A dynamic cross-linked hydrogel and a preparation method and application thereof

Abstract

The application belongs to the technical field of hydrogel and provides a dynamic cross-linking hydrogel and a preparation method and application thereof, and comprises the following steps: cross-linking oxidized dextran and carboxymethyl chitosan containing a phenylboronic acid modified group; adding dextranase before hydrogel formation, and introducing polydopamine-coated calcium peroxide nanoparticles; wherein the mass concentration of the oxidized dextran is 2-10% w / v, the mass concentration of the carboxymethyl chitosan containing the phenylboronic acid modified group is 2% w / v, the dextranase concentration is 10-100 U, and the concentration of the polydopamine-coated calcium peroxide nanoparticles is 0.1-1% w / v. The hydrogel of the application shows good cell compatibility, pro-angiogenic capacity and biological activity for promoting dental pulp regeneration in a plurality of in-vitro and in-vivo experiments, is suitable for the field of tissue engineering such as dental pulp injury repair, and effectively solves technical problems such as insufficient cell survival support of conventional hydrogels in three-dimensional culture.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of hydrogels, and particularly relates to a dynamic crosslinking hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Dental pulp tissue plays an irreplaceable role in maintaining tooth vitality, perception and repair. However, due to reasons such as deep caries and trauma, the traditional treatment of dental pulp necrosis or periapical lesion adopts a root canal treatment method to remove infected tissue, which can preserve the dental tissue but loses the biological activity of the dental pulp, and long-term existence of the tooth is fragile and easy to break.

[0003] In recent years, dental pulp regeneration has become an important direction of tissue engineering research. Through the synergistic effect of stem cells, scaffold materials and biological signals, it is expected to realize the reconstruction of the structure and function of natural dental pulp tissue. Functional hydrogels have been widely used in the construction of dental pulp regeneration scaffolds due to their similar three-dimensional structure to the natural extracellular matrix and controllable physical and chemical properties. An ideal dental pulp scaffold material should not only have suitable biocompatibility and biodegradability, but also provide a stable microenvironment to support the adhesion, growth and differentiation of stem cells, and relieve the extreme pathological environment such as ischemia and hypoxia; however, the unique closed structure of the dental pulp cavity makes the regeneration process prone to hypoxia and low energy state, which seriously affects the survival of stem cells and tissue repair. At present, some studies have attempted to introduce oxygen donors or sugar sources to improve the local metabolic environment, but often face problems such as low release efficiency, strong stimulation and unstable scaffolds; in addition, traditional hydrogels mostly rely on physical or chemical crosslinking structures, which lack dynamic response ability and cannot adapt to changes in tissue stress and environment.

[0004] Therefore, it is urgent to develop a hydrogel material that has stable sugar / oxygen production capacity, good mechanical properties and dynamic self-repairing performance, to support the survival of stem cells while reconstructing the dental pulp microenvironment, promoting angiogenesis and tissue regeneration. SUMMARY

[0005] The purpose of the embodiment of the application is to provide a preparation method of a dynamic crosslinking hydrogel, which aims to solve the problems raised in the background.

[0006] The embodiment of the application is implemented as follows: a preparation method of a dynamic crosslinking hydrogel, comprising the following steps:

[0007] Crosslinking oxidized dextran (ODex) and carboxymethyl chitosan (CMCS-PBA) containing a phenylboronic acid modified group;

[0008] Adding dextranase before the formation of the hydrogel, and introducing polydopamine-coated calcium peroxide (PDA@CPO) nanoparticles;

[0009] The concentration of oxidized dextran is 2-10% w / v (preferably 5%), the concentration of carboxymethyl chitosan containing phenylboronic acid modified groups is 2% w / v, the concentration of dextranase is 10-100 U (preferably 100 U), and the concentration of polydopamine-coated calcium peroxide nanoparticles is 0.1-1% w / v (preferably 0.5%).

[0010] Another objective of this invention is to provide a dynamically cross-linked hydrogel prepared using the above-described preparation method.

[0011] Another objective of this invention is to provide an application of dynamically cross-linked hydrogels in the preparation of dental pulp regeneration materials.

