Iris element-based live marrow preservative as well as preparation method and application thereof

Abstract

The invention discloses an irisin-based live marrow preservative and a preparation method and application thereof, the irisin-based live marrow preservative comprises irisin, cerium-containing mesoporous bioactive glass nanoparticles and hydrogel, the irisin is loaded on the mesoporous structure of the cerium-containing mesoporous bioactive glass nanoparticles, and the hydrogel is loaded on the mesoporous structure of the cerium-containing mesoporous bioactive glass nanoparticles. And hydrogel is filled among the cerium-containing mesoporous bioactive glass nanoparticles loaded with the irisin. The Irisin / Ce-MBGNs live marrow preservative disclosed by the invention is stable in physical and chemical properties, and shows good Irisin slow release and enrichment capabilities. In-vitro cell experiments and animal experiments prove that the Irisin / Ce-MBGNs live pulp preservative can regulate and control polarization of macrophages by reducing overexpressed ROS (reactive oxygen species) in a targeted manner, so that the pulpitis reaction is remarkably relieved, and the Irisin / Ce-MBGNs live pulp preservative has the effect of positively promoting dentin differentiation of dental pulp cells.

Description

Technical Field

[0001] The present invention relates to an irisin-based vital pulp preservative and a preparation method and application thereof, belonging to the technical field of oral biomaterials. Background Art

[0002] At present, the clinical treatment of pulpitis is mainly root canal therapy, but root canal therapy may lead to a complete loss of the immune function of the tooth and a significant decrease in the fracture resistance of the remaining tooth tissue. In recent years, vital pulp preservation therapy has gradually attracted attention as a minimally invasive alternative therapy. Its core goal is to preserve the vitality of the dental pulp through minimal intervention and maintain the physiological sensation and metabolic function of the tooth. This therapy is mainly suitable for the treatment of injured dental pulp caused by caries, trauma, etc., and vital pulp preservation therapy is also widely used in mature permanent teeth with irreversible pulpitis. Among them, the preservatives used in vital pulp preservation therapy have always been an important research direction in this field.

[0003] Calcium hydroxide (CH) and mineral trioxide aggregate (MTA) are classic pulp preservation agents, widely used as the gold standard for clinical pulp preservation. However, they have side effects such as promoting persistent chronic inflammation, pulp necrosis, tooth discoloration, and poor dentin repair. Other pulp preservation agents, such as iRoot BP, although rapidly coagulating, lack significant anti-inflammatory effects. Furthermore, Biodentine, a novel bioceramic material, has a certain chance of developing diffuse pulp cavity calcification when used in pulp preservation treatments, resulting in poor long-term efficacy. Therefore, there is an urgent need to develop new pulp preservation agents to address these issues, thereby maintaining the vitality and function of dental pulp tissue while inhibiting inflammation and promoting the regeneration of hard tissues such as dentin.

[0004] Irisin is a myokine that has been shown to possess excellent biocompatibility, anti-inflammatory, and antioxidant properties. It holds great potential for the treatment of systemic inflammatory diseases, significantly reducing the immune response induced by macrophages in vitro and promoting bone regeneration. However, as an exogenous polypeptide, irisin exhibits poor stability in vivo and is susceptible to proteolysis. Therefore, it is necessary to identify suitable carriers and matrix materials to enhance its stability and enable precise targeting and controlled release to areas of inflammation. Summary of the Invention

[0005] The present invention provides an irisin-based pulp preservation agent, a preparation method, and an application thereof, aiming to solve the problems that traditional pulp preservation agents easily promote persistent chronic inflammation of pulp tissue, pulp necrosis, tooth discoloration, and poor dentin repair ability.

[0006] In order to achieve the above-mentioned objectives, the present invention adopts an irisin-based vital pulp preservation agent, which includes irisin, cerium-containing mesoporous bioactive glass nanoparticles and hydrogel. The irisin is loaded on the mesoporous structure of the cerium-containing mesoporous bioactive glass nanoparticles, and the hydrogel is filled between the cerium-containing mesoporous bioactive glass nanoparticles loaded with irisin.

