A method for preparing a shed-huvec organoid, an injectable composite and applications thereof

Abstract

This invention discloses a method for preparing SHED-HUVEC organoids, an injectable complex, and their applications. The preparation method includes the following steps: a) SHED extraction and culture; b) preparation of a microporous disc culture model of spherical bodies; c) construction of SHED spherical bodies; d) activity detection; e) construction of SHED-HUVEC organoids; and f) cell dynamic monitoring. This preparation method constructs SHED-HUVEC organoids without the need for additional growth factors, thus reducing the defects associated with the use of growth factors. SHED-HUVEC organoids significantly promote the regeneration of vascularized dental pulp tissue, thereby enabling the regenerated dental pulp tissue to form more mature blood vessels, improving the vascularization level and functional recovery of the regenerated dental pulp tissue. The injectable complex is prepared by mixing SHED-HUVEC organoids with a biocompatible hydrogel, and this injectable complex is used in dental pulp regeneration therapy.

Description

Technical Field

[0001] This invention relates to the field of dental pulp regeneration technology, and in particular to a method for preparing SHED-HUVEC organoids, an injectable complex, and their applications. Background Technology

[0002] Traditional treatments for pulpitis and periapical periodontitis mainly include apical barrier surgery and root canal treatment. These methods primarily involve removing necrotic pulp tissue and sealing the root canal system with biomaterials to control inflammation and promote healing of periapical lesions. However, while these procedures can alleviate symptoms, the treated teeth are at risk of secondary infection and longitudinal root canal fractures due to a lack of nutrient supply, immune protection, and the ability to sense external stimuli. This risk is particularly pronounced in young permanent teeth.

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

[0004] It should be noted that the success of tissue engineering depends on the rapid vascularization of the graft. Due to the elongated anatomy of the root canal system, the growth of new capillaries is slow, making it difficult to meet the nutritional and oxygen requirements of the graft. To promote rapid graft vascularization, current techniques achieve in vitro prevascularization by adding endothelial cells. However, this method still relies on the addition of angiogenic factors, which have drawbacks such as high cost, difficulty in preservation, short in vivo half-life, and potential adverse reactions. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing SHED-HUVEC organoids, an injectable complex, and its applications. Specifically: This method for preparing SHED-HUVEC organoids enables the construction of SHED-HUVEC organoids based on human exfoliated deciduous tooth pulp stem cells (SHED) and human umbilical vein endothelial cells (HUVEC) without the need for additional growth factors. By constructing SHED spheroids and utilizing their paracrine products to stabilize and chemotact with human umbilical vein endothelial cells, SHED-HUVEC organoids significantly promote the regeneration of vascularized dental pulp tissue, thereby enabling the regenerated dental pulp tissue to form more mature blood vessels, thus improving the vascularization level and functional recovery of the regenerated dental pulp tissue. Since no additional growth factors are required, this method for preparing SHED-HUVEC organoids effectively overcomes the limitations of existing technologies that rely on growth factors. This injectable compound is prepared by mixing SHED-HUVEC organoids with biocompatible hydrogel. This injectable compound is used in pulp regeneration therapy and can effectively improve the convenience and adaptability of clinical operation during pulp regeneration therapy.