[0012] The present invention provides a simple method for preparing a dynamically cross-linked hydrogel, with widely available and cost-controllable raw materials. Using CMCS-PBA and ODex as the backbone, the hydrogel rapidly gels under mild conditions, forming a three-dimensional network structure. Furthermore, dextranase and PDA@CPO nanoparticles are introduced into this gel network to achieve simultaneous regulation of glucose and oxygen release in the local microenvironment, significantly improving its overall performance in tissue repair. On one hand, dextranase continuously degrades the dextran structural units in the hydrogel, releasing glucose to provide a carbon source for nutrient-deficient cells and regulate their metabolic activities. On the other hand, PDA@CPO nanoparticles effectively alleviate hypoxia and improve the cell proliferation and survival environment by decomposing and releasing oxygen in the physiological environment. Both components work synergistically in a gentle and controllable manner within a dynamic hydrogel network, forming a dual-function regulatory mechanism of "glucose production + oxygen production." This provides cells with a more suitable metabolic environment, promoting tissue repair and regeneration in ischemic and hypoxic microenvironments. The functional hydrogel also possesses excellent injectability, self-healing properties, and biocompatibility, making it suitable for constructing and repairing complex spatial structures. The rational combination of nanoparticles and enzymes, along with the encapsulation capacity of the hydrogel network, effectively prevents nanoparticle aggregation and enzyme activity loss, achieving uniform distribution and synergistic release of the dual-function components. It shows significant application potential in promoting dental pulp tissue regeneration. Attached Figure Description

[0013] Figure 1 The characterization diagram provided in Example 1 of the present invention (a is the ratio of ODex to unmodified dextran) 1 H NMR spectrum, b is CMCS-PBA and CMCS 1 (H NMR spectrum, c is a photograph of the appearance of ODex and CMCS-PBA in solution).

[0014] Figure 2Characterization figures provided for the embodiment 2 of the present application (a is the actual image of CPO and PDA@CPO nanoparticles, b is the transmission electron microscope (TEM) image, c is the X-ray diffraction (XRD) pattern of CPO and PDA@CPO, d is the Zeta potential measurement result);

[0015] Figure 3 The physicochemical properties and glucose production performance test results of the hydrogel provided for the embodiment 3 of the present application (a is the inclined state photo of Gel, b is the frequency scanning graph of Gel, c is the strain-recovery test result of Gel, d is the glucose concentration result released by Gel within 3 days and 7 days);

[0016] Figure 4 The physicochemical properties and oxygen production performance test results of the oxygen production hydrogel provided for the embodiment 4 of the present application (a is the inclined state photo of the oxygen production hydrogel, b is the frequency scanning graph of the oxygen production hydrogel, c is the dissolved oxygen concentration column chart of the oxygen production hydrogel measured every two days within 14 days, d is the cumulative oxygen production amount comparison result of the 0.5% PDA@CPO group and the 0.5% CPO group within 14 days);

[0017] Figure 5 The influence result graph of different hydrogel systems on the activity of DPSCs cells provided for the embodiment 5 of the present application;

[0018] Figure 6 The influence analysis graph of different hydrogel systems on the survival state of DPSCs provided for the embodiment 6 of the present application (a is the live / dead staining imaging, b is the statistical column chart corresponding to a, c is the live / dead cell double staining imaging, d is the statistical column chart corresponding to c, f is the statistical pie chart);

[0019] Figure 7 The influence result of the SGO-Gel hydrogel on the function of angiogenesis related cells provided for the embodiment 7 of the present application (a is the wound healing experiment image, b is the quantitative statistical result of the migration area, c is the tube lumen formation experiment image, d is the quantitative graph of the total tube lumen length);

[0020] Figure 8 The in vivo experiment result of the SGO-Gel hydrogel for promoting the regeneration of dental pulp tissue provided for the embodiment 8 of the present application (a is the immunohistochemical staining result of VEGF and DSPP, b is the quantitative graph of the immunohistochemical positive area). DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application is further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0022] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0023] Example 1: The preparation methods of oxidized dextran (ODex) and carboxymethyl chitosan containing phenylboronic acid modified groups (CMCS-PBA) are as follows:

[0024] 1.4 g of dextran (molecular weight 70,000) was dissolved in 14 mL of deionized water, and then 4 mL of sodium periodate (100 mg / mL) solution was added dropwise. The reaction was carried out at room temperature for 4 h in the dark, and then 500 μL of ethylene glycol was added to quench the reaction. The product was dialyzed in deionized water in a 3500 molecular weight dialysis bag in the dark for 72 h and then lyophilized to obtain ODex.

