DNMTi (at) ZIF-8 nano-composite and application thereof

Abstract

The invention relates to the technical field of oral medicine, in particular to a DNMTi and ZIF-8 nano-complex and application thereof.The DNMTi and ZIF-8 nano-complex is technically characterized in that in-situ synthesized zeolite imidaze-8 (ZIF-8) is adopted for delivering a DNA methyltransferase inhibitor (DNMTi), dental pulp stem cell (DPSC) cell spheres are combined, the DNMTi and ZIF-8 nano-complex can be used for bionic therapy, the activity of medicine is prolonged, and the effect of treating dental pulp stem cells is achieved. And zinc ions are continuously released to activate a PI3K-AKT signal channel, so that the dentin differentiation of stem cells and the generation of blood vessels are promoted. Through accurate simulation of a tooth development microenvironment, the biomimetic therapy can realize regeneration of a dental pulp-dentin complex. The therapy for simulating the tooth development process not only solves the key problems in the dental tissue engineering, but also opens up a new direction for treating the dental pulp and periapical diseases, and highlights the wide application prospect in regenerative medicine.

Description

Technical Field

[0001] This invention relates to the field of oral medicine technology, and more specifically, to a DNMTi@ZIF-8 nanocomposite and its applications. Background Technology

[0002] The ultimate goal of modern endodontic treatment is to regenerate the functional pulp-dentin complex, thereby restoring the biological function of teeth. Currently, only regenerative endodontic treatments (REPs) involving induced intracanal bleeding are widely used clinically. REPs are suitable for young permanent teeth with or without periapical necrosis of the pulp. However, histological evidence shows that due to the uncontrolled introduction of stem cells and cytokines by blood clots, the regenerated connective tissue resembles non-pulp-like structures such as ectopic bone, cementum, and periodontal ligaments. To address these limitations, stem cell-based pulp regeneration strategies have evolved from traditional REPs, involving the transplantation of exogenous stem cells into the root canal. Transplantation of human dental pulp stem cells (DPSCs) has been shown to successfully regenerate vascularized pulp tissue, demonstrating the promising potential of this approach. However, the lack of sufficient regenerative signals and limited vascularization still restrict the widespread application of stem cell-based regeneration methods. Furthermore, effective pulp regeneration requires a regenerative environment that promotes the migration, proliferation, and differentiation of mesenchymal stem cells; therefore, the development of biomimetic pulp regeneration therapies is urgently needed.

[0003] Epigenetic factors play a crucial role in cell differentiation and stem cell fate determination during tooth development. DNA methylation regulates key signaling pathways during tooth development. Studies of whole-genome DNA methylation sequencing of miniature pig tooth germs have revealed that differentially methylated genes are involved in tooth morphogenesis, odontoblast differentiation, ameloblast differentiation, and mineralization. Dynamic DNA methylation patterns have also been observed in developing mouse incisors. Inhibition of DNA methyltransferase inhibitors (DNMTi) has been reported to increase the expression of odontoblast differentiation markers, suggesting that DNA methylation plays a regulatory role in promoting odontoblast differentiation. However, the dynamic expression of DNA methylation during dentin-pulp complex regeneration and its role in this regeneration remain largely unexplored. The application of epigenetic regulators in stem cell-based pulp regeneration requires suitable scaffold materials to achieve sustained drug release and long-term induction of odontoblast differentiation and angiogenesis in DPSCs. Zeolite imidazole framework-8 (ZIF-8) is a type of metal-organic framework (MOF) material. Due to its porous structure, biocompatibility, and biodegradability, it is considered a promising regenerative therapy platform. However, its application in dentin-pulp complex regeneration has not yet been explored.

[0004] Furthermore, a key challenge in biomimetic pulp regeneration therapy lies in replicating the physiological environment of mesenchymal stem cells (MSCs). MSC spheroids, as a tool for mimicking the spatial structure of the developmental microenvironment, have become an important method in regenerative therapy. Compared to dispersed single cells, MSC spheroids exhibit enhanced biological and physiological relevance, capable of secreting tissue-specific extracellular matrix, cytokines, and extracellular vesicles. Three-dimensionally cultured DPSC spheroids demonstrate stronger odontoblastic differentiation capacity than monolayer cultures. Three-dimensional culture of DPSCs promotes the multi-lineage differentiation potential of stem cells and the expression of genes related to stem cell characteristics, highlighting their application in biomimetic regeneration methods.

[0005] Therefore, this invention develops a DNMTi@ZIF-8 nanocomposite and its applications to solve the problems in the prior art. Summary of the Invention

[0006] The purpose of this invention is to solve the existing technical problems mentioned in the background section above, and to provide a DNMTi@ZIF-8 nanocomposite and its applications.

[0007] The above-mentioned objective of the present invention is achieved as follows:

[0008] One aspect of the present invention provides a DMNTi@ZIF-8 nanocomposite, wherein the nanocomposite uses ZIF-8 as the delivery system for DMNTi and combines it with DPSC cell spheres for simulating the tooth development microenvironment.