[0006] To achieve the above objectives, the present invention is implemented through 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: SHED was extracted from human deciduous teeth for primary culture and passage expansion. P3-P6 generation cells were selected for subsequent experiments. SHED is human deciduous tooth pulp stem cells. Step b, Preparation of spherical culture model microplates: Pour the 3% agarose solution, sterilized by high temperature and high pressure, into the polydimethylsiloxane microcolumn positive mold. After the 3% agarose solution cools and solidifies naturally, a low-adhesion agarose culture model is obtained. The polydimethylsiloxane microcolumn positive mold contains microcolumns with a diameter of 200 µm and a depth of 150 µm, and the spacing 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, using a cylindrical cutter of the same diameter as the well of a 24-well plate, the low-adhesion agarose culture model was cut into cylindrical agarose microplates. The cut cylindrical agarose microplates were then placed into each well of a 24-well plate, with one cylindrical agarose microplate in each well. Next, 500 μL of PBS buffer was added to each agarose microplate in the 24-well plate, and the plates were sterilized under ultraviolet light for 1 hour before use. Step c, Constructing SHED spheres: After sterilizing a 24-well plate with agarose microplates using UV light, aspirate PBS from each well. Then, add 800 μL of α-MEM PM medium to each well. Next, use the P3-P6 SHEDs obtained in step a, and fill each well with 5 × 10⁶ spheroids. 5 One cell was resuspended in 200 μL of α-MEM PM medium and slowly added dropwise to an agarose microplate. After standing for 10 minutes, the plate was placed in a 37°C incubator for 24 hours. After the incubation, the α-MEM PM medium in the agarose microplate was replaced with DMEM OM medium, and the plate was placed in a 37°C incubator for 14 days. Half of the DMEM OM medium was replaced daily to induce the formation of SHED spheroids with a diameter of 50 μm-80 μm. Step d, Activity detection: The activity and cytoskeleton changes of SHED spheroids were detected by live-dead staining and DAPI / Phalloidin fluorescence staining to verify their survival status and structural stability during culture. Step e: Construct SHED-HUVEC organoids: Step e1: Culture human umbilical vein endothelial cells: Purchase commercial primary HUVECs, seed HUVECs into 96-well plates at a cell density of 3000 cells / well, add 100 μL of endothelial cell culture medium to each well of the 96-well plate, and then incubate in a cell culture incubator at 37°C in the dark. Step e2, Delayed addition of human umbilical vein endothelial cells: On day 4 of SHED spheroid culture in step c, the HUVECs cultured and incubated in step e1 were resuspended in endothelial cell culture medium to 3.2 × 10⁻⁶ cells. 5 cells / mL, and then the HUVEC suspension was seeded at 500 μL / well onto the agarose microplate of the 24-well plate in step c, and continued to be co-cultured in DMEM OM medium until day 14, with half of the DMEM OM medium being replaced daily to form SHED-HUVEC organoids. Step f, Cell dynamic monitoring: The fusion process of HUVEC and SHED spheroids was observed by PKH26 / PKH67 live cell tracking staining or CD31 immunohistochemical staining to verify the self-organization of SHED-HUVEC organoids.

[0008] In step a, the culture 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 solution. Furthermore, the SHED culture environment in step a is: constant temperature culture in a 37℃ constant temperature incubator.

[0009] In step c, the α-MEM PM medium specifically consists of 44.5 mL of α-MEM medium containing 5 mL of fetal bovine serum (FBS) and 0.5 mL of penicillin / streptomycin solution.

[0010] In step c, the DMEM OM medium specifically comprises: 50 mL of DMEM OM medium containing 5 mL of fetal bovine serum (FBS), 0.5 mL of penicillin / streptomycin solution, 2.5 μL of dexamethasone solution at a concentration of 100 mmol / L, 0.5 mL of sodium β-glycerophosphate solution at a concentration of 10 mmol / L, 0.5 mL of vitamin C solution at a concentration of 50 mg / L, and the remainder being high-glucose DMEM medium.

[0011] In 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 supplement (ECGS), and 5 ml of penicillin / streptomycin solution.

[0012] An injectable complex is formed by mixing SHED-HUVEC organoids cultured and constructed by the above-described method for preparing SHED-HUVEC organoids with a biocompatible hydrogel.

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

[0014] Application of the above-mentioned injection compound in pulp regeneration treatment.

[0015] Compared with the prior art, the present invention has the following beneficial effects, specifically: 1. The SHED-HUVEC organoids prepared by the method of the present invention can effectively promote the regeneration of functional dental pulp tissue and restore the nutritional supply, immune protection and sensory ability of dental pulp tissue, that is, can effectively solve the problem of loss of biological function of diseased teeth in the prior art. 2. The method for preparing 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, it can effectively solve the problem of dependence on growth factors in the prior art.

[0016] 3. The method for preparing SHED-HUVEC organoids of the present invention achieves rapid vascularization of the graft by constructing in vitro prevascularized SHED-HUVEC organoids, thus effectively solving the problem of low survival rate of grafts in the prior art; 4. The injectable compound of the present invention can effectively improve the convenience and adaptability of clinical operation in the process of pulp regeneration treatment; 5. Therefore, the preparation method of the SHED-HUVEC organoid, the injectable complex, and its application in this invention have significant clinical application value, providing a novel regenerative treatment strategy for pulpitis and periapical periodontitis. By achieving functional pulp regeneration, this valve core can not only effectively restore the biological function of the affected tooth but also significantly improve its long-term survival rate, making it particularly suitable for the treatment of young permanent teeth. Furthermore, because this invention avoids the use of growth factors, it effectively reduces the costs and potential adverse reaction risks associated with the use of growth factors, thereby improving the clinical feasibility and safety of pulp regeneration. Attached Figure Description

[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 on the present invention.

[0018] Figure 1 This is a flowchart illustrating the fabrication process of a low-adhesion agarose microporous disk.

