A preparation method of lithium chloride-induced SHEDs three-dimensional microspheres, injectable composite and application thereof

Abstract

The application discloses a preparation method of lithium chloride-induced SHEDs three-dimensional microspheres, an injection compound and application of the lithium chloride-induced SHEDs three-dimensional microspheres. The preparation method comprises the following steps: a, cell extraction and culture; b, screening of optimal LiCl concentration for inducing SHEDs dentin differentiation; c, construction of SHEDs three-dimensional microspheres by adopting an agarose microporous three-dimensional culture method; and d, detection of the activity of the SHEDs three-dimensional microspheres. The preparation method avoids the use of exogenous growth factors, induces SHEDs to differentiate into dentinogenic cells by lithium chloride, and combines a three-dimensional culture technology to construct SHEDs three-dimensional microspheres, so that the differentiation efficiency and regeneration capacity of the dentinogenic cells are significantly improved by simulating a microenvironment in vivo. The injection compound is prepared by mixing the SHEDs three-dimensional microspheres and a biocompatible hydrogel, and the injection compound can be applied to pulp regeneration treatment.

Description

Technical Field

[0001] This invention relates to the field of dental pulp regeneration technology, and in particular to a method for preparing lithium chloride-induced SHEDs three-dimensional microspheres, an injectable complex, and its applications. Background Technology

[0002] Regeneration of the dental pulp-dentin complex is a crucial goal in the treatment of endodontic diseases. An ideal pulp regeneration strategy requires restoring the odontoblastic function of the pulp while simultaneously promoting angiogenesis to maintain the survival and function of the regenerated tissue. However, current technologies face numerous challenges in achieving this goal, and specifically suffer from the following shortcomings:

[0003] Defect 1: In terms of odontoblast differentiation, existing technologies mostly rely on exogenous growth factors (such as BMP-2, TGF-β, etc.) to differentiate into odontoblasts. Although promoting cell differentiation through exogenous growth factors can promote the formation of mineralized nodules to a certain extent, the high cost, poor stability, and short in vivo half-life of exogenous growth factors will increase costs and potential immune response risks, and it is difficult to achieve long-term stable odontoblast differentiation in vivo.

[0004] Defect 2: The two-dimensional culture mode used in the existing technology cannot fully simulate the in vivo microenvironment, which limits the multi-directional differentiation potential of stem cells. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing lithium chloride-induced SHEDs three-dimensional microspheres, an injectable complex, and its applications. Specifically:

[0006] The method for preparing lithium chloride-induced SHEDs three-dimensional microspheres involves inducing SHEDs to differentiate into odontoblasts with lithium chloride and constructing SHEDs three-dimensional microspheres using three-dimensional culture technology. By simulating the in vivo microenvironment, the method significantly improves the differentiation efficiency and regeneration capacity of odontoblasts. This method avoids the use of exogenous growth factors, thus reducing the treatment costs and potential risks associated with exogenous growth factors.

[0007] This injectable compound is prepared by mixing induced SHEDs three-dimensional microspheres 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.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions.

[0009] A method for preparing lithium chloride-induced SHEDs three-dimensional microspheres includes the following steps:

[0010] Step a, Cell extraction and culture: SHEDs were extracted from human deciduous teeth for primary culture and passage expansion. P3-P6 generation cells were selected for subsequent experiments. SHEDs are human deciduous tooth pulp stem cells.

[0011] Step b: Screening for the optimal LiCl concentration to induce odontoblastic differentiation of SHEDs:

[0012] Step b1: Select SHEDs cells with good growth status obtained in step a from the P3-P6 generation, and induce odontogenic differentiation of SHEDs by setting osteogenic induction medium with different LiCl concentrations.

[0013] Step b2: Compare and analyze the effects of osteogenic induction medium with different LiCl concentrations on the odontogenic differentiation of SHEDs, in order to analyze the influence of LiCl on the proliferation and odontogenicity of SHEDs and screen out the optimal LiCl concentration;

[0014] Step c: Construct SHEDs three-dimensional microspheres using the agarose microporous three-dimensional culture method:

[0015] Step c1: Preparation of culture model microplates: A 3% agarose solution, sterilized by high temperature and pressure, is poured into a polydimethylsiloxane microcolumn positive mold. After the 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, with a spacing of 200 μm between adjacent microcolumns. 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, one microplate per well. Finally, 500 μL of PBS buffer is added to each agarose microplate in the 24-well plate, and the plates are sterilized under ultraviolet light before use.