[0009] Another aspect of the present invention provides a method for preparing DNMTi@ZIF-8 nanocomposite, comprising the following steps:

[0010] S1. In-situ synthesis of nano-ZIF-8: 812.5 mg of 2-methylimidazole (2-MIM) and 50 mg of DMNTi were dissolved in 10 mL of methanol and stirred at room temperature for 30 minutes until a homogeneous solution A was formed; then 375 mg of zinc nitrate hexahydrate was added to 5 mL of methanol, shaken at room temperature for 5 minutes, and gently stirred for 1 hour to form a homogeneous solution B; finally, solution A was added dropwise to solution B, and the mixture was gently stirred at room temperature for 2 hours to form the DMNTi@ZIF-8 drug delivery system.

[0011] S2. Synthesis of DPSC cell spheres using an agarose microarray: DPSC spheres were generated using an agarose microarray according to previously reported methods. First, a hydrogel microarray was fabricated using a silicone mold with a smooth 3D microstructure and hemispherical protrusions. 2 mL of 3% agarose solution was poured into the mold and allowed to solidify at room temperature. Then, the agarose substrate was carefully separated from the mold to obtain an agarose mold with a diameter of 200 μm. P3 generation DPSC cells were harvested from the culture plate, and a cell suspension was prepared. The agarose microarray was placed in a 6-well cell culture plate, with 1 × 10⁻⁶ cells added to each well. 5 Add 2 mL of cell culture medium to each well; place the cells in a cell culture incubator at 37°C and 5% CO2, where they will spontaneously form spheroids within 24 hours.

[0012] S3, Bionic therapy combining DNMTi@ZIF-8 with DPSC cell spheres: 20 μg / mL DNMTi@ZIF-8 and 5000 / ml dental pulp stem cell microspheres were suspended in GelMA hydrogel for the regeneration of the dental pulp-dentin complex.

[0013] Another aspect of the present invention provides the application of DNMTi@ZIF-8 nanocomposite or DNMTi@ZIF-8 drug delivery system prepared by the aforementioned preparation method in the preparation of drugs that promote dentin-pulp complex regeneration.

[0014] The difficulty and significance of the technical problem solved by this invention lie in:

[0015] Regenerating functional dentin-pulp complex remains a major challenge in oral medicine. Traditional pulp regeneration treatments often result in non-pulp-like tissue due to uncontrolled stem cell and cytokine introduction from periapical hemorrhage. Mimicking developmental processes to promote regeneration represents a promising but challenging approach in regenerative medicine. This invention develops a biomimetic regeneration therapy that combines DNMTi@ZIF-8 nanocomposites with DPSC spheres to effectively regenerate the dentin-pulp complex.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention utilizes in-situ synthesized ZIF-8 to deliver DMNMTi and combines it with DPSC cell spheres for regenerative therapy in the development of biomimetic teeth. This biomimetic therapy prolongs drug activity and enhances the angiogenesis-promoting effect by continuously releasing zinc ions to activate the PI3K-AKT signaling pathway, demonstrating a significantly enhanced ability to promote odontoblast differentiation. The application of this invention in regenerative therapy for the development of biomimetic teeth enables the regeneration of the pulp-dentin complex. This biomimetic tooth development therapy not only solves a key problem in dental tissue engineering but also opens up new directions for the treatment of pulp and periapical diseases, highlighting its broad application prospects in regenerative medicine. Attached Figure Description

[0018] Figure 1 This invention relates to a DNMTi@ZIF-8 nanocomposite and its applications.

[0019] Figure 2 This document describes the preparation and characterization of the DNMTi@ZIF-8 complex in this invention. (A) Schematic diagram of DNMTi@ZIF-8 synthesis; (B) Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of ZIF-8 and DNMTi@ZIF-8; (C) X-ray diffraction (XRD) patterns of simulated ZIF-8, synthesized ZIF-8, and DNMTi@ZIF-8; (D) Fourier transform infrared (FT-IR) spectra of DNMTi, ZIF-8, and DNMTi@ZIF-8; (E) Drug release characteristics of DNMTi in DNMTi@ZIF-8.

[0020] Figure 3 This document describes the preparation and characterization of dental pulp stem cell (DPSC) spheres in this invention. (a) Schematic diagram of DPSC spheres synthesized using an agarose microwell array. (b) Scanning electron microscope (SEM) image of the silica mold. (c, d) Optical images of DPSC spheres spontaneously formed on the agarose microwell array, taken under a microscope, and the diameter of the spheres formed after three days of culture. (e) Confocal microscope image of DPSC spheres stained with CellTracker Green CMFDA and Hoechst 33342.