[0019] Figure 2 This image shows the process of cells transitioning from uniform suspension and aggregation to spherical formation at different time points after SHED plating under an inverted optical microscope.

[0020] Figure 3 The staining status of SHED spheroids at 6d, 8d, 10d, and 14d shows their liveness and deadness.

[0021] Figure 4 The staining of SHED spheroids with phalloid peptides at 6d, 8d, 10d, and 14d.

[0022] Figure 5 This is a comparative diagram showing the effect of SHED spheroid supernatant on the proliferation of HUVECs.

[0023] Figure 6 A comparative diagram showing the promoting effect of SHED spheroid supernatant on the horizontal migration of HUVECs.

[0024] Figure 7 A comparative diagram showing the promoting effect of SHED spheroid supernatant on the vertical migration of HUVECs.

[0025] Figure 8 This is a comparative graph showing the promoting effect of SHED spheroid supernatant on the tube-forming ability of HUVECs.

[0026] Figure 9 This is a diagram illustrating the culturing process of SHED-HUVEC organoids.

[0027] Figure 10 The live and dead staining status of SHED-HUVEC organoids at 6d, 8d, 10d, and 14d.

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

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

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

[0031] Figure 14 The results of H&E staining of sagittal sections of the tooth segment.

[0032] Figure 15 This is the result of Masson staining a sagittal section of a tooth segment. Detailed Implementation

[0033] The present invention will now be described in conjunction with specific embodiments.

[0034] Example 1: A method for preparing SHED-HUVEC organoids, comprising the following steps: Step a, SHED extraction and culture: SHED was extracted from human deciduous teeth for primary culture and passage expansion. P3-P6 generation cells were selected for subsequent experiments. SHED is human deciduous tooth pulp stem cells. Step b: Preparation of microplates for spherical culture model: A 3% agarose solution sterilized by high temperature and pressure is poured into a polydimethylsiloxane micropillar positive mold. After the 3% agarose solution cools and solidifies naturally, a low-adhesion agarose culture model is obtained. The polydimethylsiloxane micropillar positive mold contains micropillars with a diameter of 200 µm and a depth of 150 μm, with a spacing of 200 μm between adjacent micropillars. Each 100 mL of 3% agarose solution contains 3.0 g of agarose powder and 100 mL of ultrapure water. Then, using a cylindrical cutter of the same diameter as a single well of a 24-well plate, the low-adhesion agarose culture model is cut into cylindrical agarose microplates. The cut cylindrical agarose microplates are then placed into each well of a 24-well plate. Finally, 500 μL of [unspecified liquid] is added to each agarose microplate in the 24-well plate. Prepare PBS buffer and sterilize it under ultraviolet light for 1 hour before use; Step c, Constructing SHED spheres: After sterilizing a 24-well plate with agarose microplates using UV light, aspirate PBS from each well. Then, add 800 μL of α-MEM PM medium to each well. Next, use the P3-P6 SHEDs obtained in step a, and fill each well with 5 × 10⁶ spheroids. 5 One cell was resuspended in 200 μL of α-MEM PM medium and slowly added dropwise to an agarose microplate. After standing for 10 minutes, the plate was placed in a 37°C incubator for 24 hours. After the incubation, the α-MEM PM medium in the agarose microplate was replaced with DMEM OM medium, and the plate was placed in a 37°C incubator for 14 days. Half of the DMEM OM medium was replaced daily to induce the formation of SHED spheroids with a diameter of 50 μm-80 μm. Step d, Activity detection: The activity and cytoskeleton changes of SHED spheroids were detected by live-dead staining and DAPI / Phalloidin fluorescence staining to verify their survival status and structural stability during culture. Step e: Construct SHED-HUVEC organoids: Step e1: Culture human umbilical vein endothelial cells: Purchase commercial primary HUVECs, seed HUVECs into 96-well plates at a cell density of 3000 cells / well, add 100 μL of endothelial cell culture medium to each well of the 96-well plate, and then incubate in a cell culture incubator at 37°C in the dark. Step e2, Delayed addition of human umbilical vein endothelial cells: On day 4 of SHED spheroid culture in step c, the HUVECs cultured and incubated in step e1 were resuspended in endothelial cell culture medium to 3.2 × 10⁻⁶ cells. 5 cells / mL, and then the HUVEC suspension was seeded at 500 μL / well onto the agarose microplate of the 24-well plate in step c, and continued to be co-cultured in DMEM OM medium until day 14, with half of the DMEM OM medium being replaced daily to form SHED-HUVEC organoids. Step f, Cell dynamic monitoring: The fusion process of HUVEC and SHED spheroids was observed by PKH26 / PKH67 live cell tracking staining or CD31 immunohistochemical staining to verify the self-organization of SHED-HUVEC organoids.

