Repair injection preparation containing dental pulp stem cells and preparation method thereof

Abstract

The invention belongs to the field of biological medicine manufacturing, and particularly relates to a repairing injection preparation containing dental pulp stem cells and a preparation method of the repairing injection preparation. The repair injection preparation comprises a microcarrier compound and a dental pulp stem cell mixed solution, and the microcarrier compound is formed by combining a substance 1 synthesized by citric acid lactone, angelica lactone and pentaerythritol, a substance 2 obtained by reacting calcium nitrate tetrahydrate with diammonium hydrogen phosphate, chitosan and shikonin. The dental pulp stem cell mixed solution is formed by combining passaged dental pulp stem cells and an activity maintaining reagent, the activity maintaining reagent is formed by extracting hypericum monogynum by adopting a green solvent and mixing the obtained filtrate with trehalose after high-voltage electric field treatment, and the microcarrier compound can effectively strengthen the repairing effect of the dental pulp stem cell mixed solution. Experiments prove that the obtained repair injection preparation has a good repair effect on dental pulp, dentin and cartilage, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biological medicine manufacturing, and specifically 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 show different degrees of tissue reconstruction ability. Among them, dental pulp stem cells are pluripotent stem cells derived from dental pulp tissue. They are abundant in source, the acquisition process is relatively painless and low-risk, have the ability to self-replicate, can maintain proliferation activity for a long time in in vitro culture, have low immunogenicity, and can also secrete growth factors to stimulate angiogenesis, thereby promoting tissue repair and regeneration. They can also release a series of anti-inflammatory factors to reduce inflammatory responses. Dental pulp stem cells have high genetic 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 many aspects such as tissue repair. Stem cell injection preparations are a more common form of preparation, which has the advantages of simple operation and easy control of dosage. At the same time, they also put forward higher requirements for the maintenance of stem cell purity and activity. Summary of the invention

[0003] Based on the above problems, the present invention provides a repair injection preparation containing dental pulp stem cells, comprising a microcarrier complex and a dental pulp stem cell mixture, wherein the microcarrier complex is composed of a combination of multiple effective ingredients, such as chitosan, lithospermum officinale, etc., and the dental pulp stem cell mixture is composed of a combination of dental pulp stem cells after passage and an activity-maintaining agent, and the activity-maintaining agent is obtained by extracting Hypericum perforatum flowers with a green solvent, and after combining high-voltage electric field treatment, the obtained filtrate is mixed with trehalose. The repair injection preparation containing dental pulp stem cells obtained by the present invention has been experimentally verified to have a good repair effect on both dental pulp dentin and cartilage, and the repair effect is stronger than that of a single dental pulp stem cell preparation, and has a good application prospect.

[0004] The 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 a microcarrier complex and 10-15 parts of a dental pulp stem cell mixture.

[0005] The preparation method of the microcarrier complex is as follows: L1. Weigh pentaerythritol, angelica lactone, isocitrate lactone, and stannous isooctanoate in 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 to 80-90° C., then add pentaerythritol, angelica lactone and isocitrate lactone, and continue stirring at a speed of 100-150 rpm for 18-20 hours under a nitrogen atmosphere, and then obtain solution 2; L2. The solution 2 obtained in step L1 was naturally cooled to room temperature, chloroform in an amount of 8-10 times the volume of solution 2 was added, and the mixture was stirred until uniformly mixed to obtain solution 3, anhydrous ethanol in an amount of 3-5 times the volume of solution 3 was added, and the mixture was allowed to stand for 2-3 hours, and a precipitate was obtained by filtration. The precipitate was vacuum dried at 60-70°C 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, drop solution 5 into solution 4, and stir at a speed of 150-200 rpm for 40-50 minutes to obtain solution 6, 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 at a power of 500-600 W for 15-20 min, and then obtain solution 8, and vacuum freeze-dry solution 8 to obtain a freeze-dried powder; L5. Weigh the lyophilized powder obtained in step L4 and shikonin at a mass ratio of 30-35:1, dissolve shikonin with 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.

[0006] Preferably, in step L1, the CAS number of pentaerythritol is 115-77-5, the CAS number of isocitrate lactone is 4702-32-3, the CAS number of angelica lactone is 591-12-8, the CAS number of stannous isooctanoate is 301-10-0, and the amount of toluene used is 4-5 times the volume of stannous isooctanoate.