[0021] Figure 4This invention focuses on the detection and mechanistic analysis of the angiogenesis-promoting ability of the DNMTi@ZIF-8 complex. (A) Representative images of the scratch test; (B) Quantitative analysis of the scratch test; (C) Chicken embryo allantoic membrane (CAM) test; (D) Statistical analysis of the CAM test; (E) Fluorescence microscopy images of the tubule formation test; (F) Quantitative analysis of the tubule formation test; (G, H) RT-PCR analysis of Vegfa and Hif1a gene expression in HUVECs treated with Blank, DNMTi, ZIF-8, and DNMTi@ZIF-8; (I) GO enrichment bubble diagram of differentially expressed genes (DEGs) between the DNMTi and DNMTi@ZIF-8 groups; (J) KEGG enrichment bubble diagram of DEGs between the DNMTi and DNMTi@ZIF-8 groups; (K) Western blotting verification of DNMTi@ZIF-8 activation of the PI3K-AKT pathway. (L) Relative expression ratios of p-PI3K, PI3K, p-AKT, and AKT;

[0022] Figure 5This is an example of the efficacy testing of a DNMTi@ZIF-8 nanocomposite in the biomimetic regeneration therapy of tooth development in this invention. (A) Schematic diagram of the efficacy testing of the DNMTi@ZIF-8-based biomimetic regeneration therapy of tooth development by subcutaneous transplantation experiment in nude mice. DPSC spheres and DNMTi@ZIF-8 / DNMTi are co-encapsulated in GelMA hydrogel and injected into the dentin matrix. After photocrosslinking and curing, they are subcutaneously transplanted to the back of nude mice. (B) H&E staining images of DPSC spheres, DPSC spheres + D group, and DPSC spheres + D@Z group. The newly formed dentin-like structures are outlined by red dashed lines. Blue arrows: odontoblast-like cells; red arrows: blood vessels; nd: newly formed dentin. (C) Masson staining images of DPSC spheres, DPSC spheres + D group, and DPSC spheres + D@Z group. (D) Quantitative analysis of the number of microvessels formed in DPSC spheres, DPSC sphere + D group, and DPSC sphere + D@Z group (calculated in H&E stained sections, n=3 per group). (E) Quantitative analysis of the relative area of ​​newly formed dentin in DPSC spheres, DPSC sphere + D group, and DPSC sphere + D@Z group (calculated in H&E stained sections, n=3 per group). (F) Representative images of DSPP, COL-1, and EMCN immunofluorescence staining and DMP-1 immunohistochemical staining in DPSC spheres, DPSC sphere + D group, and DPSC sphere + D@Z group. Cell nuclei: DAPI. (G,H,I) Mean fluorescence intensity of COL-1, DSPP, and EMCN in DPSC spheres, DPSC sphere + D group, and DPSC sphere + D@Z group. (j) Semi-quantitative analysis of the proportion of DMP-1 positive areas (in immunohistochemical staining images). Red arrow: DMP-1 positive odontoblast-like cells; nd: newly formed dentin. DNMTi: DNA methyltransferase inhibitor; ZIF-8: zeolite imidazole framework-8; DPSC: dental pulp stem cells; TDM: dentin matrix; H&E: hematoxylin-eosin staining. Detailed Implementation

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0024] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0025] Reference Figure 1-Figure 5 The image shows a preferred embodiment of the present invention.

[0026] The present invention provides a DNMTi@ZIF-8 nanocomposite and its application in the preparation of a drug that promotes the regeneration of a biomimetic dentin-pulp complex.

[0027] The following are specific experimental implementations of the embodiments of the present invention:

[0028] 1. Method

[0029] 1.1 Preparation of nano-ZIF-8 and DMNTi@ZIF-8 composite

[0030] Nano-ZIF-8 was synthesized in situ. First, 2-MIM (812.5 mg) and DMNTi (50 mg) were dissolved in 10 mL of methanol and stirred at room temperature for 30 minutes until a homogeneous solution (solution A) was formed. Then, 375 mg of zinc nitrate hexahydrate was added to 5 mL of methanol, and the mixture was shaken at room temperature for 5 minutes and gently stirred for 1 hour to form a homogeneous solution (solution B). Finally, solution A was added dropwise to solution B, and the mixture was gently stirred at room temperature for 2 hours to form the DMNTi@ZIF-8 drug delivery system. After washing the DMNTi@ZIF-8 solution with methanol and centrifuging three times (10,000 rpm, 15 minutes), the supernatant was collected, and the drug loading of DMNTi was determined by UV-Vis absorption spectroscopy. The white precipitate (nano-DNMTi@ZIF-8) was collected and dried under vacuum for 24 hours. The synthesis method for nano-ZIF-8 was similar, but DMNTi was not added to solution A.

[0031] 1.2 Characterization of nano ZIF-8 and DMNTi@ZIF-8

[0032] The morphology of ZIF-8 and DNMTi@ZIF-8 was observed using scanning electron microscopy (SEM) (KYKY Technology Development Ltd., China) and transmission electron microscopy (TEM) (Tecnai G2 F20S-TWIN, USA). For SEM analysis, the samples were mounted on conductive carbon double-sided tape and coated with a thin layer of gold using a spray-coating machine. They were then placed in the SEM chamber, and the microstructure was observed at 10 kV. For TEM analysis, the samples were dispersed in ethanol to form a transparent solution. A small amount of sample was then dropped onto a 400-mesh ultrathin carbon film copper grid. After the solution evaporated, the grid was placed on a stage for observation. The field of view and voltage were adjusted, and the electron beam was accelerated, focused, and projected onto the samples to obtain TEM images of ZIF-8 and DNMTi@ZIF-8. X-ray diffraction (XRD) patterns of nano-ZIF-8 and DNMTi@ZIF-8 nanocomposites were collected using a D4 ENDEAVO X-ray diffractometer (Bruker, Germany) with Cu Kα radiation (15 mA and 40 kV) at a scan rate of 1°min. -1 The scans were performed over a wide angular range (2θ = 5–50°). Zeta potentials were measured using a Malvern Instruments Zetasizer Nano ZS90. Functional groups in the samples were analyzed using potassium chloride plates on a Fourier transform infrared (FT-IR) spectrometer (Thermo, Nicolet 6700, USA) with a measurement range of 400–4000 cm⁻¹. -1 .