[0016] Step c2, Construction of SHEDs microspheres: Add 800 μL of PM medium to each well of a 24-well plate with agarose microplates, and then add the SHEDs cultured in step a at a rate of 5 × 10⁻⁶. 5 The microspheres were inoculated at a density of 1 microsphere per well in agarose microplates, allowed to stand for 10 minutes, and then incubated at 37°C for 24 hours. The culture medium in the agarose microplates was then replaced with osteogenic induction medium containing the optimal LiCl concentration obtained in step b, and the microspheres were incubated at 37°C. The osteogenic induction medium was replaced every 2 days. During this process, the formation of microspheres was observed using an optical microscope. SHEDs three-dimensional microspheres were obtained after 14 days of culture.

[0017] Step d: The activity of the obtained SHEDs three-dimensional microspheres was detected by live-dead staining method and DAPI / Phalloidin staining method;

[0018] The odontogenic differentiation effects of SHEDs cultured in two dimensions, SHEDs microspheres induced by conventional osteogenic induction medium, and SHEDs microspheres induced by LiCl-containing osteogenic induction medium were analyzed and compared using RT-PCR, Western blotting immunofluorescence, or alizarin red staining.

[0019] The optimal LiCl concentration obtained in step b is 2.5 mmol / L.

[0020] In step a, the culture medium used for SHEDs culture contains 44.5 mL of α-MEM medium, 5 mL of fetal bovine serum (FBS), and 0.5 mL of penicillin / streptomycin solution.

[0021] Furthermore, the SHEDs culture environment in step a is: constant temperature culture in a 37℃ constant temperature incubator.

[0022] In step b1, the culture environment for LiCl-induced dentin differentiation of SHEDs is: cultured in a constant temperature incubator at 37℃, with the culture medium changed every 2 days.

[0023] The comparative analysis method in step b2 is CCK-8 cell viability analysis, ALP staining analysis, alizarin red staining analysis, semi-quantitative analysis of calcified nodules, qRT-PCR analysis, or Western Blot immunofluorescence analysis.

[0024] An injectable complex is formed by mixing SHEDs three-dimensional microspheres prepared by the above-described method for preparing lithium chloride-induced SHEDs three-dimensional microspheres with a biocompatible hydrogel.

[0025] The mixing steps of SHEDs three-dimensional microspheres and hydrogel include:

[0026] Prepare a 10% GelMA hydrogel and store it in a 37°C water bath in the dark.

[0027] SHEDs three-dimensional microspheres were directly resuspended and mixed with 10% GelMA hydrogel.

[0028] Application of an injection compound in dental pulp regeneration.

[0029] Compared with the prior art, the present invention has the following beneficial effects, specifically:

[0030] 1. The method for preparing lithium chloride-induced SHEDs three-dimensional microspheres of the present invention constructs SHEDs three-dimensional microspheres through agarose micropore three-dimensional culture, which can better simulate the in vivo microenvironment, significantly enhance the multi-directional differentiation ability of cells, and enhance cell activity and regeneration effect;

[0031] 2. The method for preparing lithium chloride-induced SHEDs three-dimensional microspheres of the present invention innovatively uses LiCl to induce odontoblast differentiation of SHEDs microspheres. SHEDs are significantly superior to traditional two-dimensional culture methods in terms of odontoblast-related gene and protein expression, have stronger mineralized nodule formation ability, can better simulate the function of odontoblasts in vivo, and can avoid the high cost and potential risks of exogenous growth factors.

[0032] 3. The injectable complex of the present invention is prepared by mixing induced SHEDs three-dimensional microspheres with biocompatible hydrogel. The injectable complex is applied to pulp regeneration treatment, and the injectable complex can effectively improve the convenience and adaptability of clinical operation during pulp regeneration treatment. Attached Figure Description

[0033] 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.

[0034] Figure 1 Comparison of ALP staining analysis results after culturing SHEDs with different concentrations of LiCl.

[0035] Figure 2 Comparison of Alizarin Red staining analysis results after 14 days of culturing SHEDs with different concentrations of LiCl.

[0036] Figure 3 This is a comparison of the semi-quantitative analysis results of calcified nodules in SHEDs induced by different concentrations of LiCl for 14 days.