[0025] 2 g of chitosan with a molecular weight of 200,000 was soaked in 25 mL of 50 wt% NaOH solution for 8 h for alkalization, and then reacted with 5 g of monochloroacetic acid in 25 mL of isopropanol for 24 h. The resulting product was adjusted to pH 7, centrifuged, precipitated, and dried to obtain carboxymethyl chitosan (CMCS).

[0026] 100 mg of 4-carboxyphenylboronic acid, 170 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 160 mg of 1-hydroxybenzotriazole, 103.5 mg of N-hydroxysuccinimide and 0.2 mL of triethylamine were dissolved in 5 mL of DMSO and activated for 12 h. The solution was then slowly added to 50 mL of CMCS (concentration 10 mg / mL) dissolved in PBS and reacted for 24 h. After the reaction was completed, the solution was dialyzed in deionized water for 72 h using a 14000 molecular weight dialysis bag and then lyophilized to obtain CMCS-PBA.

[0027] Analysis of ODex and CMCS-PBA revealed the relationship between ODex and unmodified dextran (Dextran). 1 H NMR spectrum as shown Figure 1 As shown in Figure a, the characteristic peak of aldehyde protons at 9.5 ppm of ODex can be observed, verifying that dextran is oxidized to form oxidized dextran (ODex); CMCS-PBA and unmodified carboxymethyl chitosan (CMCS) 1 H NMR spectrum as shown Figure 1 As shown in Figure b, the characteristic aromatic ring proton peak of phenylboronic acid appears in the 7.5-8.2 ppm region, indicating that 4-carboxyphenylboronic acid has been successfully grafted onto the carboxymethyl chitosan molecular chain; the appearance of ODex and CMCS-PBA in solution is shown in the following photographs. Figure 1 As shown in Figure c, all of them are clear and transparent solutions, indicating that they have good water solubility and dispersibility, which is beneficial for subsequent uniform mixing and gel formation.

[0028] Example 2, the preparation method of polydopamine-coated calcium peroxide (PDA@CPO) nanoparticles is as follows:

[0029] 4 g of polyvinylpyrrolidone (PVP) was dissolved in 30 mL of anhydrous ethanol, 2 g of CaCl2 was added, after complete dissolution, 10 mL of 0.8 M ammonia water was added dropwise and stirred for 10 min, then 4 mL of 1 M H2O2 was added dropwise at a speed of 100 μL / min, and reacted for 6 h in the dark, the obtained precipitate was collected by centrifugation at 15000 rpm and washed twice with anhydrous ethanol to obtain calcium peroxide (CPO) nanoparticles;

[0030] The CPO nanoparticles were resuspended in 100 mL of anhydrous ethanol (concentration 1 mg / mL), 1 mL of dopamine hydrochloride (150 mg / mL) was added and the pH was adjusted to 8.5 with 1 M NaOH, and after 12 h of reaction in the dark, centrifugal purification and drying were performed to obtain PDA@CPO.

[0031] The PDA@CPO was analyzed, and the results are shown in Figure 2 The actual images of CPO and PDA@CPO nanoparticles are shown in Figure 2 The left side of the figure is the CPO nanoparticles without polydopamine coating, which appears as a white powder, and the right side is PDA@CPO after polydopamine coating, which appears as a typical dark black, indicating that the polydopamine coating is successful; the transmission electron microscope (TEM) image is shown in Figure 2 The left image shows that the surface of the uncoated CPO nanoparticles is relatively smooth, and the right image shows that the surface of PDA@CPO is coated with a layer of obvious polydopamine film, and the edge profile is blunt, further confirming the success of the surface modification; the X-ray diffraction (XRD) patterns of CPO and PDA@CPO are shown in Figure 2 Both of them show the characteristic diffraction peaks of CPO, while the peak intensity of PDA coating is slightly weakened and the peak width is increased, reflecting the influence of the coating layer on the crystal structure signal; the Zeta potential measurement results are shown in Figure 2 The surface of CPO particles is positively charged, while after coating PDA, the surface potential of PDA@CPO particles turns to negative, further indicating the success of the surface polydopamine modification, which improves the dispersion stability in the hydrogel.

