Preparation method of lithium chloride induced SHEDs three-dimensional microspheres, injection type compound and application of injection type compound

Abstract

The invention discloses a preparation method of lithium chloride induced SHEDs three-dimensional microspheres, an injection type compound and application of the injection type compound. The preparation method of the lithium chloride induced SHEDs three-dimensional microspheres comprises the following steps: a, extracting and culturing cells; step b, screening the optimal LiCl concentration for inducing the dentin differentiation of the SHEDs; c, constructing the SHEDs three-dimensional microspheres by adopting an agarose micropore three-dimensional culture method; and d, detecting the activity of the SHEDs three-dimensional microspheres. According to the preparation method, the use of exogenous growth factors is avoided, the SHEDs are induced to differentiate into the dentin cells through lithium chloride, the SHEDs three-dimensional microspheres are constructed in combination with a three-dimensional culture technology, and the differentiation efficiency and regeneration capacity of the dentin cells are remarkably improved by simulating an in-vivo microenvironment. The injection type compound is prepared by mixing the SHEDs three-dimensional microspheres and biocompatible hydrogel, and can be applied to dental pulp regeneration treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of dental pulp regeneration, and particularly to a preparation method of lithium chloride-induced SHEDs three-dimensional microspheres, an injectable composite and its application. Background Art

[0002] The regeneration of the dental pulp-dentin complex is an important goal in the treatment of endodontic diseases. An ideal dental pulp regeneration strategy needs to restore the odontoblastic function of the dental pulp, and at the same time promote angiogenesis to maintain the survival and function of the regenerated tissue. However, the existing technologies face many challenges in achieving this goal, and the existing technologies specifically have the following defects: Defect 1: In terms of odontoblast differentiation, the existing technologies mostly rely on exogenous growth factors (such as BMP-2, TGF-β, etc.) to differentiate into odontoblasts. Although promoting cell differentiation by exogenous growth factors can promote the formation of mineralized nodules to a certain extent, the problems of high cost, poor stability and short in vivo half-life of exogenous growth factors will increase the cost and the risk of potential immune reactions, and it is difficult to achieve long-term and stable odontoblast differentiation in vivo; Defect 2: The two-dimensional culture mode adopted by the existing technologies cannot fully simulate the in vivo microenvironment, which limits the multi-directional differentiation potential of stem cells. Summary of the Invention

[0003] The purpose of the present invention is to provide a preparation method of lithium chloride-induced SHEDs three-dimensional microspheres, an injectable composite and its application in view of the deficiencies of the existing technologies. Specifically: The preparation method of the lithium chloride-induced SHEDs three-dimensional microspheres induces SHEDs to differentiate into odontoblasts by lithium chloride, and combines three-dimensional culture technology to construct SHEDs three-dimensional microspheres, and by simulating the in vivo microenvironment, it can significantly improve the differentiation efficiency and regeneration ability of odontoblasts; the preparation method of the lithium chloride-induced SHEDs three-dimensional microspheres avoids the use of exogenous growth factors, that is, it can reduce the treatment cost and potential risks caused by exogenous growth factors; The injectable composite is prepared by mixing the induced SHEDs three-dimensional microspheres 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 operation in the process of dental pulp regeneration treatment.

[0004] To achieve the above purpose, the present invention is realized through the following technical solutions.