[0037] Figure 4 This is a comparison of protein expression results after 10 days of treatment with different concentrations of LiCl on SHEDs.

[0038] Figure 5 Comparison of Runx-2 and DSPP expression results after 10 days of treatment with different concentrations of LiCl on SHEDs.

[0039] Figure 6 This is a comparison of the effects of different concentrations of LiCl on the relative expression levels of odontoblast-related genes in SHEDs.

[0040] Figure 7 This shows the process of cell aggregation to polymerization within 0h, 0.5h, and 5h after cell seeding during the construction of SHEDs microspheres.

[0041] Figure 8 This study describes the differentiation process of SHEDs three-dimensional microspheres induced by different conditions during the construction of SHEDs microspheres.

[0042] Figure 9 The activity and staining status of SHEDs three-dimensional microspheres under different culture conditions at 5h, 24h, 7day, and 14day were shown during the activity assay of SHEDs three-dimensional microspheres.

[0043] Figure 10 Three-dimensional reconstructed confocal images of DAPI (nucleus) and Phalloidin (F-actin) staining over time during SHEDs three-dimensional microsphere activity assay.

[0044] Figure 11 The relative expression levels of odontoblast-related genes in SHEDs and SHEDs 3D microspheres under different culture conditions.

[0045] Figure 12 The results of H&E staining of coronal sections of the tooth segment. Detailed Implementation

[0046] The present invention will now be described in conjunction with specific embodiments.

[0047] Example 1: A method for preparing lithium chloride-induced SHEDs three-dimensional microspheres, characterized by comprising the following steps:

[0048] Step a, Cell extraction and culture: SHEDs were extracted from human deciduous teeth for primary culture and passage expansion. P3-P6 generation cells were selected for subsequent experiments. SHEDs are human deciduous tooth pulp stem cells.

[0049] Step b: Screening for the optimal LiCl concentration to induce odontoblastic differentiation of SHEDs:

[0050] Step b1: Select SHEDs cells with good growth status from the P3-P6 generation obtained in step a, and induce odontoblast differentiation of SHEDs by setting osteogenic induction medium with different LiCl concentrations. The culture environment is: cultured in a constant temperature incubator at 37℃, and the culture medium is changed every 2 days.

[0051] Step b2: Comparative analysis was conducted on the effects of osteogenic induction medium with different LiCl concentrations on the odontogenic differentiation of SHEDs, in order to analyze the influence of LiCl on the proliferation and odontogenesis of SHEDs and to screen out the optimal LiCl concentration. The comparative analysis methods used were CCK-8 cell viability analysis, ALP staining analysis, Alizarin Red staining analysis, semi-quantitative analysis of calcified nodules, qRT-PCR analysis, or Western Blot immunofluorescence analysis.

[0052] Step c: Construct SHEDs three-dimensional microspheres using the agarose microporous three-dimensional culture method:

[0053] Step c1: Preparation of culture model microplates: A 3% agarose solution, sterilized by high temperature and autoclave, is poured into a polydimethylsiloxane micropillar positive mold. After the 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. 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, one microplate per well. Next, 500 μL of PBS buffer is added to each agarose microplate in the 24-well plate, and the plates are sterilized under ultraviolet light before use. Each 100 mL of the 3% agarose solution contains 3.0 g of agarose powder. mL of ultrapure water;

[0054] Step c2, Construction of SHEDs microspheres: Add 800 μL of PM medium to each well of a 24-well plate with agarose microplates, and then add the SHEDs cultured in step a at a rate of 5 × 10⁻⁶. 5 The microspheres were inoculated at a density of 1 microsphere per well in agarose microplates, allowed to stand for 10 minutes, and then incubated at 37°C for 24 hours. The culture medium in the agarose microplates was then replaced with osteogenic induction medium containing the optimal LiCl concentration obtained in step b, and the microspheres were incubated at 37°C. The osteogenic induction medium was replaced every 2 days. During this process, the formation of microspheres was observed using an optical microscope. SHEDs three-dimensional microspheres were obtained after 14 days of culture.

[0055] Step d: The activity of the obtained SHEDs three-dimensional microspheres was detected by live-dead staining method and DAPI / Phalloidin staining method;

[0056] The odontogenic differentiation effects of SHEDs cultured in two dimensions, SHEDs microspheres induced by conventional osteogenic induction medium, and SHEDs microspheres induced by LiCl-containing osteogenic induction medium were analyzed and compared using RT-PCR, Western blotting immunofluorescence, or alizarin red staining.