[0032] Example 3, a kind of ODex / CMCS-PBA hydrogel (Gel) without loaded nanoparticles, the preparation method comprises the following steps:

[0033] The mass concentration of ODex and CMCS-PBA prepared in Example 1 was adjusted to 5% w / v and 2% w / v respectively, and mixed according to the volume ratio of 1:1 to obtain Gel;

[0034] The preparation method of the Gel incorporating dextranase is as follows: a dextranase mother liquor with a concentration of 1000 U is configured, different volumes of the mother liquor are incorporated into a 5% w / v ODex solution in advance, vortexed and mixed uniformly, and then the ODex and CMCS-PBA solutions are mixed to form a gel, so that the final concentration of dextranase in the hydrogel is 10 U, 20 U, 50 U or 100 U.

[0035] The Gel is analyzed, and the photos of the inclined state of the Gel are as shown in Figure 3 a, showing its good gel-forming ability and formability; the frequency scanning diagram of the Gel is as shown in Figure 3 b, the storage modulus (G') is greater than the loss modulus (G''), indicating that it has a typical elastic network structure; the strain-recovery test results of the Gel are as shown in Figure 3 c, indicating that it exhibits good self-recovery performance in multiple shear-release cycles; the glucose production performance of the Gel incorporating different concentrations of dextranase (10 U, 20 U, 50 U, 100 U) is detected by a glucose detection kit, and the glucose concentration released by the Gel incorporating different concentrations of dextranase within 3 days and 7 days is as shown in Figure 3 d.

[0036] Example 4, an oxygen-producing hydrogel (a composite hydrogel incorporating 0.5% PDA@CPO nanoparticles), the preparation method thereof comprises the following steps:

[0037] The mass concentrations of the ODex and CMCS-PBA prepared in Example 1 are adjusted to 5% w / v and 2% w / v respectively, CPO and PDA@CPO nanoparticles prepared in Example 2 are incorporated into the ODex solution in advance respectively, and then mixed uniformly with the CMCS-PBA to form a gel, so that the final concentration of the two kinds of nanoparticles is 0.5% w / v, and the oxygen-producing hydrogels incorporating CPO and PDA@CPO are obtained respectively.

[0038] The oxygen-producing hydrogel is analyzed, and the photos of the composite hydrogel incorporating 0.5% PDA@CPO nanoparticles in an inclined state are as shown in Figure 4 a, showing obvious color change, proving that it is successfully loaded and gelled; the frequency scanning diagram thereof is as shown in Figure 4 b, showing a higher G' value, indicating that the nanoparticles improve the mechanical properties of the hydrogel; the dissolved oxygen concentration of different system hydrogels is measured every two days within 14 days, and the column chart is as shown in Figure 4 c, indicating that the 0.5% PDA@CPO group has more stable oxygen production capacity; the comparison of the cumulative oxygen production of the 0.5% PDA@CPO and 0.5% CPO groups within 14 days is as shown in Figure 4 d, further illustrating that PDA coating can effectively improve the sustainability of oxygen release.

[0039] Example 5, evaluation of the influence of different hydrogel systems on the cell activity of dental pulp stem cells (DPSCs):

[0040] DPSCs were derived from the dental pulp tissue of the third molar or orthodontic premolar of a healthy young donor. After sterile separation, mesenchymal stem cells in the dental pulp tissue were obtained by combined enzyme digestion with collagenase I and trypsin. The obtained cells were inoculated in α-MEM medium containing fetal bovine serum and cultured for expansion under the condition of 37°C and 5% CO2. When the passage number reached 3-5, the cells were used for subsequent experiments.