[0005] A preparation method of lithium chloride-induced SHEDs three-dimensional microspheres includes the following steps. Specifically: Step a, Cell extraction and culture: Extract SHEDs from human exfoliated deciduous teeth for primary culture and subculture amplification. Select cells at passages P3-P6 for subsequent experiments. SHEDs are dental pulp stem cells from human exfoliated deciduous teeth; Step b, Screen the optimal LiCl concentration for inducing odontogenic differentiation of SHEDs: Step b1, Select SHEDs cells at passages P3-P6 with good growth status obtained in step a, and induce odontogenic differentiation of SHEDs by setting osteogenic induction media with different LiCl concentrations; Step b2, Compare and analyze the effects of osteogenic induction media with different LiCl concentrations on inducing odontogenic differentiation of SHEDs, to analyze the effects of LiCl on the proliferation and odontogenic differentiation of SHEDs, and screen out the optimal LiCl concentration; Step c, Construct SHEDs three-dimensional microspheres by agarose microporous three-dimensional culture method: Step c1, Prepare the culture model microporous plate: Pour the 3% agarose solution sterilized by high temperature and high pressure into the polydimethylsiloxane microcolumn positive mold. After the 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; 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, with one cylindrical agarose microporous plate 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 standby after ultraviolet disinfection; Step c2, Construct SHEDs microspheres: Add 800 μL of PM medium to each well of the 24-well plate with the agarose microporous plate laid, and then inoculate the SHEDs cultured in step a at a density of 5×10 5 cells / well into the agarose microporous plate. After standing for 10 minutes, place it in a 37°C constant temperature incubator for constant temperature culture for 24 hours; then change the medium of the agarose microporous plate to the osteogenic induction medium containing the optimal LiCl concentration screened in step b, and continue constant temperature culture in a 37°C constant temperature incubator, and change the osteogenic induction medium every 2 days. During this process, observe the formation of microspheres through an optical microscope, and obtain SHEDs three-dimensional microspheres after culturing for 14 days; Step d, Detect the activity of the obtained SHEDs three-dimensional microspheres by live / dead staining method and DAPI / Phalloidin staining method; Analyze and compare the odontogenic differentiation effects among two-dimensional cultured SHEDs, SHEDs microspheres induced by conventional osteogenic induction medium, and SHEDs microspheres induced by osteogenic induction medium containing LiCl through RT-PCR analysis method, Western Blot immunofluorescence analysis method, or alizarin red staining analysis method.

[0006] Among them, the optimal LiCl concentration obtained by screening in step b is 2.5 mmol / L.

[0007] Among them, in step a, the medium used for culturing SHEDs 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 SHEDs in step a is: constant temperature culture in a 37°C incubator.

[0008] Among them, the culture environment for LiCl-induced odontogenic differentiation of SHEDs in step b1 is: culture in a 37°C incubator, and the culture solution is changed every 2 days.

[0009] Among them, the comparative analysis method in step b2 is CCK-8 cell viability analysis method, ALP staining analysis method, alizarin red staining analysis method, semi-quantitative analysis method of calcified nodules, qRT-PCR analysis method, or Western Blot immunofluorescence analysis method.

[0010] An injection composite, which is composed of SHEDs three-dimensional microspheres obtained by the preparation method of the above-mentioned lithium chloride-induced SHEDs three-dimensional microspheres and a biocompatible hydrogel.

[0011] Among them, the step of mixing SHEDs three-dimensional microspheres and hydrogel includes: Prepare 10% GelMA hydrogel and keep it in reserve under the condition of 37°C water bath in the dark; Resuspend and mix SHEDs three-dimensional microspheres and 10% GelMA hydrogel directly.

[0012] An application of an injection composite in dental pulp regeneration.

[0013] Compared with the prior art, the present invention has the following beneficial effects: specifically: 1. The preparation method of the lithium chloride-induced SHEDs three-dimensional microspheres of the present invention constructs SHEDs three-dimensional microspheres through agarose microporous three-dimensional culture method, which can better simulate the in vivo microenvironment, significantly improve the multi-directional differentiation ability of cells, and enhance cell activity and regeneration effect; 2. The preparation method of the lithium chloride-induced SHEDs three-dimensional microspheres of the present invention innovatively uses LiCl to induce the odontoblastic differentiation of SHEDs microspheres. SHEDs are significantly superior to the traditional two-dimensional culture method in terms of the expression of odontoblast-related genes and proteins, have a stronger ability to form mineralized nodules, can better simulate the functions of odontoblasts in vivo, and can avoid the high cost and potential risks of exogenous growth factors. 3. The injectable composite of the present invention is prepared by mixing the induced SHEDs three-dimensional microspheres 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 the dental pulp regeneration treatment. Brief Description of the Drawings

[0014] The present invention will be further described below with the aid of the drawings, but the embodiments in the drawings do not constitute any limitation to the present invention.