[0007] 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.

[0008] Preferably, in step L4, the mass percentage concentration of the acetic acid solution is preferably 2%.

[0009] Preferably, the CAS number of shikonin in step L5 is 54952-43-1.

[0010] The dental pulp stem cell mixture is composed of 3rd generation, 4th generation, or 5th generation dental pulp stem cells and an activity-maintaining agent, wherein the preparation method of the activity-maintaining agent is as follows: S1. freeze-drying the flowers of Hypericum perforatum in vacuum, grinding them into powder, passing them through a 2-mesh sieve 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 to 40-50° C., stirring them continuously at a speed of 150-200 rpm for 45-60 min, treating them at a field strength of 3-5 kV / cm for 5-10 min, 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.

[0011] Preferably, the primary dental pulp stem cells are sequentially subcultured to obtain the third, fourth, or fifth generation dental pulp stem cells, and the third, fourth, or fifth generation dental pulp stem cells are placed in an activity-maintaining agent such that the concentration of the subcultured dental pulp stem cells in the activity-maintaining agent is 1×10 6 -10 9 / mL, and the dental pulp stem cell mixture was obtained.

[0012] The present invention also provides a method for preparing a repair injection preparation containing dental pulp stem cells, the specific steps of which are as follows: The microcarrier complex and the dental pulp stem cell mixture are obtained according to the corresponding mass fractions, and after mixing the microcarrier complex and the dental pulp stem cell mixture, ultrasonic treatment is performed at a power of 200-250W for 1-2 minutes, and a repair injection preparation containing dental pulp stem cells is obtained after the treatment.

[0013] Preferably, the above-mentioned repair injection preparation containing dental pulp stem cells needs to be prepared and used immediately.

[0014] The beneficial effects of the present invention are as follows: The repair injection preparation containing dental pulp stem cells of the present invention comprises a microcarrier complex and a dental pulp stem cell mixture, wherein the microcarrier complex is composed of a plurality of effective components. Pentaerythritol is used as an initiator, and in the presence of stannous isooctanoate, citric acid lactone and angelica lactone are ring-opened and further connected to obtain Figure 1The substance 1 shown in the figure has good biodegradability and low toxicity, which is better than the common PGCL and other complexes; calcium nitrate tetrahydrate is reacted with diammonium hydrogen phosphate to obtain substance 2 with certain mechanical support, which 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 adhesion, promotes the uniform mixing of various materials, and helps the subsequent dental pulp stem cells to maintain biological activity; lithospermum officinale, as the final coating, can enhance the induced differentiation and repair potential of dental pulp stem cells. In addition, the dental pulp stem cell mixture is composed of dental pulp stem cells after passage and an activity-maintaining agent. The presence of the activity-maintaining agent can maintain the in vitro activity of dental pulp stem cells to the greatest extent. The activity-maintaining agent is extracted from Hypericum perforatum flowers using a green solvent, and after high-voltage electric field treatment, the obtained filtrate is mixed with trehalose.

[0015] The repair injection preparation containing dental pulp stem cells obtained by the present invention has 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, wherein the microcarrier complex can effectively enhance the repair effect of the dental pulp stem cell mixture. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 is a schematic diagram of the structure of substance 1; Figure 2 Live-dead staining fluorescence microscopy images (40×), (A), (B), (C), and (D) correspond to Examples 4-7, respectively; Figure 3 This is the result graph of cell survival rate; Figure 4 This is the result of the thickness test of the new periodontal ligament-like tissue; Figure 5 This is the test result diagram of blood biochemical indexes ALT and AST; Figure 6 This is the test result diagram of blood biochemical indexes BUN and CRE; Figure 7 This is the ICRS general assessment score result chart. DETAILED DESCRIPTION

[0017] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided 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 technical features well known in the art are not described.