[0041] The different hydrogels prepared in Examples 3 and 4 were respectively co-cultured with DPSCs for 1, 3 and 7 days. Cell activity detection was performed by cell counting kit (CCK-8). The influence of different treatment groups (Ctrl, Gel, 100 U dextranase doped group, 0.5% PDA@CPO doped group) on the activity of DPSCs at 1 day, 3 days and 7 days was analyzed. The experimental results are shown in Figure 5 As can be seen, each group of hydrogels did not significantly inhibit cell proliferation, indicating that the materials have good biocompatibility and non-cytotoxicity characteristics.

[0042] Example 6, evaluation of the support ability of different hydrogel systems on the cell activity of dental pulp stem cells (DPSCs):

[0043] DPSCs were derived from the dental pulp tissue of the third molar or orthodontic premolar of a healthy young donor. After sterile separation, mesenchymal stem cells in the dental pulp tissue were obtained by combined enzyme digestion with collagenase I and trypsin. The obtained cells were inoculated in α-MEM medium containing 10% fetal bovine serum and cultured for expansion under the condition of 37°C and 5% CO2. When the passage number reached 3-5, the cells were used for subsequent experiments. The survival of cells in different hydrogels was compared under different conditions by live / dead cell staining experiment.

[0044] DPSCs were encapsulated in hydrogels containing 0 (Control), 10 U, 50 U and 100 U dextranase, respectively, and placed in α-MEM medium without glucose. Live / dead staining was observed at 3 days and 7 days. The results are shown in Figure 6 As shown in a, the images show that the number of green living cells in the hydrogel gradually increases with the increase of enzyme concentration, indicating that the increase of enzyme concentration helps to maintain the cell activity of the hydrogel. The corresponding statistical bar chart is shown in Figure 6As shown in FIG. 3B, the proportions of living cells in each group on the 3rd day were 58%, 67%, 75%, and 94%, respectively; by the 7th day, the proportions of living cells in each group were 40%, 56%, 65%, and 71%, respectively, and the cell survival rate of the high-concentration enzyme treatment group was significantly higher than that of the control group. The proportions of living cells in the 100 U group on the 3rd day and the 7th day were 94% and 71%, respectively, which were significantly better than those of other concentrations;

[0045] The DPSCs were embedded in the ordinary Gel and the oxygen-producing hydrogel containing 0.5% PDA@CPO, respectively, and placed in a hypoxic environment (0.1% O2) provided by a Mitsubishi MGC sealed culture tank (2.5 L) and an anaerobic gas bag. Live / dead cell staining was performed on the 3rd day and the 7th day, and the results are shown in FIG. 3C. Figure 6 As shown in FIG. 3C, the effects of Gel and oxygen-producing hydrogel containing 0.5% PDA@CPO on cell viability are shown. Green represents living cells, and red represents dead cells. Compared with the Gel group, the PDA@CPO group showed a higher proportion of living cells on the 3rd day and the 7th day. The corresponding bar chart statistical results are shown in FIG. 3D. Figure 6 As shown in FIG. 3D, the results show that on the 3rd day, the proportion of living cells in the Gel group was only 28%, while that in the 0.5% PDA@CPO group was as high as 91%. On the 7th day, the proportion of living cells in the Gel group was 15%, while that in the PDA@CPO group remained at 79%. This indicates that the sustained oxygen release provided by PDA@CPO effectively improves the hypoxic microenvironment and significantly improves the cell survival rate, showing excellent cell support capability.

[0046] To further verify the comprehensive performance of the hydrogel, a sugar / oxygen-producing hydrogel (SGO-Gel) was prepared. The preparation process was as follows: the mass concentrations of ODex and CMCS-PBA were adjusted to 5% w / v and 2% w / v, respectively. Dextranase solution and PDA@CPO nanoparticles were added to the ODex solution in advance, and then vortexed to mix. Subsequently, the ODex solution and the CMCS-PBA solution were mixed in equal volumes to form a gel, so that the final concentrations of dextranase and PDA@CPO were 100 U and 0.5%, respectively, i.e., SGO-Gel.