[0015] Figure 1 It is a comparative diagram of the results of ALP staining analysis after culturing SHEDs with different concentrations of LiCl.

[0016] Figure 2 It is a comparative diagram of the results of alizarin red staining analysis after culturing SHEDs with different concentrations of LiCl for 14 days.

[0017] Figure 3 It is a comparative diagram of the semi-quantitative analysis results of calcified nodules of SHEDs induced with different concentrations of LiCl for 14 days.

[0018] Figure 4 It is a comparative diagram of the protein expression results after treating SHEDs with different concentrations of LiCl for 10 days.

[0019] Figure 5 It is a comparative diagram of the expression results of Runx-2 and DSPP after treating SHEDs with different concentrations of LiCl for 10 days.

[0020] Figure 6 It is a comparative diagram of the effects of different concentrations of LiCl on the relative expression levels of odontoblast-related genes of SHEDs.

[0021] Figure 7 It shows the situation of cells aggregating and polymerizing from 0 h, 0.5 h, to 5 h after cell seeding during the construction of SHEDs microspheres.

[0022] Figure 8 It shows the differentiation process of SHEDs three-dimensional microspheres after induction under different conditions during the construction of SHEDs microspheres.

[0023] Figure 9Live / dead staining of SHEDs three-dimensional microspheres at 5h, 24h, 7 days, and 14 days under different culture conditions during the activity detection of SHEDs three-dimensional microspheres.

[0024] Figure 10 Three-dimensional reconstruction confocal images of DAPI (nucleus) and Phalloidin (F-actin) staining over time during the activity detection of SHEDs three-dimensional microspheres.

[0025] Figure 11 Relative expression levels of dentinogenesis-related genes in SHEDs and SHEDs three-dimensional microspheres under different culture conditions.

[0026] Figure 12 H&E staining results of coronal sections of tooth segments. Detailed implementation methods

[0027] The present invention will be described below in conjunction with specific implementation methods.

[0028] Example 1, A method for preparing lithium chloride-induced SHEDs three-dimensional microspheres, characterized by comprising the following steps: specifically: Step a, Cell extraction and culture: Extract SHEDs from human exfoliated deciduous teeth for primary culture and subculture amplification, and select P3-P6 generation cells for subsequent experiments. SHEDs are human exfoliated deciduous tooth pulp stem cells; Step b, Screen the optimal LiCl concentration for inducing dentinogenesis of SHEDs: Step b1, Select the P3-P6 generation SHEDs cells with good growth state obtained in step a, and induce the dentinogenesis of SHEDs by setting osteogenic induction media with different LiCl concentrations. The culture environment is: culture in a 37°C constant temperature incubator, and change the culture medium every 2 days; Step b2, Conduct a comparative analysis on the effect of osteogenic induction media with different LiCl concentrations on inducing dentinogenesis of SHEDs to analyze the effects of LiCl on the proliferation and dentinogenesis of SHEDs, and screen out the optimal LiCl concentration; among them, the comparative analysis methods used are CCK-8 cell viability analysis method, ALP staining analysis method, alizarin red staining analysis method, semi-quantitative analysis method of calcified nodules, qRT-PCR analysis method or Western Blot immunofluorescence analysis method; Step c, Construct SHEDs three-dimensional microspheres by agarose microporous three-dimensional culture method: Step c1: Prepare the culture model microwell plate: Pour the 3% agarose solution sterilized by high-temperature and high-pressure into the polydimethylsiloxane microcolumn positive mold. After the agarose solution naturally cools and solidifies, 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 distance between adjacent microcolumns is 200 µm. 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, with one cylindrical agarose microwell plate 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 keep it for use after ultraviolet disinfection. Among them, every 100 mL of 3% agarose solution contains 3.0 g of agarose powder and 100 mL of ultrapure water. Step c2: Construct SHEDs microspheres: Add 800 μL of PM medium to each well of the 24-well plate with the agarose microwell plates laid. Then, inoculate the SHEDs cultured in step a into the agarose microwell plates at a density of 5×10 5 cells / well. After standing for 10 minutes, place it in a 37°C constant-temperature incubator for constant-temperature culture for 24 hours. Then, change the medium of the agarose microwell plates to the osteogenic induction medium containing the optimal LiCl concentration screened in step b, and continue the constant-temperature culture in a 37°C constant-temperature incubator, and change the osteogenic induction medium every 2 days. During this process, observe the formation of microspheres through an optical microscope, and SHEDs three-dimensional microspheres can be obtained after 14 days of culture. Step d: Detect the activity of the obtained SHEDs three-dimensional microspheres by the live / dead staining method and the DAPI / Phalloidin staining method. Analyze and compare the odontoblastic differentiation effects among two-dimensional cultured SHEDs, SHEDs microspheres induced by conventional osteogenic induction medium, and SHEDs microspheres induced by osteogenic induction medium containing LiCl by RT-PCR analysis method, Western Blot immunofluorescence analysis method or alizarin red staining analysis method.