[0035] In step a, the culture 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 solution; and the SHED culture environment in step a is constant temperature culture in a 37℃ incubator.

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

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

[0038] It should be noted that in 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 supplement (ECGS), and 5 ml of penicillin / streptomycin solution.

[0039] During the preparation of the spherical culture model microporous disk in step b above, such as Figure 1 As shown in the fabrication flowchart of the low-adhesion agarose microplate, micropillars with a diameter of 200 μm and a depth of 150 μm are visible on the polydimethylsiloxane micropillar positive mold fabricated using SU-8 photolithography, with a spacing of 200 μm between the micropillars. During the preparation process, a 3% agarose solution sterilized at high temperature and pressure and below 60°C is poured onto the polydimethylsiloxane micropillar positive mold to obtain the negative mold, i.e., the agarose microplate. Then, the microplate is cut with a cutter with a diameter matching that of a 24-well culture plate and placed in the well plate for observation under an inverted microscope, where micropores with a diameter of 200 μm are visible.

[0040] When observing the formation process of SHED spheroids in step c above using an inverted optical microscope, such as Figure 2 The diagram shown is an inverted optical microscope illustrating the process of SHED cells at different time points after plating, from uniform suspension and aggregation to the formation of spherical bodies. It can be seen that after 0.5 h of SHED plating, cells uniformly aggregate into microwells, and between 5 h and 1 d, they initially self-aggregate into spherical bodies. As the culture time changes, the volume gradually decreases and tends to become a regular spherical shape; at the same time, the connections between cells gradually become tighter, and the surface becomes smoother; after 14 days of culture, the diameter of the SHED spherical bodies is approximately 50 μm-80 μm.

[0041] In the process of detecting the activity of SHED spheroids using the live / dead staining method, such as Figure 3 The images show the live and dead cell staining of SHED spheroids at 6, 8, 10, and 14 days. Green fluorescent dye (Calcein-AM) can penetrate the cell membrane and be released by intracellular lipases to label live cells. Red fluorescent dye (PI) can bind to the DNA double helix in the exposed nucleus of inactive cells, labeling dead cells. According to laser confocal microscopy, the volume of SHED spheroids gradually decreased with increasing culture time, reaching a relatively stable size at 10 days. Simultaneously, the morphology of the spheroids tended towards regular spheres, and the surface became smoother. A large amount of green fluorescence from live cells was observed in the SHED spheroids at all time points. However, no significant red fluorescence was observed with increasing time, indicating that the spheroids received sufficient nutrient supply at their center during the 14-day culture period, and no significant necrosis occurred.

[0042] During the activity detection of SHED spheroids using the DAPI / Phalloidin fluorescent staining method, such as Figure 4 The images show the phalloidin staining of SHED spheroids at 6, 8, 10, and 14 days. FITC (fibrillated filamentous protein monomers) specifically binds to eukaryotic cell fibrillary monomers to stain the cytoskeleton; DAPI (dark blue fluorescent dye) stains the cell nucleus. Laser confocal microscopy reveals that as culture time increases, cell adhesion within the SHED spheroids increases and they gradually become more tightly connected. This indicates that the SHED spheroids aggregate more densely over 14 days of culture, increasing the intercellular communication area.