[0047] The DPSCs were encapsulated in the ordinary Gel and the SGO-Gel, respectively, and cultured in a combined sugar-deficient and hypoxic environment. The cell state was observed on the 7th day, and the results are shown in FIG. 3E. Figure 6 As shown in FIG. 3E, 3D imaging shows the performance of the finally constructed sugar / oxygen-producing hydrogel (SGO-Gel) in cell survival. Compared with the Gel group, the red dead cells were significantly reduced. The statistical analysis is shown in FIG. 3F. Figure 6As shown in FIG. 7, the results show that the green living cells in the SGO-Gel group are densely and uniformly distributed, and there are few dead cells. The proportion of living cells in the SGO-Gel group is 95%, and the proportion of dead cells is only 5%. The proportion of living cells in the Gel group is 92%, and the proportion of dead cells is 8%. Although the ordinary Gel also has certain biocompatibility, the SGO-Gel further optimizes the cell survival microenvironment through the synergistic mechanism of "sugar production + oxygen production", and exhibits a more optimal cell survival promotion effect.

[0048] Example 7, analysis of the promotion effect of SGO-Gel hydrogel on the function of angiogenesis-related cells:

[0049] The cell migration under different culture conditions was evaluated by scratch test, and the results are shown in FIG. 8a. Figure 7 The conditioned media under different conditions were collected, i.e., two-dimensional DPSCs cell culture, SGO-Gel hydrogel (without cells), SGO-Gel embedded DPSCs, and human umbilical vein endothelial cells (purchased from the Chinese Academy of Sciences Cell Library (number: EAhy926)) were respectively cultured in the three kinds of conditioned media. The changes in the cell migration area at 0 h and 24 h were compared, and the results showed that the scratch area of the SGO-Gel (DPSCs) group after 24 h was significantly reduced, and the cell migration to the scratch center was more significant. The corresponding relative migration area quantitative statistical results are shown in FIG. 8b. Figure 7 As shown in FIG. 8b, the SGO-Gel (DPSCs) group was significantly higher than the 2D group and the SGO-Gel alone group, indicating that the hydrogel embedded with DPSCs can promote cell migration, which may be helpful for subsequent dental pulp regeneration.

[0050] The angiogenesis ability of DPSCs was evaluated by tube formation experiment. The human umbilical vein endothelial cells were cultured in three kinds of conditioned media prepared by 2D condition, SGO-Gel hydrogel, and SGO-Gel (DPSCs), respectively, and then the formation of blood vessel-like structure was observed. The results are shown in FIG. 9c. Figure 7 As shown in FIG. 9c, there are obvious differences in the number of capillary-like structure tube networks and the complexity of branches formed under different culture conditions. The capillary-like structure formed in the SGO-Gel (DPSCs) group is more abundant, has more branches, and the tubular network is more stable. The corresponding total tube lumen length quantitative statistical results are shown in FIG. 9d. Figure 7 As shown in FIG. 9d, the tube lumen length formed in the SGO-Gel (DPSCs) group is significantly higher than that in the other two groups, indicating that it has a positive effect on promoting angiogenesis and effectively activates the angiogenesis potential of DPSCs.

[0051] Example 8, SGO-Gel hydrogel promotes dental pulp tissue regeneration in vivo experiment:

[0052] SGO-Gel embedding DPSCs, using a heterotopic pulp regeneration model in Balb / c nude mice (Balb / c nude mice (4-6 weeks old, male) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), first prepare dentin segments, the preparation process is: the dentin segments are derived from healthy young donors' premolar teeth removed before orthodontic treatment, and the dentin segments are prepared into several 5 mm long scaffolds using a high-speed turbine, with an inner cavity diameter of 2-4 mm, then the dentin segments are treated with 17% and 5% EDTA solution for 10 min, then washed with deionized water for 3 times, each time for 5 min, and the prepared dentin segments are stored in PBS containing 1% double antibody for standby, the blank control group is not added, the DPSCs cell suspension is added to the dentin segments for the two-dimensional DPSCs culture group, the SGO-Gel embedding DPSCs is injected into the dentin segments for the SGO-Gel group, and the blank control group, the two-dimensional DPSCs culture group and the SGO-Gel group are implanted subcutaneously in nude mice, respectively, and the samples are taken after 12 weeks, VEGF (vascular endothelial growth factor) and DSPP (dentin sialoprotein) are used as markers, and the expression levels of new blood vessels and dentin-like tissues in each group are evaluated by immunohistochemical staining, such as Figure 8 As shown in FIG. 8a, the results show that the SGO-Gel group shows more significant positive signals in the staining of the two markers, the staining area is wider, and the cell staining degree is deeper, indicating that SGO-Gel significantly promotes angiogenesis and dentin-like tissue formation; the staining area is quantified by image analysis software, and the proportion of positive expression area in the total area in different treatment groups is calculated, such as Figure 8 As shown in FIG. 8b, the results show that the VEGF positive area of the SGO-Gel group is about 0.5%, and the DSPP positive area is about 0.9%, which is significantly higher than that of the 2D cell culture group and the control group. In summary, SGO-Gel hydrogel can significantly up-regulate the expression of markers related to blood vessel neogenesis and dentin regeneration, and prove that it has good application potential in promoting pulp tissue regeneration.

