074] Comparative Example 1: This comparative example provides an injection preparation containing dental pulp stem cells. The specific steps are as follows:

[0075] The injection preparation of this comparative example is different from that of Example 9 in that Substance 1 is used to replace the microcarrier complex, and other steps are the same as those of Example 9, and then the injection preparation of this comparative example is obtained.

[0076] Comparative Example 2: This comparative example provides an injection preparation containing dental pulp stem cells. The specific steps are as follows:

[0077] The injection preparation of this comparative example is different from that of Example 9 in that Substance 2 is used to replace the microcarrier complex, and other steps are the same as those of Example 9, and then the injection preparation of this comparative example is obtained.

[0078] Comparative Example 3: This comparative example provides an injection preparation containing dental pulp stem cells. The specific steps are as follows:

[0079] The injection preparation of this comparative example is different from that of Example 9 in that freeze-dried powder is used to replace the microcarrier complex, and other steps are the same as those of Example 9, and then the injection preparation of this comparative example is obtained.

[0080] Experimental test:

[0081] Structural analysis of Substance 1:

[0082] Perform hydrogen spectrum and carbon spectrum analysis on Substance 1 obtained when preparing the microcarrier complex in Example 1, and the obtained results are shown as follows:

[0083] 11H NMR: δ 2.13 - 2.23 (12H, 2.18 (s), 2.18 (s)), 2.87 - 3.10 (16H, 2.93 (d, J = 6.8 Hz), 2.93 (d, J = 6.8 Hz), 2.93 (d, J = 6.8 Hz), 2.93 (d, J = 6.8 Hz), 3.04 (d, J = 7.2 Hz), 3.04 (d, J = 7.2 Hz), 3.04 (d, J = 7.2 Hz), 3.04 (d, J = 7.2 Hz)), 3.44 - 3.57 (4H, 3.50 (td, J = 6.8, 5.4 Hz), 3.50 (td, J = 6.8, 5.4 Hz)), 4.29 - 4.39 (8H, 4.34 (s), 4.34 (s), 4.34 (s), 4.34 (s)), 4.70 - 4.81 (4H, 4.76 (d, J = 5.4 Hz), 4.76 (d, J = 5.4 Hz)), 5.44 - 5.55 (4H, 5.50 (t, J = 7.2 Hz), 5.50 (t, J = 7.2 Hz)).

[0084] 13 13C NMR: δ 18.0 - 18.1 (4C, 18.0 (s), 18.0 (s)), 33.4 - 33.4 (4C, 33.4 (s), 33.4 (s)), 33.5 - 33.6 (4C, 33.6 (s), 33.6 (s)), 41.9 (1C, s), 49.5 - 49.6 (4C, 49.6 (s), 49.6 (s)), 62.4 - 62.5 (4C, 62.4 (s), 62.4 (s)), 73.6 - 73.7 (4C, 73.6 (s), 73.6 (s)), 123.9 - 124.0 (4C, 123.9 (s), 123.9 (s)), 163.0 - 163.1 (4C, 163.1 (s), 163.1 (s)), 170.8 - 170.9 (4C, 170.8 (s), 170.8 (s)), 172.1 - 172.2 (4C, 172.1 (s), 172.1 (s)), 174.8 - 174.9 (4C, 174.8 (s), 174.8 (s)), 175.8 - 175.9 (4C, 175.8 (s), 175.8 (s)).