[0018] Example 1: This example provides a method for preparing a microcarrier complex, and the specific steps are as follows: L1. Pentaerythritol, angelica lactone, isocitrate lactone, and stannous isooctanoate were weighed in a mass volume ratio of 27.2 mg: 139.3 mg: 78.5 mg: 0.8 mL, and stannous isooctanoate was added to toluene in an amount of toluene that was 4 times the volume of stannous isooctanoate to obtain solution 1. Solution 1 was heated to 80° C., and then pentaerythritol, angelica lactone, and isocitrate lactone were added. Stirring was continued at a speed of 100 rpm for 18 h under a nitrogen atmosphere, and solution 2 was obtained after the stirring was completed. L2. The solution 2 obtained in step L1 was naturally cooled to room temperature, chloroform in an amount of 8 times the volume of solution 2 was added, and the mixture was stirred until uniformly mixed to obtain solution 3, anhydrous ethanol in an amount of 3 times the volume of solution 3 was added, and the mixture was allowed to stand for 2 hours, and a precipitate was obtained by filtration. The precipitate was vacuum dried at 60°C to obtain substance 1; L3. Weigh calcium nitrate tetrahydrate and diammonium hydrogen phosphate in a mass ratio of 2:1, and prepare calcium nitrate tetrahydrate and diammonium hydrogen phosphate into solutions with distilled water to obtain solution 4 and solution 5, wherein the concentration of solution 4 is 0.2 g / mL and the concentration of solution 5 is 0.1 g / mL. Add solution 5 dropwise into solution 4 and stir at a speed of 150 rpm for 40 minutes to obtain solution 6. Filter solution 6, wash the obtained precipitate with distilled water, and dry to obtain substance 2. L4. Dissolve chitosan in acetic acid solution, the mass percentage concentration of the acetic acid solution is 2%, and obtain a solution 7 with a concentration of 3g / mL. Weigh the substance 1 obtained in step L2, the substance 2 obtained in step L3 and the solution 7 according to the mass volume ratio of 3g:4g:15mL, mix and ultrasonically treat at a power of 500W for 15min, and then obtain a solution 8. The solution 8 is vacuum freeze-dried to obtain a freeze-dried powder; L5. Weigh the lyophilized powder obtained in step L4 and shikonin at a mass ratio of 30:1, 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.

[0019] Example 2: This example provides a method for preparing a microcarrier complex, and the specific steps are as follows: L1. Pentaerythritol, angelica lactone, isocitrate lactone, and stannous isooctanoate were weighed in a mass volume ratio of 27.2 mg: 139.3 mg: 78.5 mg: 0.8 mL, and stannous isooctanoate was added to toluene in an amount of toluene that was 4.5 times the volume of stannous isooctanoate to obtain solution 1. Solution 1 was heated to 85° C., and then pentaerythritol, angelica lactone, and isocitrate lactone were added. Stirring was continued at a speed of 120 rpm for 19 h under a nitrogen atmosphere, and solution 2 was obtained after the stirring was completed. L2. The solution 2 obtained in step L1 was naturally cooled to room temperature, chloroform in an amount of 9 times the volume of solution 2 was added, and the mixture was stirred until uniformly mixed to obtain solution 3, anhydrous ethanol in an amount of 4 times the volume of solution 3 was added, and the mixture was allowed to stand for 2.5 hours, and a precipitate was obtained by filtration. The precipitate was vacuum dried at 65°C to obtain substance 1; L3. Weigh calcium nitrate tetrahydrate and diammonium hydrogen phosphate in a mass ratio of 2.2:1, and prepare calcium nitrate tetrahydrate and diammonium hydrogen phosphate into solutions with distilled water to obtain solution 4 and solution 5, wherein the concentration of solution 4 is 0.25 g / mL, and the concentration of solution 5 is 0.15 g / mL. Solution 5 is dropped into solution 4 and stirred at a speed of 180 rpm for 45 minutes to obtain solution 6. Solution 6 is filtered, and the obtained precipitate is washed with distilled water and dried to obtain substance 2; L4. Dissolve chitosan in acetic acid solution, the mass percentage concentration of the acetic acid solution is 2%, and obtain a solution 7 with a concentration of 4 g / mL. Weigh the substance 1 obtained in step L2, the substance 2 obtained in step L3 and the solution 7 according to the mass volume ratio of 4g:6g:18mL, mix them, and then ultrasonically treat them at a power of 550W for 18min. After the end, obtain solution 8, and vacuum freeze-dry solution 8 to obtain a freeze-dried powder; L5. Weigh the lyophilized powder obtained in step L4 and shikonin at a mass ratio of 32:1, dissolve shikonin with 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.

