Preparation method of SHED-HUVEC organoid, injection type compound and application of injection type compound

Abstract

The invention discloses a preparation method of an SHED-HUVEC organoid, an injection type compound and application of the injection type compound. The preparation method comprises the following steps: a, extracting and culturing SHED; b, preparing a spheroid culture model micropore disc; c, constructing an SHED spheroid; d, activity detection; e, an SHED-HUVEC type organ is constructed; and f, dynamically monitoring the cells. According to the preparation method, the SHED-HUVEC organoid is constructed under the condition of not additionally adding growth factors, that is, the defects caused by using the growth factors can be reduced, the SHED-HUVEC organoid can remarkably promote the regeneration of vascularized dental pulp tissues, so that the regenerated dental pulp tissues form more mature blood vessels, and the regeneration rate of the dental pulp tissues is increased. The vascularization level and functional recovery of regenerated dental pulp tissues are improved. The injection type compound is prepared by mixing an SHED-HUVEC organ and a biocompatible hydrogel, and the injection type compound is applied to dental pulp regeneration treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of dental pulp regeneration, and in particular to a method for preparing SHED-HUVEC organoids, an injectable composite, and their applications. Background Art

[0002] Traditional treatment methods for pulp diseases and periapical diseases mainly include apical barrier technique and root canal treatment, and mainly control inflammation and promote the healing of periapical lesions by removing necrotic dental pulp tissue and using biomaterials to seal the root canal system. However, although the above-mentioned apical barrier technique and root canal treatment can relieve symptoms, the treated teeth are at risk of secondary infection and root canal longitudinal fracture due to the lack of biological functions such as nutrient supply, immune protection, and perception of external stimuli, especially in young permanent teeth.

[0003] With the development of regenerative endodontic procedures (REP), cell-free regenerative endodontic treatment based on the homing of endogenous stem cells has been widely used, but the regenerated tissue structure is not similar to that of natural dental pulp, and the physiological functions of dental pulp have not been fully restored.

[0004] It should be noted that the success of tissue engineering depends on the rapid vascularization of grafts. Due to the long and narrow anatomical morphology of the root canal system, the growth of new capillaries is slow, making it difficult to meet the nutritional and oxygen requirements of grafts. To promote the rapid vascularization of grafts, the existing technology realizes in vitro prevascularization by adding endothelial cells, but this method still relies on the addition of pro-angiogenic growth factors, and the additional addition of growth factors has defects such as high cost, difficult preservation, short in vivo half-life, and possible adverse reactions. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing SHED-HUVEC organoids, an injectable composite, and their applications in view of the deficiencies of the prior art. Specifically: The method for preparing SHED-HUVEC organoids can construct SHED-HUVEC organoids based on human shed deciduous dental pulp stem cells (SHED) and human umbilical vein endothelial cells (HUVEC) without the need to additionally add growth factors. By constructing SHED spheroids and using their paracrine products to achieve the stabilization and chemotaxis of human umbilical vein endothelial cells, the SHED-HUVEC organoids can significantly promote the regeneration of vascularized dental pulp tissue, and then enable the regenerated dental pulp tissue to form more mature blood vessels, so as to improve the vascularization level and functional recovery of the regenerated dental pulp tissue. Since there is no need to additionally add growth factors, the method for preparing SHED-HUVEC organoids can effectively solve the limitations of the prior art that rely on growth factors; The injectable composite is prepared by mixing SHED-HUVEC organoids with a biocompatible hydrogel. The injectable composite is applied to dental pulp regeneration treatment, and the injectable composite can effectively improve the convenience and adaptability of clinical operations during dental pulp regeneration treatment.

[0006] To achieve the above object, the present invention is realized by the following technical solutions.