[0007] As an improvement, the hydrogel is a PNI / CS / GP hydrogel loaded with 5-FU.

[0008] The second aspect of the present invention further provides a method for preparing the irisin-based marrow preservation agent, comprising the following steps:

[0009] (1) Preparation of mesoporous bioactive glass nanoparticles by sol-gel method;

[0010] (2) Ce ions were incorporated into mesoporous bioactive glass nanoparticles to obtain Ce-MBGNs powder;

[0011] (3) Irisin was loaded into the mesoporous structure of Ce-MBGNs to obtain Irisin / Ce-MBGNs powder;

[0012] (4) preparing a hydrogel;

[0013] (5) The Irisin / Ce-MBGNs powder and the hydrogel are mixed and stirred to obtain a vital bone marrow preservation agent.

[0014] As an improvement, in step (1), ammonium bromide is dissolved in deionized water, ethyl acetate is added, and the mixture is stirred at room temperature. Ammonia water is added to adjust the pH value, and stirring is continued. Then, ethyl orthosilicate and calcium nitrate tetrahydrate are added in sequence, and stirring is continued to carry out a sol-gel reaction.

[0015] The precipitate was collected by centrifugation, washed, dried at low temperature and calcined at high temperature to obtain MBGNs powder with a mesoporous structure.

[0016] As an improvement, in step (2), the MBGNs powder is immersed in a cerium nitrate ethanol solution, stirred at room temperature, washed with ethanol, dried, and calcined at high temperature to obtain Ce-MBGNs powder.

[0017] As an improvement, in step (3), the Ce-MBGNs powder is dissolved in PBS, Irisin is added, the mixture is shaken vigorously, vortexed, and incubated at room temperature. After the incubation, the mixture is frozen and lyophilized to obtain Irisin / Ce-MBGNs powder, which is then stored at -20°C.

[0018] As an improvement, in step (4), the hydrogel is a PNI / CS / GP hydrogel loaded with 5-FU, and the preparation steps are as follows:

[0019] NIPAAm and IA were dissolved in THF, stirred under nitrogen atmosphere, AIBN was added, and polymerization reaction was carried out in an oil bath. After cooling to room temperature, the hydrogel precursor was purified and vacuum dried to obtain poly (N-isopropylacrylamide-co-IA), denoted as PNI.

[0020] CS was dissolved in acetic acid at room temperature to prepare a CS solution, and PNI was then added and stirred to obtain a PNI / CS solution. GP solution was prepared by magnetically stirring the PNI / CS solution and the GP solution in an ice-water bath, and the pH was adjusted to 7-8. Finally, the mixture was heated to 37°C to form a PNI / CS / GP hydrogel.

[0021] 5-FU was added to the PNI / CS / GP hydrogel and stirred until completely dissolved to obtain the 5-FU-loaded PNI / CS / GP hydrogel.

[0022] As an improvement, 100 mg of CS was dissolved in 5 ml of 0.1 mol / L acetic acid solution to prepare a CS solution, and then 150 mg of PNI was added and stirred for thorough mixing. After preparing a 40% w / v GP solution in distilled water, the PNI / CS solution and the GP solution were mixed in an ice-water bath at a ratio of 5:1 with continuous magnetic stirring. The 5-FU was added to the PNI / CS / GP hydrogel solution at a concentration of 3% w / w.

[0023] As an improvement, in step (5), the Irisin / Ce-MBGNs powder and the hydrogel are mixed and stirred at 4°C.

[0024] The third aspect of the present invention further provides a use of the irisin-based pulp preservation agent or the irisin-based pulp preservation agent prepared by the preparation method in the preparation of a drug for preventing or treating pulpitis.