[0043] In the process of detecting the effect of SHED spheroid supernatant on the proliferation, migration, and angiogenesis of HUVECs, the results of the CCK-8 cytotoxicity assay showed that, as Figure 5 As shown, continuous cell proliferation was observed in both the SHED 2D group and the SHEDSpheroid group at 24h, 48h, 72h, and 96h. At 24h and 72h, the OD value of the SHED Spheroid group was higher than that of the SHED 2D group, and the difference was statistically significant (P < 0.05), indicating that the SHED spheroid supernatant has a certain cell proliferation-promoting effect. Based on the scratch assay results, it can be seen that... Figure 6 As shown, after 12 h of treatment with the supernatant, compared with the SHED2D group, the scratch width of HUVEC cells in the SHED Spheroid group was significantly narrower, the cells were almost confluent, and the boundaries were blurred; quantitative results showed that under the action of SHED spheroid supernatant, the horizontal migration rate of HUVEC cells was higher than that in the SHED 2D group, and the difference was statistically significant (P<0.05). Based on the Transwell chamber experiment results, as... Figure 7As shown, the cell density that migrated to the submural membrane in the SHED Spheroid group was significantly greater than that in the SHED 2D group. Under the influence of the SHED spheroid supernatant, the vertical migration rate of HUVECs was higher in the SHED 2D group than in the SHED spheroid group, and the difference was statistically significant (P<0.05). Based on the results of the Matrigel lumen formation assay, as... Figure 8 As shown, the number of tubular connection points, the number of nodes, and the total length of the main lumen in the SHED Spheroid group were all higher than those in the SHED 2D group, as demonstrated by quantitative statistics. Figure 8 B) Consistent with the above observations, the difference was statistically significant (P < 0.05). Therefore, SHED spheroids can secrete effective components that promote angiogenesis, and SHED spheroid paracrine products (e.g., supernatant) can enhance the proliferation, migration, and tube formation ability of human umbilical vein endothelial cells. Furthermore, experimental results show that HUVECs stimulated with SHED spheroid supernatant are significantly superior to traditionally cultured SHED cells in multiple indicators, indicating their significant advantage in promoting angiogenesis.

[0044] During the construction of SHED-HUVEC organoids in step e above, such as Figure 9 As shown, observations using an inverted optical microscope revealed that, under the influence of DMEM OM medium, pre-coated SHEDs gradually self-aggregated to form smooth spherical bodies. After 4 days of culture, HUVECs were added later. As the culture time increased, HUVECs gradually aggregated on the outer layer of the original SHED spherical bodies and gradually fused with them. The connections between cells became tighter, and the overall volume gradually decreased. After 10 days, the volume remained basically unchanged, and by 14 days, the diameter was approximately 70 μm. During the culture period from 7 to 14 days, unlike the smooth surface of the SHED spherical bodies, the SHED-HUVEC organoids exhibited an irregular surface morphology.

[0045] In the process of detecting SHED-HUVEC organoids using the live-death staining method, the survival status of SHED-HUVEC organoids at 6, 8, 10, and 14 days of culture was evaluated using live-death staining technology. Three-dimensional imaging was performed using a laser confocal microscope, and the results are as follows: Figure 10As shown; at all time points, a large number of green fluorescently labeled live cells were observed on the surface of SHED-HUVEC organoids, and no obviously red fluorescently labeled dead or low-activity cells were found; with the extension of culture time, the volume of SHED-HUVEC organoids gradually decreased, and the internal cell distribution was relatively loose, while the cells of SHED spheroids were tightly connected; SHED-HUVEC organoids exhibited an irregular surface morphology, which was consistent with the morphology observed under an inverted microscope; at day 14, the green fluorescence intensity in the central part of the SHED-HUVEC organoids decreased, suggesting the possible presence of cavities.

[0046] In the process of detecting SHED-HUVEC organoids using DAPI / Phalloidin fluorescence staining, the cytoskeleton morphology of SHED-HUVEC organoids at 6, 8, 10, and 14 days of culture was tracked using DAPI / Phalloidin staining. The three-dimensional imaging results after laser confocal microscopy are shown in Figure 11. With the extension of culture time, the cytoskeleton inside the SHED-HUVEC organoids gradually shrinks, intercellular adhesion increases, and the connections gradually become tighter, exhibiting characteristics similar to SHED spherical bodies. By day 10 of culture, the volume of SHED-HUVEC organoids is basically stable, which is verified by inverted microscopy and live / dead staining results. This indicates that within 14 days of culture, SHED-HUVEC organoids undergo a cell migration process, the binding between SHED and HUVEC gradually strengthens, and the intercellular communication area increases.

[0047] In step f above, during the cell dynamic monitoring of SHED-HUVEC organoids using the PKH26 / PKH67 live-cell tracking staining method, PKH dyes are cell membrane dyes with extremely low cytotoxicity, labeling cells while preserving their biological and proliferative activity. PKH dyes have a half-life of over 100 days and are commonly used for in vivo cell tracking and studying cell-cell interactions. In this cell dynamic monitoring process, 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 laser confocal microscopy are shown below. Figure 12As shown; at 6 days, that is, 2 days after the delayed addition of HUVEC, the SHED-HUVEC organoids generally exhibited a distinct double-layer structure, with the central region consisting of red fluorescently labeled SHED spheres surrounded by a green fluorescently labeled HUVEC layer; by 8 days, the surface of the SHED spheres began to diffuse outwards and fuse with HUVECs; by 10 days, the distribution of red and green fluorescent signals largely overlapped, and SHED and HUVECs were basically fused together. At this point, the overall volume of the organoids shrank, and a thin layer of HUVECs was still visible on the surface; by 14 days, the positions of red and green fluorescent signals completely overlapped, and SHED and HUVECs were completely fused together. This indicates that under the chemical induction of paracrine components from SHED spheres, HUVECs can aggregate on the surface of SHED spheres and subsequently fuse, eventually spontaneously organizing into a unified structure.