[0057] It should be noted that the optimal LiCl concentration obtained in step b is 2.5 mmol / L.

[0058] In step a, the culture medium used for SHEDs 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 SHEDs culture environment in step a is constant temperature culture in a 37℃ incubator.

[0059] When comparing the effects of different LiCl concentrations in osteogenic induction media on the odontogenic differentiation of SHEDs, the ALP (alkaline phosphatase) staining method was used for analysis. Figure 1 As shown, at day 4, the 1.0 mmol / L, 2.5 mmol / L, and 5.0 mmol / L LiCl groups stained more deeply than the control group; at day 7, the 2.5 mmol / L LiCl group stained the deepest, while the 1.0 mmol / L and 5.0 mmol / L LiCl groups showed no significant difference from the control group; at day 10, the 2.5 mmol / L LiCl group stained slightly more deeply than the control group, the 1.0 mmol / L LiCl group showed no significant difference in staining depth compared to the control group, and the 5.0 mmol / L and 10 mmol / L LiCl groups stained lighter than the control group. The above comparative analysis indicates that low concentrations of LiCl can increase ALP activity in SHEDs, with the 2.5 mmol / L LiCl group showing the best effect, while high concentrations of LiCl inhibit ALP activity in SHEDs.

[0060] When comparing the effects of different LiCl concentrations of osteogenic induction media on the odontogenic differentiation of SHEDs, such as... Figure 2 The results of alizarin red staining of SHEDs after 14 days of culture with different concentrations of LiCl are shown. The LiCl groups with 1.0 mmol / L, 2.5 mmol / L, and 5.0 mmol / L had more red mineralized nodules and were stained more deeply than the control group. The LiCl group with 10 mmol / L formed more mineralized nodules than the control group, but the nodules were smaller.

[0061] When comparing the effects of osteogenic induction media with different LiCl concentrations on the odontogenic differentiation of SHEDs using a semi-quantitative analysis method of calcified nodules, such as... Figure 3 The semi-quantitative results of calcified nodules in SHEDs induced by different concentrations of LiCl for 14 days shown above are consistent with the above. Figure 2 The results of alizarin red staining were consistent. The mineralization levels of the 1.0 mmol / L, 2.5 mmol / L, and 5.0 mmol / L LiCl groups were significantly higher than those of the control group, with the 5.0 mmol / L LiCl group showing the highest mineralization level. The results were statistically significant (P < 0.05). There was no statistically significant difference in mineralization levels between the 10 mmol / L LiCl group and the control group (P > 0.05).

[0062] When comparing the effects of different LiCl concentrations in osteogenic induction media on the odontogenic differentiation of SHEDs, such as... Figure 4 The results of protein expression in SHEDs treated with different concentrations of LiCl for 10 days are shown, as well as... Figure 5 The results of Runx-2 and DSPP expression in SHEDs treated with different concentrations of LiCl 10 days prior are shown. On day 10, Runx-2 expression in the 1.0 mmol / L, 2.5 mmol / L, 5.0 mmol / L, and 10 mmol / L LiCl groups was significantly higher than that in the control group (P < 0.05). Compared with the control group and other concentration groups, the 2.5 mmol / L LiCl group showed the highest DSPP expression (P < 0.05), while the 10 mmol / L LiCl group showed lower DSPP expression than the control group (P < 0.05). There was no significant difference in DSPP expression between the 1.0 mmol / L and 5.0 mmol / L LiCl groups and the control group (P > 0.05). In summary, low concentrations of LiCl can promote the expression of DSPP and Runx-2 in SHEDs and promote their differentiation into odontoblasts.