[0007] A method for preparing SHED-HUVEC organoids, characterized by comprising the following steps, specifically: Step a, SHED extraction and culture: Extract SHED from human exfoliated deciduous teeth for primary culture and subculture expansion, and select cells of passages P3-P6 for subsequent experiments. SHED is human exfoliated deciduous tooth dental pulp stem cells; Step b, preparing a microplate for spheroid culture model: Pour the autoclaved 3% agarose solution into the polydimethylsiloxane microcolumn male mold. After the 3% agarose solution naturally cools and solidifies, a low-adhesion agarose culture model is obtained; wherein, the polydimethylsiloxane microcolumn male mold contains microcolumns with a diameter of 200 µm and a depth of 150 µm, and the distance between adjacent microcolumns is 200 µm; each 100 mL of the 3% agarose solution contains 3.0 g of agarose powder and / 100 mL of ultrapure water; Then use a cylindrical cutter with the same diameter as a single well of a 24-well plate to cut the low-adhesion agarose culture model into cylindrical agarose microplates, and place the cut cylindrical agarose microplates into the 24-well plate respectively, with one cylindrical agarose microplate placed in each well of the 24-well plate; then add 500 μL of PBS buffer to each agarose microplate in the 24-well plate, and after ultraviolet disinfection for 1 hour, set aside for use; Step c, constructing SHED spheroids: After the 24-well plate with the agarose microplates laid is disinfected by ultraviolet light, aspirate the PBS in each well of the 24-well plate, then add 800 μL of α-MEM PM medium to each well of the 24-well plate, and then select the SHED of passages P3-P6 obtained by culturing in step a, and resuspend 5×10 5 cells per well in 200 μL of α-MEM PM medium and slowly drop it onto the agarose microplate. After standing for 10 minutes, place it in a 37°C constant temperature incubator for constant temperature culture for 24 hours; after the constant temperature culture is completed, replace the α-MEM PM medium in the agarose microplate with DMEM OM medium, and continue to place it in a 37°C constant temperature incubator for constant temperature culture, and replace half of the DMEM OM medium every day and induce for 14 days to form SHED spheroids with a diameter of 50 μm - 80 μm; Step d, Activity detection: Detect the activity and cytoskeletal changes of SHED spheroids through live / dead staining and DAPI / Phalloidin fluorescence staining methods to verify their survival status and structural stability during the culture process; Step e, Construct SHED-HUVEC organoids: Step e1, Culture human umbilical vein endothelial cells: Purchase commercial primary HUVECs, inoculate HUVECs into a 96-well plate at a cell density of 3000 cells / well, add 100 μL of endothelial cell medium to each well of the 96-well plate, and then incubate in the dark in a 37 °C cell culture incubator; Step e2, Delayed addition of human umbilical vein endothelial cells: When the SHED spheroids in step c are cultured to the 4th day, resuspend the HUVECs cultured and incubated in step e1 with endothelial cell medium to 3.2 × 10 5 cells / mL, and then inoculate the HUVEC suspension onto the agarose microporous plate of the 24-well plate in step c at 500 μL / well, and continue to co-culture in DMEM OM medium for 14 days, changing half of the DMEM OM medium every day to form SHED-HUVEC organoids; Step f, Cell dynamic monitoring: Observe the fusion process of HUVECs and SHED spheroids through PKH26 / PKH67 live cell tracking staining or CD31 immunohistochemical staining methods to verify the self-organization formation of SHED-HUVEC organoids.

[0008] Among them, in the step a, the medium used for SHED culture contains 44.5 mL of α-MEM medium, 5 mL of fetal bovine serum (FBS), and 0.5 mL of penicillin / streptomycin double antibody solution; And the SHED culture environment in step a is: constant temperature culture in a 37 °C incubator.

[0009] Among them, in the step c, the α-MEM PM medium specifically is: 44.5 mL of α-MEM medium contains 5 mL of fetal bovine serum (FBS) and 0.5 mL of penicillin / streptomycin double antibody solution.

[0010] Among them, in the step c, the DMEM OM medium specifically is: 50 mL of DMEM OM medium contains 5 mL of fetal bovine serum (FBS), 0.5 mL of penicillin / streptomycin double antibody solution, 2.5 μL of dexamethasone solution with a concentration of 100 mmol / L, 0.5 mL of β-glycerophosphate sodium solution with a concentration of 10 mmol / L, 0.5 mL of vitamin C solution with a concentration of 50 mg / L, and the balance is high-glucose DMEM medium.

[0011] Wherein, in the step e1, the endothelial cell culture medium consists of 500 ml of basal culture medium, 25 ml of fetal bovine serum FBS, 5 ml of endothelial cell growth additive ECGS, and 5 ml of penicillin / streptomycin double antibody solution.

[0012] An injection complex is prepared by mixing the SHED-HUVEC organoids cultured and constructed by the above-mentioned SHED-HUVEC organoid preparation method with a biocompatible hydrogel.

[0013] The steps of preparing the injection compound include: Prepare 10% GelMA hydrogel and store in a 37°C water bath in the dark. SHED-HUVEC organoids were directly resuspended and mixed with 10% GelMA hydrogel.

[0014] A use of the above injection compound in dental pulp regeneration treatment.

[0015] Compared with the prior art, the present invention has the following beneficial effects, specifically: 1. The SHED-HUVEC organoids cultured and constructed by the preparation method of the SHED-HUVEC organoids of the present invention can effectively promote the regeneration of functional dental pulp tissue, and can restore the nutritional supply, immune protection and perception ability of dental pulp tissue, that is, it can effectively solve the problem of biological function loss of diseased teeth in the prior art; 2. The preparation method of SHED-HUVEC organoids of the present invention achieves the stabilization and chemotaxis of human umbilical vein endothelial cells through the paracrine effect of SHED spheroids. Since no additional growth factors are required, the problem of growth factor dependence in the prior art can be effectively solved.

[0016] 3. The preparation method of the SHED-HUVEC organoid of the present invention realizes the rapid vascularization of the transplant by constructing in vitro pre-vascularized SHED-HUVEC organoid, thus effectively solving the problem of low survival rate of the transplant in the prior art; 4. The injectable composite of the present invention can effectively improve the convenience and adaptability of clinical operation during pulp regeneration treatment; 5. Therefore, the method for preparing SHED-HUVEC organoids, the injectable composite and their applications of the present invention have important clinical application values and can provide a new regenerative treatment strategy for the treatment of pulp diseases and periapical diseases. By achieving functional pulp regeneration, the present valve core can not only effectively restore the biological function of the affected tooth, but also significantly improve the long-term survival rate of the affected tooth, especially suitable for the treatment of young permanent teeth. In addition, since the present invention avoids the use of growth factors, it can effectively reduce the costs and potential risks of adverse reactions brought about by the use of growth factors, thereby improving the clinical feasibility and safety of pulp regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation to the present invention.