[0048] In step f above, during the cell dynamics monitoring of SHED-HUVEC organoids using CD31 immunohistochemical staining, CD31, also known as the platelet-human umbilical vein endothelial cell adhesion molecule, is commonly used in immunohistochemical experiments to label human umbilical vein endothelial cells to assess angiogenesis. In this cell dynamics monitoring process, CD31 immunohistochemical staining was performed on frozen sections of SHED-HUVEC organoids to track HUVEC migration at 6, 8, 10, and 14 days of culture in DMEM OM medium. DAB chromogenic agent (brownish-colored) was used to develop the HUVECs, and hematoxylin (blue-purple) was used to develop the cell nuclei. Figure 13 As shown, at 6 days of culture, HUVECs in SHED-HUVEC organoids remained distributed in the outer layer of the organoids; at 8 days, HUVECs began to migrate into the interior of the organoids, with several small fissures around the center; at 10 days, the central fissures in the SHED-HUVEC organoids enlarged and merged, with human umbilical vein endothelial cells visible around the fissures; at 14 days, the fissures within the SHED-HUVEC organoids merged to form a cavity surrounded by a continuous layer of human umbilical vein endothelial cells, consistent with the results of live / dead staining. In contrast, no significant endothelial differentiation was observed in the control group (SHED spheroids) cultured in DMEM OM medium for 14 days. This indicates that during the 14-day culture period, coordinated migration occurred between HUVECs and SHEDs in the SHED-HUVEC organoids, gradually forming a cavity-like structure surrounded by SHEDs and lined with a complete layer of human umbilical vein endothelial cells.

[0049] In summary, the SHED-HUVEC organoid preparation method of this embodiment constructs three-dimensional organoids based on human exfoliated deciduous tooth pulp stem cells (SHED) and human umbilical vein endothelial cells (HUVEC) to achieve functional pulp tissue regeneration and restore its biological functions, such as nutrient supply, immune protection, and the ability to sense external stimuli, while reducing the risk of secondary infection and root canal fracture. The SHED-HUVEC organoids cultured and constructed using this method have significant clinical application value, providing a novel regenerative treatment strategy for pulpitis and periapical periodontitis. By achieving functional pulp tissue regeneration, it can not only effectively restore the biological function of the affected tooth but also significantly improve its long-term survival rate, making it particularly suitable for the treatment of young permanent teeth. Furthermore, in the process of culturing and constructing SHED-HUVEC organoids using this method, the use of growth factors, as found in existing technologies, is avoided. Therefore, this method reduces costs and potential adverse reaction risks, and improves the clinical feasibility and safety of pulp regeneration. Specific technical effects are as follows: 1. The SHED-HUVEC organoids prepared by the method of this embodiment can effectively promote the regeneration of functional dental pulp tissue and restore the nutritional supply, immune protection and sensory ability of dental pulp tissue, thus effectively solving the problem of loss of biological function of diseased teeth in the prior art. 2. The method for preparing SHED-HUVEC organoids in this embodiment 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, 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 this embodiment achieves rapid vascularization of the graft by constructing in vitro prevascularized SHED-HUVEC organoids, thus effectively solving the problem of low graft survival rate in the prior art.

[0051] Example 2: An injectable complex, which is composed of SHED-HUVEC organoids cultured and constructed by the above-described method for preparing SHED-HUVEC organoids and a biocompatible hydrogel.

[0052] Specifically, the preparation steps of this injection complex include: Prepare a 10% GelMA hydrogel and store it in a 37°C water bath in the dark. SHED-HUVEC organoids were directly resuspended and mixed with 10% GelMA hydrogel.

[0053] The injectable compound of this embodiment 2 is applied to pulp regeneration, and the injectable compound can be used in pulp regeneration treatment; the injectable compound can effectively improve the convenience and adaptability of clinical operation in pulp regeneration treatment.