[0029] It should be noted that the optimal LiCl concentration screened in step b is 2.5 mmol / L.

[0030] Among them, in step a, the medium used for culturing SHEDs 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 SHEDs in step a is: constant-temperature culture in a 37°C constant-temperature incubator.

[0031] When comparing the effects of osteogenic induction media with different LiCl concentrations on the odontogenic differentiation of SHEDs by ALP (alkaline phosphatase) staining analysis method, as Figure 1 shown, at 4 days, the LiCl groups with 1.0 mmol / L, 2.5 mmol / L, and 5.0 mmol / L were darker stained than the control group; at 7 days, the LiCl group with 2.5 mmol / L was the darkest stained, and there was no significant difference between the LiCl groups with 1.0 mmol / L and 5.0 mmol / L and the control group; at 10 days, the LiCl group with 2.5 mmol / L was slightly darker stained than the control group, there was no significant difference in the staining depth between the LiCl group with 1.0 mmol / L and the control group, and the LiCl groups with 5.0 mmol / L and 10 mmol / L were lighter stained than the control group. Through the above comparative analysis, it can be seen that low-concentration LiCl can increase the ALP activity in SHEDs, among which the LiCl group with 2.5 mmol / L has the best effect, and high-concentration LiCl inhibits the ALP activity in SHEDs.

[0032] When comparing the effects of osteogenic induction media with different LiCl concentrations on the odontogenic differentiation of SHEDs by alizarin red staining analysis method, as Figure 2 shown by the alizarin red staining results of SHEDs cultured with different concentrations of LiCl for 14 days, the red mineralized nodules in the LiCl groups with 1.0 mmol / L, 2.5 mmol / L, and 5.0 mmol / L were more and darker stained than those in the control group; the LiCl group with 10 mmol / L formed more mineralized nodules than the control group, but the nodules were smaller in size.

[0033] When comparing the effects of osteogenic induction media with different LiCl concentrations on the odontogenic differentiation of SHEDs by semi-quantitative analysis method of calcified nodules, as Figure 3 shown by the semi-quantitative results of calcified nodules of SHEDs induced with different concentrations of LiCl for 14 days, which were consistent with the above Figure 2 alizarin red staining results, the mineralization amounts in the LiCl groups with 1.0 mmol / L, 2.5 mmol / L, and 5.0 mmol / L were significantly higher than those in the control group, among which the LiCl group with 5.0 mmol / L had the highest mineralization amount, and the results had statistical differences (P < 0.05), and there was no statistical difference in the mineralization amount between the LiCl group with 10 mmol / L and the control group (P > 0.05).