[0018] Figure 1 It is a flowchart for fabricating a low-adhesion agarose microporous plate.

[0019] Figure 2 It is a process diagram of cells from uniform suspension, aggregation to spheroid formation at different time points after SHED plating under an inverted optical microscope.

[0020] Figure 3 It is the live / dead staining of SHED spheroids at 6d, 8d, 10d, and 14d.

[0021] Figure 4 It is the phalloidin staining of SHED spheroids at 6d, 8d, 10d, and 14d.

[0022] Figure 5 It is a comparison diagram of the proliferation effect of the supernatant of SHED spheroids on HUVEC.

[0023] Figure 6 It is a comparison diagram of the promoting effect of the supernatant of SHED spheroids on the horizontal migration of HUVEC.

[0024] Figure 7 It is a comparison diagram of the promoting effect of the supernatant of SHED spheroids on the vertical migration of HUVEC.

[0025] Figure 8 It is a comparison diagram of the promoting effect of the supernatant of SHED spheroids on the tube formation ability of HUVEC.

[0026] Figure 9 It is a culture process diagram of SHED-HUVEC organoids.

[0027] Figure 10 It is the live / dead staining of SHED-HUVEC organoids at 6d, 8d, 10d, and 14d.

[0028] Figure 11 Phalloidin staining of SHED-HUVEC organoids at 6d, 8d, 10d, and 14d.

[0029] Figure 12 PKH26 / PKH67 staining of SHED-HUVEC organoids at 6d, 8d, 10d, and 14d.

[0030] Figure 13 CD31 immunohistochemical staining of SHED-HUVEC organoids at 6d, 8d, 10d, and 14d.

[0031] Figure 14 H&E staining results of sagittal sections of dental segments.

[0032] Figure 15 Results of sagittal sections of dental segments after Masson staining. Detailed implementation manners

[0033] The present invention will be described below in conjunction with specific implementation manners.

[0034] Example 1, A method for preparing SHED-HUVEC organoids, which includes the following steps: Specifically: Step a, SHED extraction and culture: Extract SHED from human exfoliated deciduous teeth for primary culture and subculture amplification, and select P3-P6 generation cells for subsequent experiments. SHED is human exfoliated deciduous tooth pulp stem cells; Step b, Preparation of spheroid culture model microwell plates: Pour the autoclaved 3% agarose solution into the polydimethylsiloxane microcolumn positive mold. After the 3% agarose solution naturally cools and solidifies, a low-adhesion agarose culture model is obtained. Among them, the polydimethylsiloxane microcolumn positive mold contains microcolumns with a diameter of 200 µm and a depth of 150 µm, and the distance between adjacent microcolumns is 200 µm; Each 100 mL of 3% agarose solution contains 3.0 g of agarose powder and 100 mL of ultrapure water. Then, use a cylindrical cutter with the same diameter as a single well of a 24-well plate to cut the low-adhesion agarose culture model into cylindrical agarose microwell plates, and place the cut cylindrical agarose microwell plates into the 24-well plate respectively. One cylindrical agarose microwell plate is placed in each well of the 24-well plate. Then, add 500 μL of PBS buffer to each agarose microwell plate in the 24-well plate, and reserve it after ultraviolet sterilization for 1 hour; Step c, constructing SHED spheroids: After the 24-well plate with the agarose microwell plate laid is disinfected by ultraviolet light, aspirate the PBS in each well of the 24-well plate, then add 800 μL of α-MEM PM medium to each well of the 24-well plate. Then, select the SHED at P3-P6 obtained by culturing in step a, and resuspend 5×10 5 cells in 200 μL of α-MEM PM medium and slowly add it dropwise to the agarose microwell plate. After standing for 10 minutes, place it in a 37°C constant temperature incubator for constant temperature culture for 24 hours; after the constant temperature culture is completed, replace the α-MEM PM medium in the agarose microwell plate with DMEM OM medium, and continue to place it in a 37°C constant temperature incubator for constant temperature culture. Replace half of the DMEM OM medium every day and induce for 14 days to form SHED spheroids with a diameter of 50 μm - 80 μm; Step d, viability detection: Detect the viability and cytoskeleton changes of SHED spheroids by live-dead staining method and DAPI / Phalloidin fluorescence staining method to verify its survival status and structural stability during the culture process; Step e, constructing SHED-HUVEC organoids: Step e1, culturing human umbilical vein endothelial cells: Purchase commercial primary HUVEC, inoculate HUVEC into a 96-well plate at a cell density of 3000 cells / well, and add 100 μl of endothelial cell medium to each well of the 96-well plate, and then incubate it in the dark in a 37°C cell culture incubator; Step e2, delaying the addition of human umbilical vein endothelial cells: When the SHED spheroids in step c are cultured to the 4th day, resuspend the HUVEC cultured and incubated in step e1 with endothelial cell medium to 3.2 × 10 5 cells / mL, and then inoculate the HUVEC suspension onto the agarose microwell plate of the 24-well plate in step c at 500 μL / well, and continue to co-culture in DMEM OM medium for 14 days, maintaining half of the DMEM OM medium replaced every day to form SHED-HUVEC organoids; Step f, cell dynamic monitoring: Observe the fusion process of HUVEC and SHED spheroids by PKH26 / PKH67 live cell tracking staining method or CD31 immunohistochemical staining method to verify the self-organization formation of SHED-HUVEC organoids.