[0020] Example 3: This example provides a method for preparing a microcarrier complex, and the specific steps are as follows: L1. Pentaerythritol, angelica lactone, isocitrate lactone, and stannous isooctanoate were weighed in a mass volume ratio of 27.2 mg: 139.3 mg: 78.5 mg: 0.8 mL, and stannous isooctanoate was added to toluene in an amount of toluene that was 5 times the volume of stannous isooctanoate to obtain solution 1. Solution 1 was heated to 90° C., and then pentaerythritol, angelica lactone, and isocitrate lactone were added. Stirring was continued at a speed of 150 rpm for 20 h under a nitrogen atmosphere, and solution 2 was obtained after the stirring was completed. L2. The solution 2 obtained in step L1 was naturally cooled to room temperature, chloroform in an amount of 10 times the volume of solution 2 was added, and the mixture was stirred until uniformly mixed to obtain solution 3, anhydrous ethanol in an amount of 5 times the volume of solution 3 was added, and the mixture was allowed to stand for 3 hours, and a precipitate was obtained by filtration. The precipitate was vacuum dried at 70°C to obtain substance 1; L3. Weigh calcium nitrate tetrahydrate and diammonium hydrogen phosphate in a mass ratio of 2.5:1, and prepare calcium nitrate tetrahydrate and diammonium hydrogen phosphate into solutions with distilled water to obtain solution 4 and solution 5, wherein the concentration of solution 4 is 0.3 g / mL and the concentration of solution 5 is 0.2 g / mL. Add solution 5 dropwise into solution 4 and stir at a speed of 200 rpm for 50 min to obtain solution 6. Filter solution 6, wash the obtained precipitate with distilled water, and dry to obtain substance 2. L4. Dissolve chitosan in acetic acid solution, the mass percentage concentration of the acetic acid solution is 2%, and obtain a solution 7 with a concentration of 5 g / mL. Weigh the substance 1 obtained in step L2, the substance 2 obtained in step L3 and the solution 7 according to the mass volume ratio of 5g:8g:20mL, mix and ultrasonically treat at a power of 600W for 20min, and then obtain a solution 8. The solution 8 is vacuum freeze-dried to obtain a freeze-dried powder; L5. Weigh the lyophilized powder obtained in step L4 and shikonin at a mass ratio of 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.

[0021] Example 4: This example provides a method for preparing dental pulp stem cells, the specific contents are as follows: V1. Collect intact healthy third molars extracted from humans, place them in sterile PBS buffer, wipe them three times with alcohol cotton balls in a biosafety cabinet, and after disinfection, knock open the enamel and dentin to expose the pulp. The pulp tissue is repeatedly cleaned three times with sterile PBS buffer and cut into tissue fragments of 0.5 mm × 0.5 mm × 0.5 mm with ophthalmic scissors; V2. Centrifuge the tissue fragments obtained in step V1 at 4800g for 5 minutes, discard the PBS buffer, then add DispaseⅡ and collagenase type I, and place in a carbon dioxide constant temperature incubator for full digestion. After the digestion is completed, add an equal volume of 10% FBS high-glucose medium to terminate the digestion, centrifuge again at 4800g for 5 minutes, resuspend the precipitate, and obtain primary dental pulp stem cells; V3. Add the primary dental pulp stem cells obtained in step V2 into the culture medium and culture them. When the cell density reaches 80% under microscopic observation, subculture them to obtain the third, fourth, or fifth generation of dental pulp stem cells.

[0022] Example 5: This example provides a method for preparing a dental pulp stem cell mixture, and the specific steps are as follows: S1. freeze-dry the flowers of Hypericum perforatum in vacuum, grind them into powder, pass them through a 2-mesh sieve to obtain Hypericum perforatum powder, weigh Hypericum perforatum powder, proline, glucose and water according to a mass volume ratio of 5g:1g:2g:60mL, mix them evenly, heat them to 40°C, stir them continuously at a speed of 150rpm for 45min, treat them at a field strength of 3kV / cm for 10min, filter them, and obtain a filtrate; S2. Weigh trehalose and the filtrate obtained in step S1 according to a mass volume ratio of 5 mg: 10 mL, and stir until uniformly dispersed to obtain an activity-maintaining reagent; S3. Obtain the third generation of dental pulp stem cells according to the method of Example 4, and place the third generation of dental pulp stem cells in the activity-maintaining reagent obtained in step S2, so that the concentration of the third generation of dental pulp stem cells in the activity-maintaining reagent is 1×10 6 / mL, and the dental pulp stem cell mixture was obtained.