[0053] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a dynamically cross-linked hydrogel, characterized in that, Includes the following steps: Crosslinking oxidized dextran with carboxymethyl chitosan containing phenylboronic acid-modified groups; Dextranase was added before hydrogel formation, and polydopamine-coated calcium peroxide nanoparticles were introduced. The concentration of oxidized dextran is 2-10% w / v, the concentration of carboxymethyl chitosan containing phenylboronic acid modified groups is 2% w / v, the concentration of dextranase is 10-100 U, and the concentration of polydopamine-coated calcium peroxide nanoparticles is 0.1-1% w / v.

2. The method for preparing the dynamically cross-linked hydrogel according to claim 1, characterized in that, The volume ratio of the oxidized dextran to carboxymethyl chitosan containing phenylboronic acid-modified groups is 1:

1.

3. The method for preparing the dynamically cross-linked hydrogel according to claim 1, characterized in that, The preparation method of the oxidized dextran includes the following steps: Dextran was dissolved in deionized water and sodium periodate solution was added dropwise. The reaction was carried out at room temperature under light-protected conditions. Then, ethylene glycol was added to quench the reaction. After dialyzing and freeze-drying, oxidized dextran was obtained.

4. The method for preparing the dynamically cross-linked hydrogel according to claim 1, characterized in that, The preparation method of the carboxymethyl chitosan containing phenylboronic acid modified groups includes the following steps: Chitosan was alkalized by soaking it in NaOH solution, then reacted with monochloroacetic acid in isopropanol. The pH of the resulting product was adjusted, and the product was centrifuged, precipitated, and dried to obtain carboxymethyl chitosan. 4-Carboxyphenylboronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N-hydroxysuccinimide and triethylamine were then dissolved in DMSO for activation treatment. The activated mixture was then slowly added to carboxymethyl chitosan dissolved in PBS for reaction. After the reaction was completed, the mixture was dialyzed and lyophilized to obtain carboxymethyl chitosan containing phenylboronic acid modified groups.

5. The method for preparing the dynamically cross-linked hydrogel according to claim 1, characterized in that, The preparation method of the polydopamine-coated calcium peroxide nanoparticles includes the following steps: Polyvinylpyrrolidone was dissolved in anhydrous ethanol, CaCl2 was added, and after complete dissolution, ammonia water was added dropwise while stirring. Then H2O2 was added dropwise, and the reaction was carried out under light-protected conditions. The resulting precipitate was collected by centrifugation and washed to obtain calcium peroxide nanoparticles. Calcium oxide nanoparticles were resuspended in anhydrous ethanol, dopamine hydrochloride was added, and the pH was adjusted with NaOH. After reacting in the dark, the nanoparticles were purified by centrifugation and dried to obtain polydopamine-coated calcium peroxide nanoparticles.

6. A dynamically cross-linked hydrogel, characterized in that, It is prepared using the preparation method described in any one of claims 1-5.

7. The application of the dynamically cross-linked hydrogel as described in claim 6 in the preparation of dental pulp regeneration materials.

8. The application according to claim 7, characterized in that, The dynamically cross-linked hydrogel is used for three-dimensional culture of dental pulp stem cells, continuously providing glucose and oxygen.

9. The application according to claim 7, characterized in that, The dynamically cross-linked hydrogel promotes the secretion of vascular endothelial growth factor by dental pulp stem cells, thereby promoting angiogenesis and pulp regeneration.

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