[0035] Among them, in the above step a, the medium used for culturing SHED contains 44.5 mL of α-MEM medium, 5 mL of fetal bovine serum FBS, and 0.5 mL of penicillin / streptomycin double antibody solution; and the culture environment of SHED in step a is: constant temperature culture in a 37°C constant temperature incubator.

[0036] In addition, in the step c, the α-MEM PM medium specifically is: 44.5 mL of α-MEM medium contains 5 mL of fetal bovine serum (FBS) and 0.5 mL of penicillin / streptomycin double-antibody solution.

[0037] Furthermore, in the step c, the DMEM OM medium specifically is: 50 mL of DMEM OM medium contains 5 mL of fetal bovine serum (FBS), 0.5 mL of penicillin / streptomycin double-antibody solution, 2.5 μL of dexamethasone solution with a concentration of 100 mmol / L, 0.5 mL of β-glycerophosphate sodium solution with a concentration of 10 mmol / L, 0.5 mL of vitamin C solution with a concentration of 50 mg / L, and the balance is high-glucose DMEM medium.

[0038] It should be noted that in the step e1, the endothelial cell medium is composed of 500 ml of basal medium, 25 ml of fetal bovine serum (FBS), 5 ml of endothelial cell growth supplement (ECGS), and 5 ml of penicillin / streptomycin double-antibody solution.

[0039] During the process of preparing the spheroid culture model microwell plate in the above step b, as Figure 1 shown in the manufacturing flow chart of the low-adhesion agarose microwell plate, microcolumns with a diameter of 200 μm and a depth of 150 μm can be seen on the polydimethylsiloxane microcolumn master mold made by SU-8 lithography technology, and the distance between the microcolumns is 200 μm; during the preparation process, a 3% agarose solution that has been sterilized by high-temperature and high-pressure and is below 60 °C is poured onto the polydimethylsiloxane microcolumn master mold to obtain a negative mold, that is, an agarose microwell plate. Then, after cutting the microwell plate with a cutter that matches the 24-well culture plate and placing it in the well plate and observing it under an inverted microscope, microwells with a diameter of 200 μm can be seen.

[0040] When observing the formation process of SHED spheroids cultured in the α-MEM PM medium in the above step c through an inverted optical microscope, as Figure 2 shown in the process diagram of cells from uniform suspension, aggregation to polymerization into spheroids at different time points after SHED plating under an inverted optical microscope, it can be seen that SHED uniformly aggregates into the microwells 0.5 h after plating, and initially self-polymerizes into a sphere between 5 h and 1 d. As the culture time changes, the volume gradually shrinks and tends to a regular sphere shape; at the same time, the connection between cells gradually tightens and the surface also tends to be smooth; after culturing for 14 d, the diameter of the SHED spheroids is about 50 μm - 80 μm.

[0041] During the process of detecting the activity of SHED spheroids by the Live / Dead staining method, asFigure 3 Live / dead staining of the SHED spheroids at 6 d, 8 d, 10 d, and 14 d is shown. The green fluorescent dye (Calcein-AM) can penetrate the cell membrane and be cleaved by intracellular lipase to release fluorescently labeled live cells. The red fluorescent dye (PI) can bind to the DNA double helix in the nucleus exposed after cells lose their activity to label dead cells. According to the results of laser confocal microscopy, the volume of the SHED spheroids gradually decreases with the extension of the culture time and basically reaches stability at 10 d. At the same time, the morphology of the spheroids tends to be a regular sphere and the surface also tends to be smooth. A large amount of green fluorescence of live cells is visible in the SHED spheroids at all time points. At the same time, with the extension of time, no obvious red fluorescence release is seen, indicating that during the 14-d culture period of the SHED spheroids, the nutrient supply in the center of the spheroids is sufficient and no obvious necrosis occurs.

[0042] During the process of detecting the activity of SHED spheroids by DAPI / Phalloidin fluorescence staining method, as Figure 4 Phalloidin staining of the SHED spheroids at 6 d, 8 d, 10 d, and 14 d is shown. The green fluorescent dye (FITC) can specifically bind to the monomeric fibrous proteins of eukaryotic cells to stain the cytoskeleton; the blue fluorescent dye (DAPI) can stain the nucleus; according to the results after laser confocal microscopy, with the extension of the culture time, the adhesion between cells inside the SHED spheroids increases and gradually becomes closely connected, indicating that the aggregation of the SHED spheroids becomes gradually tight during the 14-d culture period and the cell communication area increases.