[0023] Example 6: This example provides a method for preparing a dental pulp stem cell mixture, and the specific steps are as follows: S1. freeze-dry the flowers of Hypericum perforatum in vacuum, grind them into powder, pass them through a 2-mesh sieve to obtain Hypericum perforatum powder, weigh Hypericum perforatum powder, proline, glucose and water according to a mass volume ratio of 8g:1.5g:2.5g:70mL, mix them evenly, heat them to 45°C, stir them continuously at a speed of 180rpm for 52min, treat them at a field strength of 4kV / cm for 8min, filter them, and obtain a filtrate; S2. Weigh trehalose and the filtrate obtained in step S1 according to a mass volume ratio of 7 mg: 12 mL, and stir until uniformly dispersed to obtain an activity-maintaining reagent; S3. Obtain the fourth generation of dental pulp stem cells according to the method of Example 4, and place the fourth generation of dental pulp stem cells in the activity-maintaining reagent obtained in step S2, so that the concentration of the fourth generation of dental pulp stem cells in the activity-maintaining reagent is 1×10 7 / mL, and the dental pulp stem cell mixture was obtained.

[0024] Example 7: This example provides a method for preparing a dental pulp stem cell mixture, and the specific steps are as follows: S1. freeze-dry the flowers of Hypericum perforatum in vacuum, grind them into powder, pass them through a 2-mesh sieve to obtain Hypericum perforatum powder, weigh Hypericum perforatum powder, proline, glucose and water according to a mass volume ratio of 10 g: 2 g: 3 g: 80 mL, mix them evenly, heat them to 50° C., stir them continuously at a speed of 200 rpm for 60 min, treat them at a field strength of 5 kV / cm for 5 min, filter them, and obtain a filtrate; S2. Weigh trehalose and the filtrate obtained in step S1 according to a mass volume ratio of 8 mg: 15 mL, and stir until uniformly dispersed to obtain an activity-maintaining reagent; S3. Obtain the fifth generation of dental pulp stem cells according to the method of Example 4, and place the fifth generation of dental pulp stem cells in the activity-maintaining reagent obtained in step S2, so that the concentration of the fifth generation of dental pulp stem cells in the activity-maintaining reagent is 1×10 9 / mL, and the dental pulp stem cell mixture was obtained.

[0025] 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: The raw materials were weighed according to the following mass proportions: 5 parts of microcarrier complex and 10 parts of dental pulp stem cell mixture. The microcarrier complex was obtained by the preparation method of Example 1, and the dental pulp stem cell mixture was obtained by the preparation method of Example 5. The microcarrier complex and the dental pulp stem cell mixture were mixed and ultrasonically treated at a power of 200 W for 1 min. After the treatment, the repair injection preparation containing dental pulp stem cells of this example was obtained.

[0026] 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: The raw materials were weighed according to the following mass proportions: 7 parts of microcarrier complex and 13 parts of dental pulp stem cell mixture. The microcarrier complex was obtained by the preparation method of Example 2, and the dental pulp stem cell mixture was obtained by the preparation method of Example 6. The microcarrier complex and the dental pulp stem cell mixture were mixed and ultrasonically treated at a power of 220 W for 1.5 min. After the treatment, the repair injection preparation containing dental pulp stem cells of this example was obtained.

[0027] Example 10: This example provides a method for preparing a repair injection preparation containing dental pulp stem cells, and the specific steps are as follows: The raw materials were weighed according to the following mass proportions: 8 parts of microcarrier complex and 15 parts of dental pulp stem cell mixture. The microcarrier complex was obtained by the preparation method of Example 3, and the dental pulp stem cell mixture was obtained by the preparation method of Example 7. The microcarrier complex and the dental pulp stem cell mixture were mixed and ultrasonically treated at a power of 250 W for 2 minutes. After the treatment, the repair injection preparation containing dental pulp stem cells of this example was obtained.