[0054] The following steps will be taken to verify the in vivo pulp regeneration performance of the above-mentioned injection complex by constructing an animal model. Step 1: Extract a single-rooted orthodontic tooth to prepare a 5 mm thick tooth segment; Step 2: Perform routine root canal preparation and disinfection on the obtained tooth segment; Step 3: Prepare 10% GelMA hydrogel and store it in a 37°C water bath away from light for later use; Step 4: Resuspend and mix SHED-HUVEC organoids directly with 10% GelMA hydrogel. The cell density and proportions for each group are as follows: ① SHED-HUVEC 2D group: 7.5 × 10⁻⁶ cells / mL 6 SHED induced by DMEM OM for 14 days and 2.5 × 10⁻⁶ mg / mL 6 ① The ratio of SHED to HUVEC cells was 3:1; ② The SHED Spheroid group was 3 × 10⁶ cells per milliliter. 4 ③ SHED spheroids induced for 14 days by OM (mineralization induction medium); ③ SHED-HUVEC Organoid group: 3×10⁶ cells / mL 4 ④ SHED-HUVEC organoids induced by OM for 14 days; ④ Control group consisted of acellular GelMA + tooth segments; Step 5: According to the experimental groups, inject 20 µL of GelMA-loaded cells, SHED spheroids, or SHED-HUVEC organoids into the root canal of the tooth segment, and expose them to 9.16 W / cm². 2 Photocrosslinking is performed under ultraviolet light for 20 seconds; Step 6: After injecting the complex into the extracted human tooth segment, it was implanted subcutaneously into the back of 5-week-old nude mice to construct a semi-in situ pulp regeneration model. Step 7, Histological evaluation: After 8 weeks of in vivo culture, the vascular content and collagen fiber composition of the newly formed dental pulp tissue were evaluated by H&E staining and Masson staining to verify the regenerative effect of SHED-HUVEC organoids in vivo.

[0055] Among them, such as Figure 14The H&E staining results of sagittal sections of the tooth segments shown indicate that in the control group, a small amount of connective tissue grew inward from the opening of the root canal, but almost no new luminal structures were observed. In the SHED-HUVEC2D group, more new connective tissue ingrowth was observed in the root canal lumen compared to the control group, and more vascular-like structures were observed within the new tissue. The diameter of the lumen varied, but almost no new blood vessels were observed within the new tissue or at its boundary with the mouse tissue. In the SHED Spheroid group, the collagen fibers in the new connective tissue were denser than in the SHED-HUVEC 2D group, with more vascular-like structures and larger lumen diameters, achieving perfusion in most areas. The boundary between the new tissue and the mouse tissue contained significantly more vascular structures than in the control and SHED-HUVEC 2D groups. In the SHED-HUVEC Organoid group, the collagen fibers in the new connective tissue were denser than in the SHED-HUVEC 2D group and denser than in the SHED Spheroid group. The group observed more newly formed mature luminal structures that achieved perfusion, and the content of new blood vessels at the boundary between the new tissue and 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 graft formed by 3D culture has more collagen fiber components, possibly due to the retention of more extracellular matrix during the culture process.

[0056] In addition, such as Figure 15The results of sagittal sections of the tooth segment after Masson staining are shown. In Masson staining, collagen fibers appear blue and muscle fibers appear red. No connective tissue vascular structures were observed in the root canals of the control group human teeth. In the SHED-HUVEC 2D group human teeth root canals, numerous immature vascular structures were observed, but few mature and perfused neovascularizations were seen. Almost no neovascularization was observed within the neoplastic tissue or at its boundary with mouse tissue. In the SHED Spheroid group, the collagen fibers in the neoplastic connective tissue were denser than in the SHED-HUVEC 2D group, and more mature and perfused neovascularizations were observed. The boundary between the neoplastic tissue and mouse tissue contained significantly more vascular structures than in the control and SHED-HUVEC 2D groups. In the SHED-HUVEC Organoid group, the collagen fibers in the neoplastic connective tissue were denser than in the SHED-HUVEC 2D group, and significantly more mature and perfused neovascularizations were observed than in the SHED Spheroid group. The content of neovascularization at the boundary between the neoplastic tissue and mouse tissue was significantly higher than in the control and SHED groups. The spheroid group was abundant. This indicates that the newly formed tissue from grafts grown using the 3D culture method contained more collagen fibers, consistent with the H&E staining results. Simultaneously, the SHED-HUVEC Organoid group was observed to form more mature blood vessels with larger luminal diameters compared to other groups, suggesting that SHED-HUVEC organoids have a stronger potential for regenerating highly vascularized dental pulp than SHED spheroids.