[0043] During the process of detecting the effects of the supernatant of SHED spheroids on the proliferation, migration, and angiogenesis ability of HUVECs, according to the results of the CCK-8 cytotoxicity experiment, as Figure 5 shown, continuous cell proliferation can be observed in both the SHED 2D group and the SHED Spheroid group within 24 h, 48 h, 72 h, and 96 h. At the 24-h and 72-h time points, the OD value of the SHED Spheroid group is higher than that of the SHED 2D group, and the difference is statistically significant (P < 0.05), indicating that the supernatant of SHED spheroids has a certain effect on promoting cell proliferation. According to the results of the scratch experiment, as Figure 6 shown, after treatment with the supernatant for 12 h, compared with the SHED2D group, the scratch width of HUVEC cells in the SHED Spheroid group becomes significantly narrower, the cells are almost fused, and the boundary is blurred; the quantitative results show that under the action of the supernatant of SHED spheroids, the horizontal migration rate of HUVEC cells is higher than that of the SHED 2D group, and the difference is statistically significant (P < 0.05). According to the results of the Transwell chamber experiment, as Figure 7As shown, the cell density of the SHED Spheroid group migrating to the lower membrane of the chamber was significantly higher than that of the SHED 2D group. Under the action of the supernatant of SHED spheroids, the vertical migration rate of HUVEC was higher than that of the SHED 2D group, and the difference was statistically significant (P<0.05). According to the results of the Matrigel lumen formation assay, as Figure 8 shown, the number of small tube connections, the number of nodes, and the total length of the tube lumen trunk in the SHED Spheroid group were all higher than those in the SHED 2D group. Quantitative statistics showed that the results ( Figure 8 B) were consistent with the above observation results, and the difference was statistically significant (P < 0.05). Therefore, SHED spheroids can secrete effective components that promote angiogenesis. The paracrine products of SHED spheroids (such as the supernatant) can enhance the proliferation, migration, and tube formation ability of human umbilical vein endothelial cells. Moreover, the experimental results showed that HUVEC stimulated by the supernatant of SHED spheroids was significantly superior to SHED cells cultured in the traditional two-dimensional manner in many indicators, indicating its significant advantage in promoting blood vessel formation.

[0044] During the construction of SHED-HUVEC organoids in step e above, as Figure 9 shown, according to the observation results of the inverted optical microscope, under the action of DMEM OM medium, the pre-plated SHED gradually self-polymerized to form smooth-surfaced spheroids; after 4 days of culture, HUVEC was added late. As the culture time extended, HUVEC gradually aggregated on the outer layer of the original SHED spheroids and gradually fused with them. The connections between cells became closer, and the overall volume gradually decreased, remaining basically unchanged after 10 days. By 14 days, the diameter was about 70 μm; during the culture period from day 7 to day 14, different from the flat surface of SHED spheroids, the SHED-HUVEC organoids showed an irregular surface morphology.

[0045] During the detection of SHED-HUVEC organoids by the live / dead staining method, the survival status of SHED-HUVEC organoids at 6 days, 8 days, 10 days, and 14 days of culture was evaluated by the live / dead staining technique, and three-dimensional imaging was performed using a laser confocal microscope. The results were as Figure 10As shown; at all time points, a large number of live cells labeled with green fluorescence were observed on the surface of SHED-HUVEC organoids, and no obvious dead cells or low-viability cells labeled with red fluorescence were found; as the culture time extended, the volume of SHED-HUVEC organoids gradually decreased, the internal cells were distributed relatively loosely, while the cells of SHED spheroids were tightly connected; SHED-HUVEC organoids presented an irregular surface morphology, which was consistent with the morphology observed under an inverted microscope; when reaching day 14, the green fluorescence intensity at the central part of SHED-HUVEC organoids decreased, suggesting the possible existence of a cavity.

[0046] During the detection of SHED-HUVEC organoids by the DAPI / Phalloidin fluorescence staining method, the cytoskeleton morphology of SHED-HUVEC organoids cultured for 6 d, 8 d, 10 d, and 14 d was traced by DAPI / Phalloidin staining, and the three-dimensional imaging results after being photographed by a laser confocal microscope were as shown in Figure 11; as the culture time extended, the internal cytoskeleton of SHED-HUVEC organoids gradually contracted, the adhesion between cells increased, and the connection gradually became tight, presenting characteristics similar to those of SHED spheroids; when cultured to day 10, the volume of SHED-HUVEC organoids was basically stable, which was verified by the results of inverted microscopy and live / dead staining; this indicated that within 14 days of culture, SHED-HUVEC organoids underwent a cell migration process, the binding between SHED and HUVEC was gradually strengthened, and the communication area between cells increased.

[0047] During the process of dynamically monitoring the cells of SHED-HUVEC organoids by the PKH26 / PKH67 live cell tracking staining method in the above step f, the PKH dye is a cell membrane dye with extremely low cytotoxicity, which retains biological activity and proliferative activity while labeling cells; the half-life of the PKH dye is up to more than 100 days, and it is commonly used for tracking cells in vivo and studying cell-cell interactions. During this process of cell dynamic monitoring, the red fluorescent dye PKH26 was used to label SHED, and the green fluorescent dye PKH67 was used to label HUVEC. The three-dimensional imaging results after being photographed by a laser confocal microscope were as Figure 12As shown in the figure; at 6 days, that is, 2 days after the delayed addition of HUVEC, the SHED-HUVEC organoids generally presented an obvious bilayer structure. The central region was the SHED spheroid labeled with red fluorescence, and the periphery was surrounded by the HUVEC layer labeled with green fluorescence; by 8 days, the surface of the SHED spheroid began to spread outward and fuse with HUVEC; by 10 days, the distribution of red and green fluorescence signals mostly overlapped, and SHED and HUVEC were basically fused. At this time, the overall volume of the organoids decreased, and a thin layer of HUVEC distribution was still visible on the surface; by 14 days, the positions of red and green fluorescence signals completely overlapped, and SHED and HUVEC were completely fused together; this indicated that under the chemical induction of the paracrine components of the SHED spheroid, HUVEC could aggregate on the surface of the SHED spheroid and then fuse, and finally spontaneously organize into an integral structure.