[0063] When comparing the effects of different LiCl concentrations in osteogenic induction media on the odontogenic differentiation of SHEDs, the following analysis was conducted using qRT-PCR (real-time quantitative PCR). Figure 6The effects of different concentrations of LiCl on the relative expression levels of odontoblast-related genes in SHEDs were shown. On day 7, there was no statistically significant difference in COL-1 gene expression between the different LiCl concentration groups and the control group (P > 0.05). The DSPP gene expression in the 1.0 mmol / L LiCl group was significantly higher than that in the control group (P < 0.05). There was no statistically significant difference in DSPP gene expression between the 2.5 mmol / L LiCl group and the control group. The DSPP gene expression levels in the other concentration groups were significantly lower than those in the control group (P < 0.05). On day 10, the COL-1 gene expression levels in the 2.5 mmol / L and 5.0 mmol / L LiCl groups were significantly higher than those in the control group (P < 0.05). There was no statistically significant difference in COL-1 gene expression levels between the other concentration groups and the control group. The DSPP gene expression level in the 2.5 mmol / L LiCl group was significantly higher than that in the control group (P < 0.05). The DSPP gene expression levels in the other concentration groups were significantly lower than those in the control group. Overall, low concentrations of LiCl upregulated the expression of odontoblast-related genes in SHEDs and promoted odontoblast differentiation in SHEDs.

[0064] In the process of constructing SHEDs three-dimensional microspheres, such as Figure 7 As shown, based on the aggregation and polymerization of cells within 0h, 0.5h, and 5h after cell seeding, it can be observed that freely diffused SHEDs slowly and uniformly aggregate into the micropores and spontaneously polymerize into a three-dimensional sphere. Additionally, as... Figure 8 As 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 shows that the volume of SHEDs three-dimensional microspheres gradually decreases with culture time and tends to be a regular spherical shape. The connections between cells are tighter, and the surface of the spheres becomes smoother. At 14 days of culture, the size of 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 size of SHEDs microspheres in the OM group and the LiCl+OM group is similar.

[0065] In the process of detecting the activity of the obtained SHEDs three-dimensional microspheres using the live-dead staining method, the survival status of the SHEDs three-dimensional microspheres at 5h, 24h, 7day, and 14d of culture was detected using live-dead staining. The results of three-dimensional imaging after laser confocal imaging are as follows: Figure 9As shown, at all time points, a large number of live cells were observed with green fluorescent markers on the surface of the SHEDs three-dimensional microspheres, with no dead or low-activity cells marked with red fluorescence. With prolonged time, the SHEDs cells became more tightly connected, the surface of the microspheres became smoother, and the SHEDs cell population formed a three-dimensional sphere. Furthermore, the size of the SHEDs three-dimensional microspheres in all three groups gradually decreased over time, with the control group's SHEDs microspheres being significantly smaller than the other two groups at 14 days. 3D reconstruction results showed that the cells at the center of the SHEDs three-dimensional microspheres in all three groups were marked with green fluorescence, and no dead or low-activity cells marked with red fluorescence were observed, indicating that the nutrient supply to the center of the microspheres was adequate during the 14-day culture period, and no significant necrosis occurred.

[0066] In the process of detecting the activity of the obtained SHEDs three-dimensional microspheres by the DAPI / Phalloidin staining method, such as Figure 10 The images show three-dimensional reconstructed confocal images of DAPI (nucleus) and Phalloidin (F-actin) staining over time. Red fluorescence represents F-actin (cytoskeleton), and blue fluorescence represents the nucleus. Cells in each group adhered well to each other and grew. Over time, the volume of SHEDs microspheres in the three groups gradually decreased. At 5 h, the morphology of the three-dimensional SHEDs microspheres varied among the three groups. At 24 h, cells aggregated into spheroids, and F-actin basically covered the entire microsphere, increasing compared to 5 h. At 7 days, F-actin on the surface of the SHEDs microspheres decreased, and the cytoskeleton in the LiCl+OM group was the sparsest. The microsphere volume in the control group was smaller than that in the OM (osteogenic induction medium) and LiCl+OM groups. At 14 days, the microsphere volume in the control group was smaller than that in the OM and LiCl+OM groups, but the volume difference among the three groups decreased compared to before.