[0034] When comparing the effects of osteogenic induction media with different LiCl concentrations on the odontogenic differentiation of SHEDs by Western Blot immunofluorescence analysis method, as Figure 4The protein expression results 10 days after the treatment of SHEDs with different concentrations of LiCl as shown, and as Figure 5 shown, the expression results of Runx-2 and DSPP 10 days after the treatment of SHEDs with different concentrations of LiCl. At the 10th day, the expression of Runx-2 in the LiCl groups of 1.0 mmol / L, 2.5 mmol / L, 5.0 mmol / L, and 10 mmol / L was higher than that in the control group, and the difference was statistically significant (P < 0.05). Compared with the control group and other concentration groups, the expression level of DSPP in the 2.5 mmol / L LiCl group was the highest, and the difference was statistically significant (P < 0.05). The expression of DSPP in the 10 mmol / L LiCl group was lower than that in the control group (P < 0.05). There was no statistically significant difference in the expression level of DSPP between the 1.0 mmol / L and 5.0 mmol / L LiCl groups and the control group (P > 0.05). Generally speaking, low-concentration LiCl can promote the expression of DSPP and Runx-2 in SHEDs and promote the differentiation of SHEDs into odontoblasts.

[0035] When comparing and analyzing the effect of osteogenic induction media with different LiCl concentrations on the odontogenic differentiation of SHEDs by qRT-PCR analysis method (real-time fluorescence quantitative PCR method), as Figure 6 shown, the effect of different concentrations of LiCl on the relative expression levels of odontogenic-related genes in SHEDs. At the 7th day, there was no statistically significant difference in the expression of COL-1 gene between the different concentration LiCl groups and the control group (P > 0.05). The expression of DSPP gene in the 1.0 mmol / L LiCl group was higher than that in the control group, and the difference was statistically significant (P < 0.05). There was no statistically significant difference in the expression of DSPP gene between the 2.5 mmol / L LiCl group and the control group. The expression levels of DSPP gene in the other concentration groups were lower than that in the control group, and the differences were statistically significant (P < 0.05). At the 10th day, the expression levels of COL-1 gene in the 2.5 mmol / L and 5.0 mmol / L LiCl groups were higher than that in the control group, and the differences were statistically significant (P < 0.05). The expression levels of COL-1 gene in the other concentration groups had no statistically significant difference from that in the control group. The expression level of DSPP gene in the 2.5 mmol / L LiCl group was higher than that in the control group, and the difference was statistically significant (P < 0.05). The expression levels of DSPP gene in the other concentration groups were all lower than that in the control group. Generally speaking, low-concentration LiCl can up-regulate the expression of odontogenic-related genes in SHEDs and promote the odontogenic differentiation of SHEDs.

[0036] During the construction of SHEDs three-dimensional microspheres, as Figure 7As shown, according to the situation of cell aggregation to polymerization within 0 h, 0.5 h, and 5 h after cell seeding, it can be seen that free-diffusing SHEDs slowly and evenly aggregate into the micropores and automatically polymerize into a three-dimensional sphere. In addition, as Figure 8 shown, after induction under different conditions, the differentiation process of SHEDs three-dimensional microspheres at 1 day, 3 days, 7 days, and 14 days after induction can be seen. It can be seen that with the change of culture time, the volume of SHEDs three-dimensional microspheres gradually decreases, and they tend to be in a regular spherical shape. The connection between cells becomes tighter, and the unevenness on the surface of the spheres becomes smoother; when cultured for 14 days, the size of the SHEDs three-dimensional microspheres in the control group is much smaller than that in the OM group (osteogenic induction medium group) and the LiCl + OM group, while the gross dimensions of the SHEDs microspheres in the OM group and the LiCl + OM group are comparable.