[0048] During the process of dynamically monitoring the cells of SHED-HUVEC organoids by CD31 immunohistochemical staining method in the above step f, CD31 is also known as platelet-human umbilical vein endothelial cell adhesion molecule, which is commonly used to label human umbilical vein endothelial cells in immunohistochemical experiments to evaluate angiogenesis. During this process of cell dynamic monitoring, CD31 immunohistochemical staining was performed on frozen sections of SHED-HUVEC organoids to track the migration of HUVEC in SHED-HUVEC organoids cultured in DMEM OM medium at 6 days, 8 days, 10 days, and 14 days; DAB chromogenic agent (brown) was used to develop the color of HUVEC, and hematoxylin (blue-violet) was used to develop the color of the cell nucleus; as Figure 13 shown, when the SHED-HUVEC organoids were cultured to 6 days, HUVEC was still distributed in the outer layer of the organoids; by 8 days, HUVEC began to migrate to the inside of the organoids, and there were several small fissures around the center of the organoids; by 10 days, the fissures in the center of the SHED-HUVEC organoids expanded and fused with each other, and the distribution of human umbilical vein endothelial cells was visible around the fissures; by 14 days, the fissures in the SHED-HUVEC organoids fused with each other to form a cavity, which was surrounded by a continuous layer of human umbilical vein endothelial cells, which was consistent with the results of live-dead staining. In the control group (SHED spheroids), no obvious endothelial differentiation was observed within 14 days of culture in DMEM OM medium; this indicated that within 14 days of culture, coordinated migration occurred between HUVEC and SHED in the SHED-HUVEC organoids, gradually forming a cavity-like structure with a complete lining of human umbilical vein endothelial cells surrounding SHED on the outside.

[0049] In summary, the method for preparing SHED-HUVEC organoids in Example 1 constructs three-dimensional organoids based on human deciduous pulp stem cells (SHED) and human umbilical vein endothelial cells (HUVEC) to achieve the regeneration of functional dental pulp tissue and restore the biological functions of dental pulp tissue, such as nutrient supply, immune protection, and the ability to sense external stimuli, while reducing the risks of secondary infection and root canal longitudinal fracture. The SHED-HUVEC organoids prepared by the method for preparing SHED-HUVEC organoids have important clinical application value and can provide a new regenerative treatment strategy for the treatment of pulpal and periapical diseases. By achieving the regeneration of functional dental pulp tissue, it can not only effectively restore the biological functions of the affected teeth but also significantly improve the long-term survival rate of the affected teeth, especially suitable for the treatment of young permanent teeth. In addition, during the process of culturing and constructing SHED-HUVEC organoids by the method for preparing SHED-HUVEC organoids, the use of growth factors in the prior art is avoided, so the method for preparing SHED-HUVEC organoids reduces the cost and the risk of potential adverse reactions and improves the clinical feasibility and safety of dental pulp regeneration. The specific technical effects are as follows: 1. The SHED-HUVEC organoids prepared by the method for preparing SHED-HUVEC organoids in Example 1 can effectively promote the regeneration of functional dental pulp tissue and restore the nutrient supply, immune protection, and sensing ability of dental pulp tissue, that is, it can effectively solve the problem of the loss of biological functions of affected teeth in the prior art; 2. The method for preparing SHED-HUVEC organoids in Example 1 realizes the stabilization and chemotaxis of human umbilical vein endothelial cells through the paracrine effect of SHED spheroids. Since no additional growth factors need to be added, it can effectively solve the problem of dependence on growth factors in the prior art.

[0050] 3. The method for preparing SHED-HUVEC organoids in Example 1 realizes the rapid vascularization of the graft by constructing in vitro pre-vascularized SHED-HUVEC organoids, so it can effectively solve the problem of low survival rate of the graft in the prior art.

[0051] Example 2, an injection composite, which is formed by mixing the SHED-HUVEC organoids prepared by the above method for preparing SHED-HUVEC organoids with a biocompatible hydrogel.

[0052] Specifically, the preparation steps of the injection composite include: Prepare 10% GelMA hydrogel and keep it in the dark at 37°C in a water bath for later use; Directly resuspend and mix the SHED-HUVEC organoids with 10% GelMA hydrogel.

[0053] The injection complex of the second embodiment is applied to dental pulp regeneration, and the injection complex can be applied to dental pulp regeneration treatment; the injection complex can effectively improve the convenience and adaptability of clinical operations during dental pulp regeneration treatment.