[0033] 1.3 Drug loading capacity and release behavior of DNMTi@ZIF-8

[0034] The encapsulation efficiency of DNMTi@ZIF-8 was evaluated by detecting the concentration of DNMTi in the washed suspension after DNMTi@ZIF-8 synthesis at a wavelength of 245 nm using UV-Vis spectrophotometry. To calculate the loading efficiency, 2 mg of DNMTi@ZIF-8 nanoparticles were dissolved in 1 M hydrochloric acid and diluted with deionized water. The concentration of DNMTi was then calculated based on a standard curve. The encapsulation efficiency (EE%) and drug loading efficiency (LE%) were calculated using the following formulas:

[0035] EE (%) = (Total amount of drug added - Remaining amount of drug) / Total amount of drug added × 100%;

[0036] LE (%) = (Amount of drug loaded in nanoparticles) / (Total weight of nanoparticles) × 100%.

[0037] Drug release behavior was studied in a 37°C isothermal shaker. 5 mg DNMTi@ZIF-8 was dispersed in 40 mL of PBS buffer and sealed in a dialysis bag with a molecular weight cutoff of 14,000 Da. At predetermined time intervals (0, 0.5, 1, 3, 5, 8, and 10 days), 2 mL of release medium was taken for UV-Vis spectrophotometric measurements, and an equal volume of fresh medium was added after each measurement. All measurements were repeated three times. Further analysis of Zn... 2+ The release of Zn was detected using inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 730, USA). ZIF-8 and DNMTi@ZIF-8 nanoparticles were immersed in 10 mL of PBS solution, respectively. Samples were taken at 37°C without shaking for 8 hours, 16 hours, 24 hours, 2 days, 3 days, 4 days, 7 days, and 10 days. The supernatant was collected at each time point, and Zn content was determined. 2+ Concentration, and plot the release curve.

[0038] 1.4 Synthesis of DPSC spheres using agarose micropore array

[0039] DPSC spheres were generated using an agarose microwell array according to a previously reported method. First, a hydrogel microwell array was fabricated using a silicone mold with a smooth 3D microstructure and hemispherical protrusions. 2 mL of 3% agarose solution was poured into the mold and allowed to solidify at room temperature. Then, the agarose substrate was carefully separated from the mold, yielding an agarose mold with a diameter of 200 μm. P3 generation DPSC cells were harvested from the culture plate, and a cell suspension was prepared. The agarose microwell array was placed in a 6-well cell culture plate, with 1 × 10⁻⁶ cells added to each well. 5 Cells were cultured in a mold, with 2 mL of cell culture medium added to each well. The cells were placed in a cell culture incubator at 37°C and 5% CO2, where they spontaneously formed spheroids within 24 hours. Cells were harvested after three days of culture in the mold for further analysis. Cell morphology was observed using a microscope (IX73, Olympus, Japan).

[0040] 1.5 RT-PCR Analysis

[0041] To determine the expression of genes associated with odontogenic differentiation, including Col1a, Dmp1, and Dspp in monolayer DPSCs, DPSC spheres, DPSC spheres cultured with DNMTi (DPSC spheroid+D group), and DPSC spheres cultured with DNMTi@ZIF-8 (DPSC spheroid+D@Z group), as well as the expression of angiogenesis-related genes (including Vegfa and Hif1a) in HUVECs after treatment with Blank, DNMTi, ZIF-8, and DNMTi@ZIF-8, RT-PCR experiments were performed. First, DPSCs were cultured at 2×10⁻⁶ in different groups. 5 Cells were cultured at densities of 7 and 14 days in differentiation medium, while HUVECs were cultured for 3 days in the corresponding medium. Total RNA was then extracted using TriQuick reagent, and the RNA was reverse transcribed into cDNA using the PrimeScript RT kit. The expression of relevant genes (Col1a, Dmp1, Dspp, Vegfa, and Hif1a) was detected according to the SYBR Green Ultramix reagent instructions. Specifically, the early odontoblastic differentiation marker Col1a was detected on day 7, and the late odontoblastic differentiation markers (Dspp and Dmp1) were detected on day 14.

[0042] 1.6 Cell viability detection

[0043] To evaluate the cell viability of SHEDs co-cultured with different concentrations of DNMTi, the CCK-8 assay was used. SHEDs were seeded in 96-well plates at different concentrations of DNMTi (0, 1, 5, 10, 15 μM), with 5 × 10⁶ cells per well. 3 Cells were collected and incubated overnight at 37°C. Subsequently, cells were treated with a specified concentration of DNMTi for 2 hours. After treatment, cells were washed twice with PBS, and 10 μL of CCK-8 reagent and 100 μL of culture medium were added to each well. After incubating the plate in the dark at 37°C for 1 hour, the absorbance was read at 450 nm using a microplate reader (Thermo, USA).