[0085] The structural schematic diagram of Substance 1 is as follows Figure 1 shown

[0086] Cell survival rate:

[0087] The cell survival rate was calculated by live-dead staining. The primary dental pulp stem cells obtained in Example 4 and the dental pulp stem cells in the mixed solution of dental pulp stem cells obtained in Examples 5-7 were used for the experiment. Among them, the dental pulp stem cells in the mixed solution of dental pulp stem cells were obtained by culturing the passaged dental pulp stem cells overnight in the coexistence of an activity-maintaining reagent, and then taken out, dispersed and resuspended. The above-mentioned dental pulp stem cells were respectively placed in a 12-well plate, with 8×10 4 cells / mL in each well, and three parallel groups were set. They were cultured in an incubator at 37°C and 5% carbon dioxide for 4 h. Then, 60 μL of propidium iodide (50 μg / mL PBS buffer) and 500 μL of fluorescein diacetate were added to each well and incubated at room temperature for 3 min. After that, they were rinsed with DPBS buffer and observed under a fluorescence microscope. The cell survival rate was calculated according to the following formula:

[0088] Cell survival rate = number of live cells (number of green-stained cells) / total number of cells × 100%.

[0089] The experimental results are as follows Figure 2 and Figure 3 shown Figure 2 is a fluorescence microscope image at 40×. It can be seen from Figure 2 that under live-dead staining, there are more green-stained cells in the dental pulp stem cells of Examples 4-7. It can be seen from Figure 3 that the activity-maintaining reagent can maintain a relatively high survival rate of passaged dental pulp stem cells, not lower than that of primary dental pulp stem cells.

[0090] III. Animal experiment I:

[0091] 1) Remove the periodontal tissue, crown, 2-mm root tip, surface enamel, cementum and predentin on the pulp cavity side of the tooth. After ultrasonic cleaning with distilled water, it was successively placed in EDTA solutions with different concentrations for gradient demineralization (18 mol / L, 10 min; 10 mol / L, 5 min; 5 mol / L, 10 min), and then ground to obtain particles with a particle size of 200 μm. After autoclaving (121°C, 30 min) and ultraviolet disinfection for 30 min, it was stored at 4°C for later use to obtain dentin matrix;

[0092] 2) Eight-week-old male ICR mice were randomly divided into ten groups, with 15 mice in each group. Except for the first group, the other nine groups corresponded to Examples 5-10 and Comparative Examples 1-3 respectively. Taking the injection preparation obtained in Example 8 as an example, the injection preparation was added to the dentin matrix, and the mass ratio of the two was 1:1. After mixing, it was quickly mixed evenly for 30 s and stored at 4 °C for later use to obtain experimental samples. The first group only involved the dentin matrix;

[0093] 3) The mice were anesthetized and treated in a laminar flow hood. The back skin of the mice was disinfected with iodophor. A 1.5-cm incision parallel to the spine was made on the back with a sterile scalpel. The skin and subcutaneous tissue on both sides of the incision were separated with forceps. The experimental samples were placed subcutaneously, one on each side. Then the wound was sutured, erythromycin ointment was applied, and a 3M sterile dressing was applied to protect the wound. At the 5th week, the experimental samples placed on both sides were collected;

[0094] 4) The experimental samples obtained in 3) were fixed with 4% paraformaldehyde for 24 h, then rinsed with running water, placed in a 10% EDTA solution for gradient demineralization. After 3 d, the sample could be penetrated by a needle. They were successively placed in ethanol solutions of 75%, 85%, 95%, 100%, and 100% for dehydration, 1 h each time, then placed in xylene for 1 h, embedded in paraffin, sectioned, with a thickness of 3 μm, baked at 60 °C, and stored at 4 °C for later use;

[0095] 5) The sections obtained in 4) were stained with HE, the staining results were photographed and recorded, and processed with ImageJ software to record the thickness of the newly formed periodontal ligament-like tissue.

[0096] 6) At the 5th week, the tail vein blood of the mice was drawn for hematological detection and blood biochemical index detection.