[0025] Mechanism of the present invention:

[0026] Irisin is a myokine with good biocompatibility, anti-inflammatory and antioxidant properties, and has good potential in the field of systemic inflammatory disease treatment. As a protein, the stability of exogenous Irisin in the body needs to be improved. In order to improve its stability and achieve its precise positioning and controlled release in the inflammatory area, the present invention adopts cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBGNs) as a drug delivery carrier. The nano-mesoporous structure of Ce-MBGNs has a large specific surface area, which is beneficial as a rigid filler or delivery platform for drugs and bioactive factors, and Ce-MBGNs has good remineralization performance and can promote the osteogenic / odontogenic differentiation of dental pulp cells, but the anti-inflammatory performance is slightly weaker. The present invention loads Irisin into the nano-mesoporous structure of Ce-MBGNs and uses hydrogel as a matrix material to synthesize a new type of vital pulp preservation preparation with a more lasting effect. Irisin slowly released from this novel vital pulp preservative is expected to better compensate for the anti-inflammatory properties of Ce-MBGNs, thereby producing more stable and long-lasting anti-inflammatory and antioxidant effects and contributing to the regeneration of reparative dentin.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) This paper firstly uses cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBGNs) as a carrier of irisin and constructs an irisin-loaded cerium-containing mesoporous bioactive glass nanoparticle drug sustained-release system (Irisin / Ce-MBGNs). This is the first study to use irisin for vital pulp preservation therapy.

[0029] (2) As a protein, exogenous irisin is easily degraded in the body, and its stability needs to be improved. The present invention utilizes the mesoporous structure of Ce-MBGNs nanoparticles as a delivery vehicle for irisin, thereby improving the bioavailability of irisin. Compared with traditional iris preservation agents, the prepared irisin / Ce-MBGNs vital pulp preservation agent has more stable and long-lasting anti-inflammatory and antioxidant effects, and also helps regenerate reparative dentin. It has important clinical significance and provides new ideas for developing new vital pulp preservation materials and improving their performance.

[0030] (3) Under inflammatory conditions, the pulp preservation agent of the present invention reduced the expression levels of proinflammatory factors in RAW264.7 cells and reduced the expression levels of ROS in dental pulp cells (DPCs) and RAW264.7 cells. Furthermore, the pulp preservation agent of the present invention promoted the proliferation of DPCs, increased the expression of mRNA related to dentin regeneration, and increased the activity of ALP, thereby promoting the odontogenic differentiation and mineralization of DPCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Scanning electron micrographs of Ce-MBGNs and Irisin / Ce-MBGNs in Example 1 of the present invention; in the figure, A is Ce-MBGNs, and B is Irisin / Ce-MBGNs (i.e., Ce-MBGNs loaded with Irisin);

[0033] Figure 2 Transmission electron microscopy images of Ce-MBGNs and Irisin / Ce-MBGNs in Example 1 of the present invention; in the figure, A is Ce-MBGNs and B is Irisin / Ce-MBGNs;

[0034] Figure 3 The pH change value and Irisin sustained-release rate of Irisin / Ce-MBGNs prepared in Example 1 of the present invention are shown in FIG. 1 ; A represents Ce-MBGNs and B represents Irisin / Ce-MBGNs;

[0035] Figure 4 Figure 1 shows the ROS-scavenging effect of Irisin / Ce-MBGNs on RAW264.7 cells under inflammatory conditions. Figure A shows a representative fluorescence image of ROS in RAW264.7 cells, and Figure B shows the intracellular ROS level detected by flow cytometry.

[0036] Figure 5 Effects of Irisin / Ce-MBGNs on the mRNA expression of inflammation-related factors in RAW264.7 cells;

[0037] Figure 6Figure 3. Effects of Irisin / Ce-MBGNs on DPCs migration. Figure A shows the migration of DPCs toward the scratch site after 0, 12, and 24 hours of culture, as imaged under an inverted microscope. Figure B shows the quantitative analysis of scratch area changes using Image J.

[0038] Figure 7 Effects of Irisin / Ce-MBGNs on the expression of genes related to osteogenesis / odontogenesis;

[0039] Figure 8 ALP staining results after DPCs were cultured with Irisin / Ce-MBGNs. In the figure, A is the ALP staining result, and B is the quantification of ALP activity.