[0044] To evaluate the cell viability of DPSC and HUVEC cells co-cultured with DNMTi@ZIF-8, the CCK-8 assay was also used. DPSC and HUVEC cells were seeded in 96-well plates at 5 × 10³ cells per well and incubated overnight at 37°C. Then, the cells were treated with different concentrations of DNMTi@ZIF-8 (0, 10, 25, 50, 75 μg / ml) for 1, 3, or 5 days. After treatment, the cells were washed twice with PBS, and 10 μL of CCK-8 reagent and 100 μL of culture medium were added to each well. After incubating the plates in the dark at 37°C for 1 hour, the absorbance was read at 450 nm using a microplate reader (Thermo, USA).

[0045] 1.7 ALP and ARS detection

[0046] To assess the effect of DNMTi on the odontogenic differentiation ability of SHEDs, SHEDs were cultured in odontogenic differentiation induction medium (OM) and different concentrations of DNMTi for 7 and 14 days, with the medium being changed every two days. On day 7, ALP activity was measured according to the manufacturer's instructions using the BCIP / NBT alkaline phosphatase colorimetric kit and alkaline phosphatase assay kit, and normalized to total protein content. On day 14, calcium deposition in the extracellular matrix was assessed using alxin red staining. Staining intensity was quantified by measuring absorbance at 542 nm after treatment with 10% (w / v) cetylpyridinium chloride at 37°C for 15 min.

[0047] To evaluate the effect of DNMTi@ZIF-8 on odontoblastic differentiation, the experimental groups included: monolayer DPSC, DPSC spheroids (DPSC spheroids), DPSC spheroids treated with 2.28 μg / ml DNMTi (DPSC spheroid+D), and DPSC spheroids treated with 20 μg / ml DNMTi@ZIF-8 (DPSC spheroid+D@Z). Monolayer DPSC served as a negative control. The culture medium was changed every two days. On day 7, ALP activity was measured using the BCIP / NBT alkaline phosphatase colorimetric kit and alkaline phosphatase assay kit, and normalized to total protein content. On day 14, calcium deposition in the extracellular matrix was assessed using Alixin Red staining, and staining intensity was quantified by measuring absorbance at 542 nm.

[0048] 1.8 RNA sequencing

[0049] DPSCs were cultured for 7 days under Blank, DNMTi, and DNMTi@ZIF-8 conditions. The DNMTi group served as a negative control. Total RNA was extracted from all three groups using an RNA extraction kit. Strand-specific libraries were constructed using the TruSeq RNA Sample Preparation Kit and sequenced on an Illumina Novaseq 6000 instrument. Raw data were processed using Skewer and quality was assessed using FastQCv0.11.2. Differentially expressed genes (DEGs) were screened using p<0.05 and an absolute fold change ≥2. Selected DEGs underwent functional and pathway enrichment analysis using the GO and KEGG databases. Pathways with p<0.05 and containing at least two related genes were considered significantly enriched. Library construction and sequencing were performed by Gene DenovoBiotechnology Co., Ltd.

[0050] 1.9 Western blot experiment

[0051] On day 3, total protein was extracted from cells in the Blank, DMNTi, ZIF-8, and DMNTi@ZIF-8 groups using a lysis kit. Cells were lysed in lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA protein quantification kit. After protein separation by SDS-PAGE, the relevant proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then immunoblotted with designated antibodies (anti-AKT antibody, anti-p-AKT antibody, anti-PI3K antibody, anti-p-PI3K antibody, and anti-β-actin antibody). After the first incubation, the membranes were washed with TBST and incubated with HRP-labeled secondary antibodies. Direct development was performed using an enhanced chemiluminescence kit. Relative quantification of proteins was performed using Fiji software (version 2.3.0). The primary and secondary antibodies used in this study are listed in Appendix Table 1.

[0052] 1.10 Scratch test and tubule formation test

[0053] HUVECs were seeded in 6-well plates, 1 × 10⁶ per well. 5Cells were cultured in groups of 1000 μL each. After 24 hours, the cells were co-cultured with Blank, DNMTi, ZIF-8, and DNMTi@ZIF-8 groups, with the DNMTi group serving as a negative control. Two days later, a “scratch” was created in the center of each well using a sterile 1000 μL pipette tip. The wells were gently washed with PBS and fresh serum-free medium was added. The cells were then fixed and photographed after 24 hours (Olympus, Japan). For the tube formation assay, Matrigel was first thawed at 4°C for several hours, then 150 μL of Matrigel was added to each well of a 48-well plate and polymerized at 37°C for 30 minutes. The plate and pipettes were pre-cooled at -20°C. After Matrigel solidification, HUVECs were seeded into each well at a ratio of 3 × 10⁶ cells / well. 4 Cells were cultured for 6 hours, after which samples were collected. To observe lumen formation, samples were washed twice with PBS, and the cytoskeleton was stained with Celltracker CM-Dil in the dark. Images were then captured using a fluorescence microscope (IX73, Olympus, Japan). The total lumen length was then statistically analyzed using Fiji software (version 2.3.0). The experiment was repeated three times.