[0054] The in vivo dental pulp regeneration performance of the above injection complex is verified by constructing an animal model below. The specific steps include: Step 1: Extract human single-root orthodontic teeth to prepare tooth segments with a thickness of 5 mm. Step 2: Perform conventional root canal preparation and disinfection treatment on the prepared tooth segments. Step 3: Prepare 10% GelMA hydrogel and keep it in the dark at 37 °C in a water bath for later use. Step 4: Resuspend and mix SHED-HUVEC organoids directly with 10% GelMA hydrogel. The cell density and ratio of each group are as follows: ① SHED-HUVEC 2D group: 7.5×10 6 SHEDs induced by DMEM OM for 14 days per milliliter and 2.5×10 6 HUVECs per milliliter. The cell ratio of SHED to HUVEC is 3:1; ② SHED Spheroid group: 3×10 4 SHED spheroids induced by OM (mineralization induction medium) for 14 days per milliliter; ③ SHED-HUVEC Organoid group: 3×10 4 SHED-HUVEC organoids induced by OM for 14 days per milliliter; ④ Control group: cell-free GelMA + tooth segments. Step 5: According to the experimental grouping, take 20 µL of GelMA-loaded cells, SHED spheroids or SHED-HUVEC organoids and inject them into the root canals of the tooth segments, and perform photocrosslinking by exposing to 9.16 W / cm 2 ultraviolet light for 20 s. Step 6: After injecting the complex into human ex vivo tooth segments, implant them subcutaneously on the back of 5-week-old nude mice to construct a semi-in situ dental pulp regeneration model. Step 7: Histological evaluation: After 8 weeks of in vivo culture, evaluate the vascular content and collagen fiber composition of the newly formed dental pulp tissue by H&E staining method and Masson staining method to verify the regeneration effect of SHED-HUVEC organoids in vivo.

[0055] Among them, as Figure 14The H&E staining results of the sagittal sections of the tooth segments are as follows. In the control group, a small amount of connective tissue could be observed growing inward from the opening in the root canal lumen of human teeth, but almost no new lumen structures were seen. In the SHED-HUVEC 2D group, more new connective tissue grew into the root canal lumen of human teeth compared with the control group. More abundant vascular-like structures could be observed in the new tissue. The diameters of the lumens varied, but almost no new blood vessels were seen in the new tissue or at the boundary between the new tissue and the mouse tissue. In the SHED Spheroid group, the collagen fibers in the new connective tissue were denser than those in the SHED-HUVEC 2D group, there were more vascular-like structures, the diameters of the lumens were larger, and perfusion was achieved in most cases. The boundary between the new tissue and the mouse tissue was significantly richer in vascular structures than that in the control group and the SHED-HUVEC 2D group. In the SHED-HUVEC Organoid group, the collagen fibers in the new connective tissue were denser than those in the SHED-HUVEC 2D group, and more perfused new mature lumen structures were observed than in the SHED Spheroid group. The content of new blood vessels at the boundary between the new tissue and the mouse tissue was significantly richer than that in the control group and the SHED Spheroid group. This indicates that the new tissue induced by the grafts formed by 3D culture methods contains more collagen fiber components, probably because more extracellular matrix is retained during the culture process.

[0056] In addition, as Figure 15The results of sagittal sections of the tooth segments shown after Masson staining. In Masson staining, collagen fibers are blue and muscle fibers are red. No connective tissue vascular structures were seen in the root canal lumen of teeth in the control group. In the SHED-HUVEC 2D group, more immature vascular structures were visible in the root canal lumen of human teeth, but few newly formed blood vessels that were mature and perfused were seen. Almost no newly formed blood vessel structures were observed in the newly formed tissue and at its boundary with mouse tissue. In the SHED Spheroid group, the collagen fibers in the newly formed connective tissue were denser than those in the SHED-HUVEC 2D group. More newly formed mature blood vessels that were perfused were visible. The boundary between the newly formed tissue and mouse tissue was significantly richer in vascular structures than in the control group and the SHED-HUVEC 2D group. In the SHED-HUVEC Organoid group, the collagen fibers in the newly formed connective tissue were denser than those in the SHED-HUVEC 2D group. Significantly more newly formed mature blood vessels that were perfused were observable compared to the SHED Spheroid group. The content of newly formed blood vessel structures at the boundary between the newly formed tissue and mouse tissue was significantly richer than in the control group and the SHED Spheroid group. This indicates that there is a greater component of collagen fibers in the newly formed tissue formed by the graft under the 3D culture method, which is consistent with the results of H&E staining. At the same time, it was observed that the SHED-HUVEC Organoid group formed more mature blood vessels with larger lumen diameters compared to other groups, indicating that SHED-HUVEC organoids have a stronger potential for regenerating pulp rich in blood vessels compared to SHED spheroids.