[0028] Comparative Example 1: This comparative example provides an injection preparation containing dental pulp stem cells, and the specific steps are as follows: 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 consistent with those of Example 9, so as to obtain the injection preparation of this comparative example.

[0029] Comparative Example 2: This comparative example provides an injection preparation containing dental pulp stem cells, and the specific steps are as follows: 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 consistent with those of Example 9, so as to obtain the injection preparation of this comparative example.

[0030] Comparative Example 3: This comparative example provides an injection preparation containing dental pulp stem cells, and the specific steps are as follows: The injection preparation of this comparative example is different from that of Example 9 in that freeze-dried powder is used instead of the microcarrier complex, and the other steps are the same as those of Example 9, so as to obtain the injection preparation of this comparative example.

[0031] Experimental test: Structural analysis of substance 1: The substance 1 obtained when preparing the microcarrier complex in Example 1 was subjected to hydrogen and carbon spectrum analysis, and the results are shown as follows: 1 H 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 = 6.8Hz), 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)).

[0032] 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)).

[0033] The structural diagram of substance 1 is shown in Figure 1 shown.

[0034] Cell Viability: 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 dental pulp stem cell mixture obtained in Examples 5-7 were used for the experiment. The dental pulp stem cells in the dental pulp stem cell mixture were obtained by culturing the dental pulp stem cells after passage and coexisting with the activity-maintaining reagent overnight, then taking them out, blowing them apart and resuspending them. The dental pulp stem cells were placed in 12-well plates, with 8×10 4 Each well was cultured in a 37°C, 5% carbon dioxide incubator for 4 h. 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 the incubation, the cells were rinsed with DPBS buffer and observed under a fluorescence microscope. The cell survival rate was calculated according to the following formula: Cell viability = number of live cells (number of green-stained cells) / total number of cells × 100%.

[0035] The experimental results are as follows Figure 2 and Figure 3 As shown, Figure 2 This is a 40× fluorescence microscope image. Figure 2It can be seen that under live-dead staining, there are more green-stained cells in the dental pulp stem cells of Examples 4-7. Figure 3 It can be seen that the activity-maintaining reagent can maintain a high survival rate of cultured dental pulp stem cells, which is not lower than that of primary dental pulp stem cells.

[0036] 3. Animal Experiment I: 1) The periodontal tissue, crown, 2mm root apex, surface enamel, cementum and pre-dentin on the pulp side of the tooth were removed, and after ultrasonic cleaning with distilled water, they were placed in different concentrations of EDTA baths 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 size of 200 μm. After high-pressure steam sterilization (121°C, 30 min) and ultraviolet disinfection for 30 min, the particles were stored at 4°C for future use to obtain the dentin matrix; 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 at a mass ratio of 1:1. After mixing, the mixture was quickly mixed for 30 seconds and stored at 4°C for later use to obtain experimental samples. The first group only involved the dentin matrix; 3) Anesthetize the mice and process them on a clean bench. Use iodine to disinfect the back skin. Use a sterile scalpel to make a 1.5 cm incision on the back parallel to the spine. Use forceps to separate the skin and subcutaneous tissue on both sides of the incision. Place the experimental samples under the skin, one on each side. Then suture the wound, apply erythromycin ointment, and apply a 3M sterile dressing to protect the wound. Collect the experimental samples on the left and right sides in the 5th week. 4) Fix the experimental samples obtained in 3) with 4% paraformaldehyde for 24 hours, then rinse with running water, place in 10% EDTA solution, demineralize in a gradient manner, after 3 days, the needle can penetrate the sample, dehydrate in 75%, 85%, 95%, 100%, and 100% ethanol solutions in sequence, each for 1 hour, then place in xylene for 1 hour, embed in paraffin, slice, 3 μm thick, bake at 60℃, and set aside at 4℃; 5) The slices obtained in 4) were stained with HE, and the staining results were recorded by taking photos. The slices were processed with ImageJ software, and the thickness of the new periodontal ligament-like tissue was recorded.