[0054] 1.12 Chicken Embryo Allantoic Membrane Experiment

[0055] Angiogenesis capacity was assessed in vivo using the chicken embryo allantoic membrane assay (CAM). Fertilized eggs were incubated for 5 days in a humid environment at 37°C to promote embryonic development. On day 5, HUVECs from different groups (n=4 per group) were applied to the surface of the yolk sac membrane. The eggshell was cut open through a 1 cm² incision, sealed with paraffin film, and secured with tape. The eggs were returned to the incubator at 37°C until day 8. Images of the allantoic membrane were taken, and vascularity was quantified using Fiji software (version 2.3.0), calculating the total number of vascular connections, vessel size, and length.

[0056] 1.13 Nude Mouse Ectopic Pulp Regeneration Model

[0057] The nude mice used in this study were 6-week-old male Balb / c nude mice (weight: 18-22g, GemPharmatech, Nanjing, China). The mice were housed in a pathogen-free environment with isolation measures and controlled light cycles. The maximum stocking density was 5 mice per cage. They were fed standardized mouse feed and provided with autoclaved drinking water. Isoflurane was used for anesthesia. For tooth fragment implantation and removal surgeries, blinding of the animals was not possible. However, all samples were numbered and labeled after removal to facilitate subsequent unbiased data analysis.

[0058] To evaluate the efficacy of combined DNMTi@ZIF-8 and DPSC sphere treatment in dentin-pulp complex regeneration, an ectopic pulp regeneration model was established in nude mice. All experimental procedures were performed in accordance with ethical guidelines and approved by the Ethics Committee of West China Hospital, Sichuan University (Approval No.: 20230926002). Thirty nude mice were randomly assigned to three groups: the DPSC sphere group, the DNMTi+DPSC sphere group, and the DNMTi@ZIF-8+DPSC sphere group (n=10 per group). Healthy premolars (N=40) were extracted from patients undergoing orthodontic treatment with informed consent to obtain processed dentin matrix (TDM). All teeth should be healthy and intact. Periodontal ligament and pulp tissue were carefully removed, and root canals were cut into 2-3 mm segments using a high-speed air turbine surgical instrument. Then, the root canal segment was subjected to three ultrasonic cleanings (5 to 6 minutes each), and treated with different concentrations of ethylenediaminetetraacetic acid (EDTA) solution (17%, 10%, and 5%) for 5 minutes each, followed by rinsing with deionized water for 5 minutes each time (three times). Finally, the treated TDM was stored in a medium containing 1% penicillin / streptomycin at 4°C.

[0059] Then, the single-layer DPSC (1×10) 6 DPSC spheres (5000 spheres / ml), DNMTi-treated DPSC spheres, and DNMTi@ZIF-8-treated DPSC spheres were mixed with GelMA (10% w / v), seeded into TDM, and cured by light. All grafts were harvested after 8 weeks, fixed with 4% paraformaldehyde for 24 hours, demineralized with 20% EDTA at 37°C, and further subjected to histological analysis. At the end of the experiment, vital organs such as the heart, liver, spleen, lungs, and kidneys of mice were collected.

[0060] 1.14. Histological observation

[0061] For TDM samples from different groups, these samples were demineralized for 3 months. The samples were then embedded in paraffin and cut into 5 μm thick sections, which were then placed on glass slides. H&E staining and Masson's trichrome staining were performed according to standard procedures. Mouse organs were also embedded in paraffin and cut into 5 μm thick sections for H&E staining. All sections were observed under a microscope (VS200, Olympus, Japan).

[0062] 1.15 Immunohistochemical staining

[0063] The antigen retrieval conditions for the samples were heating at 99°C for 20 minutes. Samples were blocked and permeated with TBST containing 5% BSA for 1 hour at room temperature, then co-incubated overnight at 4°C with the primary antibodies (anti-DMP-1 antibody, 1:200; anti-COL-1 antibody, 1:200; anti-DSPP antibody, 1:50), diluted in TBST containing 5% BSA. Secondary antibodies (donkey anti-rabbit IgG Alexa Fluor 647 / donkey anti-mouse IgG Alexa Fluor 488) were then added and incubated at room temperature for 1 hour. Cell nuclei were stained with DAPI. Images were taken using a microscope (VS200, Olympus, Japan). A list of primary and secondary antibodies used in this study is provided in Appendix Table 1.

[0064] 1.16 Immunofluorescence staining

[0065] Samples were subjected to immunofluorescence staining one month after demineralization. The immunofluorescence staining procedure followed a strict sample preparation protocol to achieve high-resolution imaging. After demineralization, samples were immersed in a solution containing 20% ​​sucrose and 2% polyvinylpyrrolidone (PVP) at 4°C for 12 hours. Then, samples were embedded in an 8% gelatin solution containing 20% ​​sucrose and 2% PVP. After embedding, samples were cut into 100 μm sections using a Leica CM1950 cryostat (Leica, 14035838382) and placed on glass slides. Sections were air-dried at room temperature for 2 hours and then stored at -20°C.