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

Claims

1. A method of preparing a SHED-HUVEC organoid, characterized by, It includes the following steps, specifically: Step a, SHED extraction and culture: SHED was extracted from human deciduous teeth for primary culture and passage expansion. P6 generation cells were selected for subsequent experiments. SHED is human deciduous tooth pulp stem cells. The culture medium used for SHED culture contained 44.5 mL of α-MEM medium, 5 mL of fetal bovine serum (FBS), and 0.5 mL of penicillin / streptomycin solution. The SHED culture environment was: constant temperature culture in a 37℃ incubator; Step b, Preparation of spherical culture model microplates: Pour the 3% agarose solution, sterilized by high temperature and high pressure, into the polydimethylsiloxane microcolumn positive mold. After the 3% agarose solution cools and solidifies naturally, a low-adhesion agarose culture model is obtained. The polydimethylsiloxane microcolumn positive mold contains microcolumns with a diameter of 200 µm and a depth of 150 µm, and the spacing 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, using a cylindrical cutter of the same diameter as the well of a 24-well plate, the low-adhesion agarose culture model was cut into cylindrical agarose microplates. The cut cylindrical agarose microplates were then placed into each well of a 24-well plate, with one cylindrical agarose microplate in each well. Next, 500 μL of PBS buffer was added to each agarose microplate in the 24-well plate, and the plates were sterilized under ultraviolet light for 1 hour before use. Step c, Constructing SHED spheres: After sterilizing a 24-well plate with agarose microplates using UV light, aspirate PBS from each well. Then, add 800 μL of α-MEM PM medium to each well. Next, use the P3-P6 SHEDs obtained in step a, and fill each well with 5 × 10⁶ spheroids. 5 One cell was resuspended in 200 μL of α-MEM PM medium and slowly added dropwise to an agarose microplate. After standing for 10 minutes, the plate was placed in a 37°C incubator for 24 hours. After the incubation, the α-MEM PM medium in the agarose microplate was replaced with DMEM OM medium, and the plate was placed in a 37°C incubator for 14 days. Half of the DMEM OM medium was replaced daily to induce the formation of SHED spheroids with a diameter of 50 μm-80 μm. The α-MEM PM medium specifically consists of 44.5 mL of α-MEM medium containing 5 mL of fetal bovine serum (FBS) and 0.5 mL of penicillin / streptomycin solution. The DMEM OM medium consists of: 50 mL of DMEM OM medium containing 5 mL of fetal bovine serum (FBS), 0.5 mL of penicillin / streptomycin solution, 2.5 μL of 100 mmol / L dexamethasone solution, 0.5 mL of 10 mmol / L sodium β-glycerophosphate solution, 0.5 mL of 50 mg / L vitamin C solution, and the remainder being high-glucose DMEM medium. Step d, Activity detection: The activity and cytoskeleton changes of SHED spheroids were detected by live-dead staining and DAPI / Phalloidin fluorescence staining to verify their survival status and structural stability during culture. Step e: Construct SHED-HUVEC organoids: Step e1: Culture human umbilical vein endothelial cells: Purchase commercial primary HUVECs, seed HUVECs into 96-well plates at a cell density of 3000 cells / well, add 100 μL of endothelial cell culture medium to each well of the 96-well plate, and then incubate in a cell culture incubator at 37°C in the dark. 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 supplement (ECGS), and 5 ml of penicillin / streptomycin antibiotic solution. Step e2, Delayed addition of human umbilical vein endothelial cells: On day 4 of SHED spheroid culture in step c, the HUVECs cultured and incubated in step e1 were resuspended in endothelial cell culture medium to 3.2 × 10⁻⁶ cells. 5 cells / mL, and then the HUVEC suspension was seeded at 500 μL / well onto the agarose microplate of the 24-well plate in step c, and continued to be co-cultured in DMEM OM medium until day 14, with half of the DMEM OM medium being replaced daily, to form SHED-HUVEC organoids with a cavity-like structure of SHED spherical bodies surrounded by a layer of human umbilical vein endothelial cells; Step f, Cell dynamic monitoring: The fusion process of HUVEC and SHED spheroids was observed by PKH26 / PKH67 live cell tracking staining or CD31 immunohistochemical staining to verify the self-organization of SHED-HUVEC organoids.

2. An injection compound, characterized in that: The injection complex is composed of SHED-HUVEC organoids cultured and constructed by the method of preparation of SHED-HUVEC organoids according to claim 1, mixed with a biocompatible hydrogel.

3. An injection compound according to claim 2, wherein, The preparation steps of this injection complex include: Prepare a 10% GelMA hydrogel and store it in a 37°C water bath in the dark. SHED-HUVEC organoids were directly resuspended and mixed with 10% GelMA hydrogel.

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