[0037] During the process of detecting the viability of the obtained SHEDs three-dimensional microspheres by the live / dead staining method, the survival status of SHEDs three-dimensional microspheres at 5 h, 24 h, 7 days, and 14 days of culture was detected by live / dead staining. The results of three-dimensional imaging after laser confocal microscopy are as Figure 9 shown; at all time points, a large number of green fluorescent-labeled live cells can be seen on the surface of the SHEDs three-dimensional microspheres, and no dead cells or low-viability cells labeled with red fluorescence are seen. With the extension of time, the connection between SHEDs cells becomes tighter, the unevenness on the surface of the microspheres becomes smoother, and the SHEDs cell population forms a three-dimensional sphere. In addition, over time, the sizes of the SHEDs three-dimensional microspheres in all three groups gradually decrease, and the SHEDs microspheres in the control group are significantly smaller than the other two groups at 14 days. The 3D reconstruction results show that the cells at the center of the SHEDs three-dimensional microspheres in all three groups are labeled with green fluorescence, and no dead cells or low-viability cells labeled with red fluorescence are seen, indicating that the nutrient supply at the center of the microspheres is sufficient and no obvious necrosis occurs during the 14-day culture period of the SHEDs microspheres in all three groups.

[0038] During the process of detecting the viability of the obtained SHEDs three-dimensional microspheres by the DAPI / Phalloidin staining method, as Figure 10The three-dimensional reconstructed confocal images of time-varying DAPI (nucleus) and Phalloidin (F-actin) staining are shown. The red fluorescence is F-actin (cytoskeleton), and the blue fluorescence is the cell nucleus. Each group of cells can adhere to each other and grow well. Over time, the volumes of the three groups of SHEDs microspheres gradually decrease; at 5 h, the three-dimensional microsphere morphologies of the three groups of SHEDs are different; at 24 h, the cells aggregate into spheroids, and F-actin basically covers the entire microsphere, increasing compared with that at 5 h; at 7 days, the F-actin on the surface of the three-dimensional SHEDs microspheres decreases, and the cytoskeleton of the LiCl+OM group is the sparest. The microsphere volume of the control group is smaller than that of the OM (osteogenic induction medium) and LiCl+OM groups; at 14 days, the microsphere volume of the control group is smaller than that of the OM and LiCl+OM groups, but the volume difference among the three is reduced compared with before.

[0039] In the process of analyzing and comparing the odontogenic differentiation effects among two-dimensional cultured SHEDs, SHEDs microspheres induced by conventional osteogenic induction medium, and SHEDs microspheres induced by osteogenic induction medium containing LiCl by RT-PCR analysis method, as Figure 11 shown are the relative expression levels of odontogenic-related genes of SHEDs and SHEDs three-dimensional microspheres under different culture conditions; at 7 days, the expression level of Runx-2 in the OM 2D group is higher than that in the OM 3D group, and the difference is statistically significant (P < 0.05), and there is no statistical difference between the OM 2D group and the LiCl+OM 3D group (P > 0.05); the expression of the DSPP gene in the OM 3D group is higher than that in the OM 2D and LiCl+OM 3D groups, and the difference is statistically significant (P < 0.05); at 10 days and 14 days, the expression levels of the Runx-2 gene in the OM 3D group and the LiCl+OM 3D group are both higher than that in the OM 2D group, and the differences are statistically significant (P < 0.05). At 10 days, the expression level of the Runx-2 gene in the OM 3D group is higher than that in the LiCl+OM 3D group, and the difference is statistically significant (P < 0.05). At 14 days, there is no statistical difference in the expression level of the Runx-2 gene between the OM 3D group and the LiCl+OM 3D group (P > 0.05); at 10 days and 14 days, the expression levels of the DSPP gene are in the order of: LiCl+OM 3D group > OM 3D group > OM 2D group, and the differences are statistically significant (P < 0.05). Therefore, generally speaking, three-dimensional culture is more conducive to the odontogenic differentiation of SHEDs than two-dimensional culture, and adding lithium chloride can promote the expression of odontogenic-related genes in SHEDs.