[0066] The immunofluorescence staining procedure was as follows: First, tissue sections were equilibrated at room temperature for 15 minutes and then hydrated with PBS. They were then infiltrated with 0.3% Triton X-100 for 10 minutes and blocked with 5% donkey serum, followed by incubation with primary antibody diluted in blocking buffer (1:150). Subsequently, sections were thoroughly washed with PBS 5–7 times, at least 3 minutes each time. Next, sections were incubated with Alexa Fluor-labeled secondary antibody (1:300) for 1–1.5 hours at room temperature. After another 5–7 PBS washes, sections were mounted with Fluoromount-G. Cell nuclei were stained with DAPI. Images were taken using a rotating confocal microscope (Andor Dragonfly 200, Oxford Instruments group, UK) and processed using Imaris software (version 9.7, Bitplane, Oxford Instruments group, UK). Mean fluorescence intensity was calculated using Fiji software.

[0067] 2. Statistical Analysis

[0068] All experiments were repeated at least three times. Results are expressed as mean ± standard deviation (SD). The normality of the distribution was tested by the D'Agostino-Pearson normality test. Statistical analysis of experimental results between two groups was performed using Student's t-test. Multiple comparisons of a single independent variable were performed using one-way ANOVA with Tukey's post-hoc test, and multiple comparisons of two independent variables were performed using two-way ANOVA. All statistical analyses were performed using GraphPad Prism software (GraphPad Software, USA, version 9.0). p < 0.05 was considered statistically significant (P < 0.05, P < 0.01, P < 0.001).

[0069] 3. Results

[0070] 3.1 Synthesis and Characterization of DMNTi@ZIF-8

[0071] DNMTi@ZIF-8 nanocomposites were synthesized in situ with an encapsulation efficiency of 94.92±1.33% and a drug loading of 11.45±4.37%. SEM and TEM showed that both ZIF-8 and DNMTi@ZIF-8 possessed regular dodecahedral structures with diameters of 275.78±35.15 nm and 281.83±41.06 nm, respectively. XRD and FT-IR confirmed that their peak values ​​were consistent with those of simulated ZIF-8. 2+ The release kinetics of DNMTi showed an initial release peak on day 1, followed by a gradual decrease and stabilization between days 8 and 10. The release of DNMTi is related to Zn. 2+ The release trend is consistent, with approximately 50% released after 24 hours.

[0072] 3.2. DNMTi@ZIF-8 promotes angiogenesis in vitro and in vivo.

[0073] CCK-8 assays showed that nano-ZIF-8 and DNMTi@ZIF-8 promoted cell proliferation at concentrations below 25 μg / mL. Based on the measured loading efficiency, the optimal concentration of DNMTi@ZIF-8 was 20 μg / mL. Scratch assays showed that ZIF-8 and DNMTi@ZIF-8 induced significantly higher levels of cell migration compared to DNMTi and the blank control group. Chicken embryo allantoic membrane assays showed that ZIF-8 and DNMTi@ZIF-8 significantly enhanced angiogenesis. Figure 4(h,i). Tube formation assays also showed that the total lumen length was longer and the tubular structure was more complete in the ZIF-8 and DNMTi@ZIF-8 groups. RT-PCR analysis confirmed the upregulation of the pro-angiogenic genes Vegfa and Hif1a in ZIF-8 and DNMTi@ZIF-8 treated HUVECs.

[0074] 3.3 Potential Mechanisms by which DNMTi@ZIF-8 Promotes Angiogenesis

[0075] To investigate the potential mechanism by which DNMTi@ZIF-8 promotes angiogenesis, RNA sequencing was performed on DPSCs (blank control group), DNMTi-treated DPSCs (DNMTi group), and DNMTi@ZIF-8-treated DPSCs (DNMTi@ZIF-8 group). RNA sequencing results showed significant differentially expressed genes (DEGs) between the DNMTi@ZIF-8 group and the control group and DNMTi-only treated cells. A total of 1432 DEGs were identified between the DNMTi and DNMTi@ZIF-8 groups, of which 753 genes were upregulated and 679 genes were downregulated in the DNMTi@ZIF-8 group. GO analysis showed that DEGs were enriched in functions related to cell migration, angiogenesis, and zinc ion binding. KEGG pathway analysis indicated that DEGs were enriched in the HIF-1, Wnt, PI3K-AKT, and MAPK signaling pathways. Western blot analysis confirmed that DNMTi@ZIF-8 activated the PI3K-AKT signaling pathway, showing that the phosphorylation level of this pathway was upregulated in the DNMTi@ZIF-8 group.

[0076] 3.4. DMNTi@ZIF-8 enhances odontoblastic differentiation of DPSC spheres

[0077] DPSC spheres were constructed using an agarose microporous array. An agarose mold with a diameter of 200 μm was created using a silica mold with smooth, hemispherical protrusions in 3D. DPSCs were inoculated onto this mold and spontaneously aggregated into spheres within 24 hours, then cultured for another 5 days.