[0040] Figure 9 Effects of Irisin / Ce-MBGNs on the expression of osteogenesis / odontoblastogenesis-related proteins;

[0041] Figure 10 Figure 1 shows the Micro-CT results of pulpitis model rats treated with Irisin / Ce-MBGNs. Figure A shows the dentin repair of rat first molars detected by Micro-CT at 4 and 8 weeks, with arrows pointing to the pulp foramen. Figure B shows the quantitative analysis of dentin volume at 4 weeks (n=3), and Figure C shows the quantitative analysis of dentin volume at 8 weeks (n=3).

[0042] Figure 11 The figure shows the effect of Irisin / Ce-MBGNs on inflammatory macrophages in the dental pulp of rats with pulpitis model. In the figure, A is the percentage of CD86-positive M1 macrophages detected by IHC staining at 4 weeks, and the scale bar represents 100 μm. B is the semi-quantitative analysis of CD86-positive cells at 4 weeks. DETAILED DESCRIPTION

[0043] In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present application are described in detail below through specific embodiments. It should be understood that the embodiments of the present application and the specific features in the embodiments are detailed descriptions of the technical solutions of the present application, rather than limitations on the technical solutions of the present application. Unless there is a conflict, the embodiments of the present application and the technical features in the embodiments can be combined with each other.

[0044] This document provides general and / or specific descriptions of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods. All reagents and instruments used, unless the manufacturer is indicated, are commercially available, conventional products and were prepared or used using conventional methods. Some of the reagents used in the following examples include: ammonium bromide (99% analytical grade, Merck, Germany), ethyl acetate (99% analytical grade, Merck, Germany), ammonia (28%, Merck, Germany), tetraethyl orthosilicate (98%, Merck, Germany), calcium nitrate tetrahydrate (99%, Merck, Germany), ethanol (96%, Sinopharm, China), and cerium nitrate (99%, Merck, Germany).

[0045] Example 1

[0046] A method for preparing an irisin-based marrow preservation agent comprises the following steps:

[0047] (1) MBGNs were synthesized by the sol-gel method: 2.8 g of ammonium bromide was dissolved in 132 mL of 35°C deionized water, 40 mL of ethyl acetate was added, the temperature was adjusted to 25°C, and the mixture was stirred for 30 min. 28 mL of ammonia water was added and stirred for 15 min. Then, 14.4 mL of ethyl orthosilicate and 9.12 g of calcium nitrate tetrahydrate were added to the mixture in sequence over 30 min and stirred for 4 h. The resulting white suspension was collected by centrifugation and washed twice with deionized water and once with 96% ethanol. The collected sediment was dried at 60°C overnight and then calcined at 700°C for 3 h at a heating rate of 2°C / min to obtain dry MBGNs.

[0048] (2) Synthesis of Ce-MBGNs: 1 g of MBGNs was immersed in 60 mL of cerium nitrate ethanol solution, stirred continuously at room temperature for 24 h, washed twice with ethanol, dried at 60 °C overnight, and finally calcined at 680 °C for 2 h to stabilize the loaded Ce ions;

[0049] (3) Synthesis of Irisin / Ce-MBGNs: 500 mg of Ce-MBGNs powder was dissolved in 50 mL of PBS, and then 100 ng / mL of irisin was added. The mixture was shaken vigorously, vortexed for 20 seconds, and incubated at room temperature for 24 hours. After incubation, the mixture was frozen at -80°C overnight and freeze-dried to powder. The obtained irisin / Ce-MBGNs powder was stored at -20°C.

[0050] (4) Synthesis of hydrogel

[0051] (4.1) Preparation of poly (N-isopropylacrylamide-co-IA) (PNI): 500 mg of NIPAAm and 65 mg of IA were dissolved in 10 ml of THF. The mixture was thoroughly mixed by continuous magnetic stirring under nitrogen for 30 min. AIBN (0.2 mmol dissolved in 2 ml of THF) was added, and polymerization was carried out in an oil bath at 65°C for 12 h. After the mixture was cooled to room temperature, the polymer compound was repeatedly purified by precipitation with excess tert-butylmethyl. Subsequently, the resulting hydrogel precursor was vacuum dried for 12 h.