[0040] Based on the above situation, for the preparation method of lithium chloride-induced SHEDs three-dimensional microspheres in Example 1 of the present invention, it has the following technical effects, specifically: 1. The three-dimensional microspheres of SHEDs were constructed by the agarose microporous three-dimensional culture method, which could better simulate the in-vivo microenvironment, significantly enhance the multi-directional differentiation ability of cells, and enhance cell activity and regeneration effect. 2. LiCl was innovatively used to induce the differentiation of SHEDs microspheres into odontoblasts. SHEDs were significantly superior to the traditional two-dimensional culture method in terms of the expression of odontoblast-related genes (such as DSPP and Runx-2) and proteins, had stronger mineralized nodule formation ability, could better simulate the function of in-vivo odontoblasts, and could avoid the high cost and potential risks of exogenous growth factors.

[0041] Example 2: An injection composite, which is formed by mixing the three-dimensional microspheres of SHEDs obtained by the preparation method of the above-mentioned lithium chloride-induced three-dimensional microspheres of SHEDs with a biocompatible hydrogel.

[0042] Specifically, the mixing step of the three-dimensional microspheres of SHEDs and the hydrogel includes: 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 three-dimensional microspheres of SHEDs with 10% GelMA hydrogel.

[0043] The injection composite of this Example 2 is applied to dental pulp regeneration, and this injection composite can be applied to dental pulp regeneration treatment; this injection composite can effectively improve the convenience and adaptability of clinical operations during dental pulp regeneration treatment.

[0044] Next, the regeneration effect of this injection composite was evaluated by constructing a subcutaneous semi-in-situ dental pulp regeneration model in nude mice. 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 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: Directly resuspend and mix the induced three-dimensional microspheres of SHEDs with 10% GelMA hydrogel. The cell density and ratio of each group are as follows: ① The 2D group is 7.5×106 OM-induced SHEDs per milliliter; ② The OM 3D group is 3×104 OM-induced SHEDs microspheres per milliliter; ③ The LiCl + OM 3D group is 3×104 SHEDs microspheres induced by 2.5 mmol / L LiCl + OM per milliliter. Step 5: Take 20 μL of 10% GelMA-loaded cells / microspheres and inject them into the root canals of the tooth segments, and perform photocrosslinking by exposing them to 9.16-W / cm2 ultraviolet light for 20 s to prepare the injection composite. Step 6: Implant the tooth segment complex under the subcutaneous tissue of the back of nude mice. After 8 weeks, take samples for histological evaluation, and observe the coronal sections of the tooth segments by H&E staining method. Specifically: as Figure 12 shown in the H&E staining image, dense connective tissue was generated in the root canals of the blank group, and no odontoblast-like cells and newly formed dentin were observed; in the control group, GelMA hydrogel that peeled off due to non-degradation was visible, and no connective tissue, newly formed odontoblast-like cells, and dentin were observed; in the 2D group, a single layer of atrophic odontoblast-like cells was visible between the pulp cavity and dentin, and the short and thin odontoblast processes extended into the original odontoblast tubules. Capillary formation was visible in the loose connective tissue; in the OM 3D group, odontoblast-like cells were generated, the odontoblast processes were longer than those in the 2D group, there was intercellular edema, the odontoblasts were squeezed and atrophied, and rich congested small blood vessels were formed on the pulp cavity side close to the odontoblasts; in the LiCl + OM 3D group, tightly arranged odontoblast-like cells were visible, the cytoplasm was rich and red-stained, the nucleus was near the basal side, the odontoblast processes were long and thin and extended into the dentin tubules, and dense connective tissue was formed on the pulp cavity side close to the odontoblasts, and congested small blood vessels were visible.

[0045] It should be noted that in the semi-in situ dental pulp regeneration model under the skin of nude mice, the SHEDs microspheres in the LiCl + OM 3D group could form a regenerated tissue with rich blood vessels and odontoblasts, and the in vivo regeneration effect was significant and significantly better than other groups, which proved its high efficiency and safety in in vivo application.

[0046] The above content is only the preferred embodiment of the present invention. For those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation manner and application scope. The content of this specification should not be construed as a limitation to the present invention.