[0057] The above content is only a preferred embodiment of the present invention. For those of ordinary skill in the art, based on the idea of the present invention, there will be changes in the specific implementation methods and application scopes. The content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing SHED-HUVEC organoids, characterized in that, It includes the following steps, specifically: Step a, SHED extraction and culture: Extract SHED from human exfoliated deciduous teeth for primary culture and subculture amplification. Select cells of passages P3-P6 for subsequent experiments. SHED is the dental pulp stem cells of human exfoliated deciduous teeth; Step b, preparation of the spheroid culture model microporous plate: Pour the autoclaved 3% agarose solution into the polydimethylsiloxane microcolumn male mold. After the 3% agarose solution naturally cools and solidifies, a low-adhesion agarose culture model is obtained. Among them, the polydimethylsiloxane microcolumn male mold contains microcolumns with a diameter of 200 µm and a depth of 150 µm, and the distance between adjacent microcolumns is 200 µm; Each 100 mL of 3% agarose solution contains 3.0 g of agarose powder and 100 mL of ultrapure water; Then, use a cylindrical cutter with the same diameter as a single well of a 24-well plate to cut the low-adhesion agarose culture model into cylindrical agarose microporous plates, and place the cut cylindrical agarose microporous plates into the 24-well plate respectively. One cylindrical agarose microporous plate is placed in each well of the 24-well plate; Then, add 500 μL of PBS buffer to each agarose microporous plate in the 24-well plate, and after ultraviolet disinfection for 1 hour, it is ready for use; Step c: Construct SHED spheroids: After the 24-well plate with the agarose microwell plate laid out is disinfected with ultraviolet light, aspirate the PBS in each well of the 24-well plate, then add 800 μL of α-MEM PM medium to each well of the 24-well plate. Then, select the SHED at P3-P6 obtained by culturing in step a, and resuspend 5×10 5 cells per well in 200 μL of α-MEM PM medium and slowly add it dropwise to the agarose microwell plate. After standing for 10 minutes, place it in a 37°C constant temperature incubator for constant temperature culture for 24 hours; after the constant temperature culture is completed, replace the α-MEM PM medium in the agarose microwell plate with DMEM OM medium, and continue to place it in a 37°C constant temperature incubator for constant temperature culture. Replace half of the DMEM OM medium every day and induce for 14 days to form SHED spheroids with a diameter of 50 μm - 80 μm; Step d, viability detection: Detect the viability and cytoskeleton changes of SHED spheroids by the live-dead staining method and the DAPI / Phalloidin fluorescence staining method to verify its survival status and structural stability during the culture process; Step e, construction of SHED-HUVEC organoids: Step e1, culture of human umbilical vein endothelial cells: Purchase commercial primary HUVEC, inoculate HUVEC into a 96-well plate at a cell density of 3000 cells / well, and add 100 μL of endothelial cell medium to each well of the 96-well plate, and then incubate in the dark in a 37 °C cell culture incubator; Step e2. Delayed addition of human umbilical vein endothelial cells: When the SHED spheroids in step c are cultured for 4 days, the HUVECs cultured and incubated in step e1 are resuspended in endothelial cell medium to 3.2 × 10 5 cells / mL. Then, the HUVEC suspension is inoculated onto the agarose microporous plate of the 24-well plate in step c at 500 μL / well, and co-cultured in DMEM OM medium for another 14 days, with half of the DMEM OM medium replaced daily to form SHED-HUVEC organoids; Step f, cell dynamic monitoring: Observe the fusion process of HUVEC and SHED spheroids by the PKH26 / PKH67 live cell tracking staining method or the CD31 immunohistochemical staining method to verify the self-organization formation of SHED-HUVEC organoids.

2. The preparation method of a SHED-HUVEC organoid according to claim 1, characterized in that: In the step a, the medium used for SHED culture contains 44.5 mL of α-MEM medium, 5 mL of fetal bovine serum FBS, and 0.5 mL of penicillin / streptomycin double antibody solution; And the SHED culture environment in step a is: constant temperature culture in a 37 °C incubator.

3. The preparation method of a SHED-HUVEC organoid according to claim 1, characterized in that: In the step c, the α-MEM PM medium specifically is: 44.5 mL of α-MEM medium contains 5 mL of fetal bovine serum FBS and 0.5 mL of penicillin / streptomycin double antibody solution.

4. The preparation method of a SHED-HUVEC organoid according to claim 1, characterized in that: In the step c, the DMEM OM medium specifically is: in 50 mL of the DMEM OM medium, there are 5 mL of fetal bovine serum (FBS), 0.5 mL of penicillin / streptomycin double-antibody solution, 2.5 μL of dexamethasone solution with a concentration of 100 mmol / L, 0.5 mL of β-glycerophosphate sodium solution with a concentration of 10 mmol / L, 0.5 mL of vitamin C solution with a concentration of 50 mg / L, and the balance is high-glucose DMEM medium.

5. The preparation method of a SHED-HUVEC organoid according to claim 1, characterized in that: In the step e1, the endothelial cell medium is composed of 500 ml of basal medium, 25 ml of fetal bovine serum (FBS), 5 ml of endothelial cell growth supplement (ECGS), and 5 ml of penicillin / streptomycin double-antibody solution.

6. An injection complex, characterized in that: The injection complex is formed by mixing SHED-HUVEC organoids cultured and constructed by the method for preparing SHED-HUVEC organoids according to any one of claims 1-5 with a biocompatible hydrogel.

7. An injection complex according to claim 6, characterized in that, The preparation steps of the injection complex include: Preparing 10% GelMA hydrogel and keeping it in reserve under the condition of 37 °C water bath in the dark; Directly resuspending and mixing the SHED-HUVEC organoids with the 10% GelMA hydrogel.

8. Use of an injection complex according to claim 6 or 7 in the treatment of dental pulp regeneration.

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