[0052] (4.2) Synthesis of PNI / CS / GP hydrogel: 100 mg of CS was dissolved in 5 ml of 0.1 mol / L acetic acid solution at room temperature to prepare a CS solution. 150 mg of PNI was then added and stirred for 3 min to mix thoroughly. A 40% w / v GP solution in distilled water was prepared. The PNI / CS solution and the GP solution were then mixed in an ice-water bath at a ratio of 5:1 with continuous magnetic stirring. The pH of the complex was adjusted to approximately 7.4. The composite was then heated to 37°C until a hydrogel was formed.

[0053] 5-FU (5-fluorouracil) was added to the PNI / CS / GP hydrogel solution at a concentration of 3% w / w and stirred continuously until completely dissolved to prepare a 5-FU-loaded PNI / CS / GP hydrogel.

[0054] Here, PNI is polyN-isopropylacrylamide-co-IA; the medium molecular weight CS (Chitosan) is 190 kDa; GP (glycerophosphate); tetrahydrofuran (THF); NIPAAm (N-isopropylacrylamide, 99% purity); azobisisobutyronitrile (AIBN); IA (Itaconic acid, 99% purity) and type I collagen were purchased from Aladdin Ltd. (Shanghai, China).

[0055] (5) Synthesis of the Pulmonary Marrow Preservative: Irisin / Ce-MBGNs powder was mixed with 5-FU-loaded PNI / CS / GP hydrogel at 4°C to prepare the Pulmonary Marrow Preservative. In this Pulmonary Marrow Preservative, Irisin / Ce-MBGNs is the core active ingredient, and the hydrogel serves only as an inert excipient. Therefore, when evaluating the pharmacological effects of this Pulmonary Marrow Preservative, its performance evaluation focused on the effects of Irisin / Ce-MBGNs.

[0056] The morphology of Ce-MBGNs and Irisin / Ce-MBGNs powder particles in Example 1 was tested by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM results showed that Ce-MBGNs were typical elliptical particles with clear nanopores visible under the microscope at 200 nm. Figure 1 A). In contrast, Irisin / Ce-MBGNs particles are more round, with a rougher surface and more protrusions. The pores inside the nanoparticles are not clear under a 200nm microscope, which confirms that Irisin has been successfully loaded ( Figure 1 B). In addition, TEM results ( Figure 2 ) showed that compared with Ce-MBGNs with uneven pores, Irisin / Ce-MBGNs had a more solid texture, a cloudy center, and clearer boundaries.

[0057] The zeta potential of Ce-MBGNs and Irisin / Ce-MBGNs was measured. The results, shown in Table 1, show that both Ce-MBGNs and Irisin / Ce-MBGNs possess negative surface charges. The absolute value of the zeta potential between the nanoparticles of Irisin / Ce-MBGNs increased, as did the electrostatic repulsion between the particles, indicating that the physical properties of Irisin / Ce-MBGNs are more stable than those of Ce-MBGNs alone. Irisin loading slightly increased the PDI of the nanoparticles, indicating that the addition of irisin slightly affected their particle size distribution.

[0058] Table 1 Zeta potential and PDI of Ce-MBGNs and Irisin / Ce-MBGNs

[0059]

[0060]

[0061] To investigate the pH changes of a pulp preservation agent and evaluate the release rate of irisin from the agent, the agent was immersed in SBF and shaken at 120 rpm on a 37°C incubator to obtain an extract. The active ingredient in the extract was essentially the extract of irisin / Ce-MBGNs particles; the hydrogel carrier, as an inert excipient, did not contribute substantially to the active ingredients in the extract. pH was measured using a pH meter on days 1, 3, 5, 7, 11, and 14, and the pH change over time was plotted. The extracts were collected on days 1, 3, 5, 7, 11, and 14, and the cumulative release of irisin was quantified using an ELISA kit. This was repeated three times.

[0062] pH value change curves at different temperatures Figure 3A), the results showed that the pH value of Irisin / Ce-MBGNs was stable at 4°C and well preserved. At 37°C, its pH value reached a maximum on the third day and then gradually decreased, and stabilized after the 11th day. In general, the pH value fluctuation of Irisin / Ce-MBGNs of the present invention was smaller than that of Ce-MBGNs. The release curve of Irisin ( Figure 3 B) shows that irisin from Irisin / Ce-MBGN is slowly and continuously released at a relatively stable rate. This result indicates that Irisin / Ce-MBGN is a sustained-release system that can function stably and effectively.