Claims

1. A preparation method of lithium chloride-induced SHEDs three-dimensional microspheres, characterized in that, It includes the following steps, specifically: Step a, cell extraction and culture: Extract SHEDs from human exfoliated deciduous teeth for primary culture and subculture amplification. Select cells at passages P3 - P6 for subsequent experiments. SHEDs are dental pulp stem cells from human exfoliated deciduous teeth; Step b, screening the optimal LiCl concentration for inducing odontogenic differentiation of SHEDs: Step b1, Select SHEDs cells at passages P3 - P6 with good growth status obtained in step a, and induce odontogenic differentiation of SHEDs by setting osteogenic induction media with different LiCl concentrations; Step b2, Conduct a comparative analysis on the effects of osteogenic induction media with different LiCl concentrations on inducing odontogenic differentiation of SHEDs to analyze the effects of LiCl on the proliferation and odontogenic differentiation of SHEDs, and screen out the optimal LiCl concentration; Step c, construct three - dimensional microspheres of SHEDs using the agarose microporous three - dimensional culture method: Step c1, Prepare a culture model microporous plate: Pour a 3% agarose solution sterilized by high - temperature and high - pressure into a polydimethylsiloxane microcolumn positive mold. After the 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. 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 each well of a 24 - well plate, with one cylindrical agarose microporous plate 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 set aside after ultraviolet disinfection; Step c2. Construct SHEDs microspheres: Add 800 μL of PM medium to each well of the 24-well plate with the agarose microporous plate laid out, and then inoculate the SHEDs cultured in step a into the agarose microporous plate at a density of 5×10 5 cells / well. After standing for 10 minutes, place it in a 37°C constant temperature incubator for constant temperature culture for 24 hours. Then, replace the medium in the agarose microporous plate with the osteogenic induction medium containing the optimal LiCl concentration screened in step b, and continue the constant temperature culture in a 37°C constant temperature incubator, and replace the osteogenic induction medium every 2 days. During this process, observe the formation of microspheres through an optical microscope, and SHEDs three-dimensional microspheres can be obtained after 14 days of culture. Step d, Detect the activity of the obtained three - dimensional microspheres of SHEDs by the live - dead staining method and the DAPI / Phalloidin staining method; Analyze and compare the odontogenic differentiation effects among two - dimensional cultured SHEDs, SHEDs microspheres induced by conventional osteogenic induction media, and SHEDs microspheres induced by osteogenic induction media containing LiCl through RT - PCR analysis method, Western Blot immunofluorescence analysis method or alizarin red staining analysis method.

2. The preparation method of a lithium chloride-induced three-dimensional microsphere of SHEDs according to claim 1, characterized in that: The optimal LiCl concentration screened in step b is 2.5 mmol / L.

3. The preparation method of a lithium chloride-induced three-dimensional microsphere of SHEDs according to claim 1, wherein: In step a, the medium used for culturing SHEDs 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 for SHEDs in step a is: constant - temperature culture in a 37 °C incubator.

4. The preparation method of a lithium chloride-induced three-dimensional microsphere of SHEDs according to claim 1, characterized in that: The culture environment for LiCl - induced odontogenic differentiation of SHEDs in step b1 is: culture in a 37 °C incubator, and the culture medium is changed every 2 days.

5. The preparation method of a lithium chloride-induced three-dimensional microsphere of SHEDs according to claim 1, characterized in that: The comparative analysis method in step b2 is the CCK-8 cell viability analysis method, ALP staining analysis method, alizarin red staining analysis method, semi-quantitative analysis method of calcified nodules, qRT-PCR analysis method or Western Blot immunofluorescence analysis method.

6. An injection complex, characterized in that: The injection composite is composed of the SHEDs three-dimensional microspheres obtained by the preparation method of lithium chloride-induced SHEDs three-dimensional microspheres according to any one of claims 1-5 and a biocompatible hydrogel.

7. An injection complex according to claim 6, wherein: The step of mixing the SHEDs three-dimensional microspheres and the hydrogel includes: 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 SHEDs three-dimensional microspheres with 10% GelMA hydrogel.

8. Use of an injection composite according to claim 6 or 7 for dental pulp regeneration.

Images