[0097] The experimental results are as Figure 4 shown. It can be seen from Figure 4 that in the nude mouse subcutaneous ectopic transplantation experiment, after the injection preparations obtained in Examples 8-10 (the fifth to seventh groups) were used, the thickness of the newly formed periodontal ligament-like tissue could be effectively increased, proving that the injection preparations obtained in Examples 8-10 could enhance the regeneration of dental pulp and dentin, thereby repairing the defects related to dental pulp and dentin. When the components of the microcarrier complex were incomplete, this effect would be severely affected, as shown by the thickness of the newly formed periodontal ligament-like tissue in Comparative Examples 1-3 (the eighth to tenth groups). At the same time, Examples 5-7 (the second to fourth groups) only involved the dental pulp stem cell mixture, and the thickness values of their newly formed periodontal ligament-like tissue were relatively close, but also higher than those of the first group.

[0098] Figure 5 Show the test results of alanine aminotransferase ALT and aspartate aminotransferase AST in blood biochemical indexes, Figure 6 Show the test results of blood urea nitrogen BUN and creatinine CRE in blood biochemical indexes. It can be seen from Figure 5 andFigure 6 It can be seen that there are no statistically significant differences in the blood biochemical test results among the groups of mice, indicating that the substances obtained in Examples 5-10 and Comparative Examples 1-3 will not have an obvious impact on the organ functions of mice.

[0099] IV. Animal Experiment II:

[0100] 1) Male New Zealand white rabbits at 2 months of age were used for the experiment. They were randomly divided into eight groups, with 15 rabbits in each group. After anesthesia, they were placed in the supine position, and their limbs were fixed. The hair was removed from the areas about 5 cm inside and outside and about 9 cm above and below the knee joint, and then disinfected and covered with drapes.

[0101] 2) A midline anterior knee incision was made to cut through the skin and subcutaneous tissue, exposing the patellar ligament and patella. Entered through the muscle space between the tendon part of the patellar ligament at the upper pole of the patella and the vastus medialis muscle to cut open the joint capsule, dislocating the patella outward to expose the trochlea of the femur. The tissues around the trochlea of the femur were retracted, and a dental bone drill was used to create a model in the middle of the trochlea of the femur covered by the patella. The diameter of the model was 5 mm and the depth was 2 mm. Except for the first group, the above treatment was performed on the remaining seven groups.

[0102] 3) The injection preparations obtained in Examples 8-10 and Comparative Examples 1-3 were injected into the defect area, slightly higher than the modeling plane during injection, and irradiated locally with a point light source to make it photocured. Finally, the local defect was repaired smoothly, the patella was reduced, the joint capsule was sutured tightly, and the skin and subcutaneous tissues were sutured intermittently in layers.

[0103] 4) At 4, 8, and 12 weeks, the rabbits were sacrificed, and the distal femur of the rabbit knee joint was intercepted. The cartilage defect area was opened to observe the repair situation of this area, including the area, depth, tissue color, morphology, smoothness of the defect area, and the integration with the surrounding normal cartilage tissue, etc. Quantitative evaluation was carried out according to the ICRS gross evaluation method for cartilage repair. The ICRS gross evaluation involves the degree of defect repair, the degree of marginal fusion, and macroscopic observation. Each item has a full score of 4 points. An ICRS gross score of 12 points indicates normal, 8-11 points indicates close to normal, 4-7 points indicates abnormal, and 1-3 points indicates extremely abnormal.

[0104] The experimental results are as Figure 7 shown. It can be Figure 7 seen that using a cartilage defect model for verification, the injection preparations obtained in Examples 8-10 (the third to fifth groups) have a good cartilage repair effect and are basically close to normal after 12 weeks. Similarly, when the components of the microcarrier complex are incomplete, the cartilage repair effect of the obtained injection preparation will be affected, as shown in the experimental results of Comparative Examples 1-3 (the sixth to eighth groups).

[0105] As described above, these are only several embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention is disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the relevant art, without departing from the scope of the technical solution of the present invention, making some changes or modifications using the disclosed technical content above is equivalent to equivalent implementation cases and all fall within the scope of protection of the technical solution of the present invention.