[0037] 6) In the 5th week, blood was drawn from the tail vein of mice for hematological tests and blood biochemical index tests.

[0038] The experimental results are as follows Figure 4 As shown by Figure 4It can be seen that, in the nude mouse subcutaneous heterotopic transplantation experiment, the injection preparations obtained in Examples 8-10 (Groups 5-7) can effectively increase the thickness of the newly formed periodontal ligament-like tissue after use, proving that the injection preparations obtained in Examples 8-10 can enhance the regeneration of dental pulp dentin, thereby repairing defects related to dental pulp dentin, and when the microcarrier complex components are incomplete, this effect will be seriously affected, see the thickness of the newly formed periodontal ligament-like tissue in Comparative Examples 1-3 (Groups 8-10). At the same time, Examples 5-7 (Groups 2-4) only involve dental pulp stem cell mixtures, and the values ​​of the newly formed periodontal ligament-like tissue thickness are relatively close, but also higher than the first group.

[0039] Figure 5 Display the test results of alanine aminotransferase ALT and aspartate aminotransferase AST in blood biochemical indicators. Figure 6 Display the test results of blood urea nitrogen BUN and creatinine CRE in blood biochemical indicators. Figure 5 and Figure 6 It can be seen that there is no statistical difference in the blood biochemical test results between the groups of mice, indicating that the substances obtained in Examples 5-10 and Comparative Examples 1-3 will not cause significant effects on the organ functions of mice.

[0040] IV. Animal Experiment II: 1) Two-month-old male New Zealand white rabbits were randomly divided into eight groups, with 15 rabbits in each group. After anesthesia, they were placed in a supine position, their limbs were fixed, and the hair was removed within 5 cm inside and outside the knee joint and 9 cm above and below. Then, the rabbits were disinfected and covered with a towel. 2) Make a midline incision in front of the knee joint, cut the skin and subcutaneous tissue, expose the patellar ligament and patella, enter the joint capsule along the tendinous part of the patellar ligament and the vastus medialis muscle space at the upper pole of the patella, dislocate the patella outward, expose the femoral trochlea, pull away the tissue around the femoral trochlea, and use a dental bone drill to create a model in the middle of the femoral trochlea covering the patella. The model diameter is 5 mm and the depth is 2 mm. Except for the first group, the other seven groups all received the above treatment; 3) Inject the injection preparations obtained in Examples 8-10 and Comparative Examples 1-3 into the defect, slightly above the modeling plane during the injection, and use a point light source for local irradiation to make it light-cured, so that the local defect is repaired smoothly, the patella is repositioned, the joint capsule is tightly sutured, and the skin and subcutaneous layer are interruptedly sutured; 4) At the 4th, 8th and 12th week, the rabbits were killed, the distal femur of the knee joint was cut off, the cartilage defect site was opened, and the repair of the site was observed, including the area, depth, tissue color, morphology, smoothness of the defect and the integration with the surrounding normal cartilage tissue. Quantitative evaluation was performed according to the ICRS macroscopic evaluation method for cartilage repair. The ICRS macroscopic evaluation involves the degree of defect repair, the degree of marginal fusion and naked eye observation. The full score for any item is 4 points. The ICRS macroscopic score of 12 points indicates normal, 8-11 points indicate close to normal, 4-7 points indicate abnormal, and 1-3 points indicate extremely abnormal.

[0041] The experimental results are as follows Figure 7 As shown by Figure 7 It can be seen that the cartilage defect model was used for verification and the injection preparations obtained in Examples 8-10 (the third to fifth groups) had a good cartilage repair effect, which was basically close to normal after 12 weeks. Similarly, when the components of the microcarrier complex were incomplete, the cartilage repair effect of the injection preparations obtained would be affected, see the experimental results of Comparative Examples 1-3 (the sixth to eighth groups).

[0042] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope 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 at a mass ratio of 30-35:1, dissolve shikonin with 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.

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. The repair injection preparation containing dental pulp stem cells according to claim 3, characterized in that: 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.

5. The repair injection preparation containing dental pulp stem cells according to claim 4, characterized in that: 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-preserving reagent.

6. A method for preparing a repair injection preparation containing dental pulp stem cells according to any one of claims 1 to 5, 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.

Images