[0067] In the process of analyzing and comparing the odontogenic differentiation effects among SHEDs cultured in two dimensions, SHED microspheres induced by conventional osteogenic induction medium, and SHED microspheres induced by LiCl-containing osteogenic induction medium using RT-PCR analysis, such as... Figure 11The relative expression levels of odontoblast-related genes in SHEDs and SHEDs 3D microspheres under different culture conditions are shown. At 7 days, the expression level of Runx-2 in the OM 2D group was higher than that in the OM 3D group (P < 0.05), but there was no statistically significant difference between the OM 2D group and the LiCl+OM 3D group (P > 0.05). The expression of DSPP gene in the OM 3D group was higher than that in the OM 2D and LiCl+OM 3D groups (P < 0.05). At 10 days and 14 days, the expression of Runx-2 gene in both the OM 3D group and the LiCl+OM 3D group was higher than that in the OM 2D group (P < 0.05). At 10 days, the expression of Runx-2 gene in the OM 3D group was higher than that in the LiCl+OM 3D group (P < 0.05). At 14 days, there was no statistically significant difference in Runx-2 gene expression between the OM 3D group and the LiCl+OM 3D group (P > 0.05). At day 1 and day 14, the expression levels of DSPP gene were as follows: LiCl+OM 3D group > OM 3D group > OM 2D group, with statistically significant differences (P < 0.05). Therefore, overall, three-dimensional culture is more conducive to the differentiation of SHEDs into odontoblasts than two-dimensional culture, and the addition of lithium chloride can promote the expression of odontoblast-related genes in SHEDs.

[0068] In summary, the preparation method of lithium chloride-induced SHEDs three-dimensional microspheres in Example 1 has the following technical effects, specifically:

[0069] 1. Constructing SHEDs three-dimensional microspheres using the agarose micropore three-dimensional culture method can better simulate the in vivo microenvironment, significantly enhance the multi-directional differentiation ability of cells, and improve cell activity and regeneration.

[0070] 2. Innovatively, LiCl is used to induce odontoblast differentiation in SHEDs microspheres. SHEDs are significantly superior to traditional two-dimensional culture methods in terms of odontoblast-related gene (such as DSPP and Runx-2) and protein expression, and have a stronger ability to form mineralized nodules. They can better simulate the function of odontoblasts in vivo and avoid the high cost and potential risks of exogenous growth factors.

[0071] Example 2: An injectable complex, which is a mixture of SHEDs three-dimensional microspheres prepared by the above-described method for preparing lithium chloride-induced SHEDs three-dimensional microspheres and a biocompatible hydrogel.

[0072] Specifically, the mixing steps of SHEDs three-dimensional microspheres and hydrogel include:

[0073] Prepare a 10% GelMA hydrogel and store it in a 37°C water bath in the dark.

[0074] SHEDs three-dimensional microspheres were directly resuspended and mixed with 10% GelMA hydrogel.

[0075] 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.

[0076] The regenerative effect of the injected complex was evaluated by constructing a nude mouse subcutaneous semi-in situ pulp regeneration model. The specific steps included:

[0077] Step 1: Extract a single-rooted orthodontic tooth to prepare a 5 mm thick tooth segment;

[0078] Step 2: Perform routine root canal preparation and disinfection on the obtained tooth segment;

[0079] Step 3: Prepare 10% GelMA hydrogel and store it in a 37°C water bath away from light for later use;

[0080] Step 4: The induced SHEDs 3D microspheres were directly resuspended and mixed with 10% GelMA hydrogel. The cell density and composition of each group were as follows: ① 2D group: 7.5 × 10⁶ OM-induced SHEDs per ml; ② OM 3D group: 3 × 10⁴ OM-induced SHEDs microspheres per ml; ③ LiCl + OM 3D group: 3 × 10⁴ 2.5 mmol / L LiCl + OM-induced SHEDs microspheres per ml.

[0081] Step 5: Inject 20 μL of 10% GelMA-loaded cells / microspheres into the root canal of the tooth segment, and perform photocrosslinking by exposing it to 9.16-W / cm2 UV light for 20 s to prepare an injectable complex.

[0082] Step 6: The tooth segment complex was implanted subcutaneously into the back of nude mice. Eight weeks later, tissue samples were collected for histological evaluation, and coronal sections of the tooth segments were observed using H&E staining. Specifically: Figure 12The H&E staining images shown indicate that in the blank group, dense connective tissue formation was observed in the root canals, but no odontoblast-like cells or newly formed dentin were seen. In the control group, GelMA hydrogel that had detached due to non-degradation was visible, but no connective tissue, newly formed odontoblast-like cells, or dentin were observed. In the 2D group, a single layer of atrophied odontoblast-like cells was visible between the pulp cavity and dentin, with short, thin odontoblast processes penetrating into the original odontoblast tubules. Capillary formation was visible in the loose connective tissue. In the OM 3D group, odontoblast-like cell formation was visible, with odontoblast processes longer than in the 2D group. Intercellular edema was present, and the odontoblasts were compressed and atrophied. Abundant congested small blood vessels were seen near the pulp cavity side of the odontoblasts. In the LiCl+OM 3D group, tightly packed odontoblast-like cells were visible, with abundant red-stained cytoplasm, nuclei near the basal side, and long, thin odontoblast processes penetrating into the dentinal tubules. Dense connective tissue formation was visible near the pulp cavity side of the odontoblasts, and congested small blood vessels were visible.