Claims

1. A restorative injection preparation containing dental pulp stem cells, characterized in that: The raw materials are comprised of the following parts by weight: 5-8 parts of microcarrier complex, 10-15 parts of dental pulp stem cell mixture; The preparation method of the microcarrier complex is as follows: L1. Weigh pentaerythritol, angelica lactone, isocitrate lactone, and stannous isooctanoate according to a mass volume ratio of 27.2 mg: 139.3 mg: 78.5 mg: 0.8 mL, add stannous isooctanoate to toluene to obtain solution 1, heat solution 1, then add pentaerythritol, angelica lactone and isocitrate lactone, continue stirring under a nitrogen atmosphere, and obtain solution 2 after completion; L2. The solution 2 obtained in step L1 is naturally cooled to room temperature, chloroform in an amount of 8 to 10 times the volume of solution 2 is added, and the mixture is stirred until uniformly mixed to obtain solution 3, anhydrous ethanol in an amount of 3 to 5 times the volume of solution 3 is added, the mixture is allowed to stand, and a precipitate is obtained by filtration. The precipitate is vacuum dried to obtain substance 1; L3. Weigh calcium nitrate tetrahydrate and diammonium hydrogen phosphate in a mass ratio of 2-2.5:1, and prepare calcium nitrate tetrahydrate and diammonium hydrogen phosphate into solutions with distilled water to obtain solution 4 and solution 5, and drop solution 5 into solution 4 while stirring to obtain solution 6, and filter solution 6, wash the obtained precipitate with distilled water, and dry to obtain substance 2; L4. Dissolve chitosan in acetic acid solution to obtain solution 7 with a concentration of 3-5 g / mL, weigh the substance 1 obtained in step L2, the substance 2 obtained in step L3 and solution 7 according to the mass volume ratio of 3-5 g: 4-8 g: 15-20 mL, mix and ultrasonically treat, and obtain solution 8 after the end, and vacuum freeze-dry solution 8 to obtain a freeze-dried powder; L5. Weigh the lyophilized powder obtained in step L4 and shikonin according to a mass ratio of 30-35:1, dissolve shikonin in anhydrous ethanol, then add the lyophilized powder, stir until evenly dispersed, and place in a ventilated environment until the anhydrous ethanol is completely evaporated to obtain a microcarrier complex; The dental pulp stem cell mixture is composed of the third generation, the fourth generation, or the fifth generation dental pulp stem cells and an activity-maintaining agent. The concentration of the third generation, the fourth generation, or the fifth generation dental pulp stem cells in the activity-maintaining agent is 1×10 6 -10 9 Pieces / mL; The preparation method of the activity-maintaining reagent is as follows: S1. freeze-drying the flowers of Hypericum perforatum in vacuum, grinding them into powder, sieving them to obtain Hypericum perforatum powder, weighing Hypericum perforatum powder, proline, glucose and water according to a mass volume ratio of 5-10 g: 1-2 g: 2-3 g: 60-80 mL, mixing them evenly, heating them, stirring them, treating them under a field strength of 3-5 kV / cm, filtering them, and obtaining a filtrate; S2. Weigh trehalose and the filtrate obtained in step S1 according to a mass volume ratio of 5-8 mg:10-15 mL, and stir until uniformly dispersed to obtain an activity-retaining reagent.

2. The repair injection preparation containing dental pulp stem cells according to claim 1, characterized in that: The amount of toluene used in step L1 is 4-5 times the volume of stannous isooctanoate.

3. The repair injection preparation containing dental pulp stem cells according to claim 2, characterized in that: The concentration of solution 4 in step L3 is 0.2-0.3 g / mL, and the concentration of solution 5 is 0.1-0.2 g / mL.

4. A method for preparing a repair injection preparation containing dental pulp stem cells according to any one of claims 1 to 3, characterized in that: The microcarrier complex and the dental pulp stem cell mixture are obtained according to the corresponding mass fractions, and after the microcarrier complex and the dental pulp stem cell mixture are mixed, ultrasonic treatment is performed, and after the treatment, a repair injection preparation containing dental pulp stem cells is obtained.

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