[0078] ALP staining results showed that the DPSC spheroid group treated with DNMTi@ZIF-8 (DPSC spheroid+D@Z group) exhibited the most intense staining compared to the DPSC spheroid group treated with DNMTi alone (DPSC spheroid+D group), while the untreated spheroid group (DPSC spheroid group) and the monolayer DPSCs group showed weaker staining. Alcinnamon staining results were consistent with ALP staining, with the DNMTi and DNMTi@ZIF-8 treatment groups showing more Alcinnamon-positive mineralized nodules. RT-PCR analysis indicated that the expression of early (Col1a) and late (Dspp and Dmp1) odontogenic markers was significantly upregulated in all spheroid groups, with the most significant upregulation in the DPSCspheroid+D@Z group.

[0079] 3.5. DMNTi@ZIF-8 promotes the regeneration of the dentin-pulp complex in vivo.

[0080] The efficacy of DMNTi@ZIF-8 in promoting dentin-pulp complex regeneration was evaluated using a semi-in situ in vivo model. DPSC spheres were co-encapsulated with either DMNTi or DMNTi@ZIF-8 in GelMA hydrogel and implanted into mice for 8 weeks. H&E staining showed no toxicity or damage in any group. H&E staining and Masson's trichrome staining were used for further analysis. Masson's trichrome staining showed significantly increased blue collagen deposition in the DPSC sphere + D and DPSC sphere + D@Z groups. Fewer microvessels were observed in the DPSC sphere + D group. Notably, the newly formed dentin in the DPSC sphere + D and DPSC sphere + D@Z groups was located away from the TDM proto-dentin tissue. Immunofluorescence and immunohistochemical staining showed the highest expression levels of angiogenesis markers (EMCN) and dental mineralization markers (COL-1 and DSPP) in the DPSC sphere + D@Z group. Immunohistochemical staining also showed that DMP-1 expression was high in newly formed dentin-like tissue.

[0081] Through the above embodiments of the present invention, the present invention developed a DNMTi@ZIF-8 nanocomposite and utilized a 3D culture system of DPSC spheres to simulate developmental signals and the developmental microenvironment. DNMTi@ZIF-8 promotes angiogenesis by releasing zinc ions, activating the PI3K-AKT signaling pathway, and enhancing dentin differentiation. This treatment successfully regenerated the dentin-pulp complex in a semi-in situ model. The combination of DNMTi@ZIF-8 and DPSC spheres in the present invention recreates the natural tooth developmental microenvironment, exhibits superior dentin-promoting and angiogenesis-promoting functions, and successfully regenerates blood vessels and dentin-like tissue. This innovative method opens up new avenues for the regeneration of the dentin-pulp complex and the treatment of pulp necrosis.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A DNMTi@ZIF-8 nanocomposite, characterized in that, The nanocomposite uses zeolite imidazole ester-8 (ZIF-8) as a delivery system for the DNA methyltransferase inhibitor (DNMTi) and combines it with dental pulp stem cell (DPSC) cell spheres to simulate the tooth development microenvironment.

2. The method for preparing a DNMTi@ZIF-8 nanocomposite according to claim 1, characterized in that, Includes the following steps: S1. In-situ synthesis of nano-ZIF-8: 812.5 mg of 2-methylimidazole (2-MIM) and 50 mg of DMNTi were dissolved in 10 mL of methanol and stirred at room temperature for 30 minutes until a homogeneous solution A was formed; then 375 mg of zinc nitrate hexahydrate was added to 5 mL of methanol, shaken at room temperature for 5 minutes, and gently stirred for 1 hour to form a homogeneous solution B; finally, solution A was added dropwise to solution B, and the mixture was gently stirred at room temperature for 2 hours to form the DMNTi@ZIF-8 drug delivery system. S2. Synthesis of DPSC cell spheres using an agarose microarray: DPSC spheres were generated using an agarose microarray according to previously reported methods. First, a hydrogel microarray was fabricated using a silicone mold with a smooth 3D microstructure and hemispherical protrusions. 2 mL of 3% agarose solution was poured into the mold and allowed to solidify at room temperature. Then, the agarose substrate was carefully separated from the mold to obtain an agarose mold with a diameter of 200 μm. P3 generation DPSC cells were harvested from the culture plate, and a cell suspension was prepared. The agarose microarray was placed in a 6-well cell culture plate, with 1 × 10⁻⁶ cells added to each well. 5 Add 2 mL of cell culture medium to each well; place the cells in a cell culture incubator at 37°C and 5% CO2, where they will spontaneously form spheroids within 24 hours. S3, Bionic therapy combining DNMTi@ZIF-8 with DPSC cell spheres: 20 μg / mL DNMTi@ZIF-8 and 5000 cells / mL DPSC cell spheres were suspended in GelMA hydrogel for the regeneration of the pulp-dentin complex.

3. The application of the DNMTi@ZIF-8 nanocomposite according to claim 1 or the DNMTi@ZIF-8 drug delivery system prepared by the preparation method according to claim 2 in the preparation of drugs that promote dentin-pulp complex regeneration.

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