[0083] It should be noted that in the nude mouse subcutaneous semi-in situ pulp regeneration model, the SHEDs microspheres in the LiCl+OM 3D group were able to form regenerative tissue with abundant blood vessels and odontoblasts, and the in vivo regeneration effect was significant and significantly better than other groups, proving its high efficiency and safety in vivo application.

[0084] 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 for preparing lithium chloride-induced SHEDs three-dimensional microspheres, characterized in that, It includes the following steps, specifically: Step a, Cell extraction and culture: SHEDs were extracted from human deciduous teeth for primary culture and passage expansion. P3-P6 generation cells were selected for subsequent experiments. SHEDs are human deciduous tooth pulp stem cells. Step b: Screening for the optimal LiCl concentration to induce odontoblastic differentiation of SHEDs: Step b1: Select SHEDs cells with good growth status from the P3-P6 generation obtained in step a, and induce odontogenic differentiation of SHEDs by setting osteogenic induction medium with different LiCl concentrations. Step b2: The effects of osteogenic induction medium with different LiCl concentrations on the odontogenic differentiation of SHEDs were compared and analyzed to analyze the influence of LiCl on the proliferation and odontogenicity of SHEDs and to screen out the optimal LiCl concentration, which was 2.5 mmol / L. Step c: Construct SHEDs three-dimensional microspheres using the agarose microporous three-dimensional culture method: Step c1: Preparation of culture model microplates: A 3% agarose solution, sterilized by high temperature and pressure, is poured into a polydimethylsiloxane microcolumn positive mold. After the 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, with a spacing of 200 μm between adjacent microcolumns. 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, one microplate per well. Finally, 500 μL of PBS buffer is added to each agarose microplate in the 24-well plate, and the plates are sterilized under ultraviolet light before use. Step c2, Construction of SHEDs microspheres: Add 800 μL of PM medium to each well of a 24-well plate with agarose microplates, and then add the SHEDs cultured in step a at a rate of 5 × 10⁻⁶. 5 The microspheres were inoculated at a density of 1 microsphere per well in agarose microplates, allowed to stand for 10 minutes, and then incubated at 37°C for 24 hours. The culture medium in the agarose microplates was then replaced with osteogenic induction medium containing the optimal LiCl concentration obtained in step b, and the microspheres were incubated at 37°C. The osteogenic induction medium was replaced every 2 days. During this process, the formation of microspheres was observed using an optical microscope. SHEDs three-dimensional microspheres were obtained after 14 days of culture. Step d: The activity of the obtained SHEDs three-dimensional microspheres was detected by live-dead staining and DAPI / Phalloidin staining methods; The odontogenic differentiation effects of SHEDs cultured in two dimensions, SHEDs microspheres induced by conventional osteogenic induction medium, and SHEDs microspheres induced by LiCl-containing osteogenic induction medium were analyzed and compared using RT-PCR, Western blotting immunofluorescence, or alizarin red staining.

2. The method for preparing lithium chloride-induced SHEDs three-dimensional microspheres according to claim 1, characterized in that: In step a, the culture medium used for SHEDs 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 SHEDs culture environment in step a is: constant temperature culture in a 37℃ constant temperature incubator.

3. The method for preparing lithium chloride-induced SHEDs three-dimensional microspheres according to claim 1, characterized in that: The culture environment for LiCl-induced dentin differentiation of SHEDs in step b1 is as follows: cultured in a constant temperature incubator at 37℃, with the culture medium changed every 2 days.

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

5. An injectable complex, characterized in that: The injection complex is composed of SHEDs three-dimensional microspheres prepared by the method of lithium chloride-induced SHEDs three-dimensional microspheres according to any one of claims 1-2 and a biocompatible hydrogel. The mixing steps of SHEDs three-dimensional microspheres and hydrogels include: Prepare a 10% GelMA hydrogel and store it in a 37°C water bath in the dark. SHEDs three-dimensional microspheres were directly resuspended and mixed with 10% GelMA hydrogel.

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