[0063] Given the similarity between the biological effects of the MMP and the pharmacological properties of Irisin / Ce-MBGNs, to ensure clarity and accuracy, subsequent descriptions of the MMP performance will focus directly on Irisin / Ce-MBGNs. This approach avoids semantic ambiguity caused by excipient interference and more accurately presents the core pharmacological mechanisms and functional properties of this invention.

[0064] Example 2

[0065] The ROS scavenging effect of Irisin / Ce-MBGNs prepared in Example 1 on RAW264.7 cells under inflammatory conditions was specifically as follows: H2DCFDA was used to measure the reactive oxygen species level in each group to evaluate the ROS scavenging ability of Irisin / Ce-MBGNs.

[0066] RAW264.7 and DPCs were plated and induced with LPS for 24 hours. The cells were then co-cultured with irisin, calcium hydroxide (CH), Ce-MBGNs, and irisin / Ce-MBGNs for another 24 hours (referred to as the irisin, CH, Ce-MBGNs, and irisin / Ce-MBGNs groups, respectively). A positive control group, cultured in standard culture medium after LPS induction, was designated the LPS group; a negative control group, untreated with LPS, was designated the CON group. H2DCFDA diluted in serum-free culture medium was then added and incubated at 37°C in the dark for 30 minutes. To further confirm the ROS-scavenging ability of irisin / Ce-MBGNs, the cells were resuspended in PBS, and ROS levels in each group were quantified by flow cytometry, with data analyzed using FlowJo software.

[0067] The results are as follows Figure 4As shown in A: Irisin / Ce-MBGNs significantly reduced the fluorescence intensity of RAW264.7 cells under inflammatory conditions. This shows that it can effectively eliminate intracellular oxygen free radicals, endow cells with resistance to endogenous oxidative stress, and is more effective than using Ce-MBGNs alone. This shows that on the basis of Ce-MBGNs, Irisin / Ce-MBGNs can further protect RAW264.7 cells from self-generated oxidative damage in an inflammatory environment. In contrast, the fluorescence intensity of the CH group was similar to that of the LPS-stimulated inflammatory group, indicating that its ability to scavenge ROS was not obvious. At the same time, the ability of Ce-MBGNs to scavenge ROS was quantitatively evaluated by flow cytometry. As shown in Figure 4 B further confirmed that Irisin / Ce-MBGNs have excellent ROS scavenging ability.

[0068] Example 3

[0069] The effects of Irisin / Ce-MBGNs prepared in Example 1 on the mRNA expression of inflammation-related factors in RAW264.7 cells are as follows:

[0070] RAW264.7 cells were evenly seeded into 6-well plates and stimulated with LPS for 24 hours. Cells were then co-cultured with Ce-MBGNs, irisin / Ce-MBGNs, and CH. After 24 hours, total RNA was extracted and purified using an RNA extraction kit, and the expression levels of inflammation-related genes were analyzed by RT-qPCR.

[0071] RT-qPCR results showed that the Irisin / Ce-MBGNs group The expression of related factors (IL-1β, iNOS, IL-6, TNF-α) was significantly reduced ( Figure 5 A~ Figure 5 E) Compared with CH and Ce-MBGNs, the Irisin and Irisin / Ce-MBGNs stimulation groups produced The expression of related factors is relatively high ( Figure 5 F~ Figure 5 I), indicating that Irisin / Ce-MBGNs also have the ability to regulate macrophage phenotype. Irisin / Ce-MBGNs has a stronger inhibitory effect on the secretion of chemokine CCL5 than Ce-MBGNs and Irisin ( Figure 5 J), indicating that Irisin / Ce-MBGNs have the ability to inhibit the infiltration and metastasis of inflammatory cells.

[0072] Example 4

[0073] The effects of Irisin / Ce-MBGNs prepared in Example 1 on DPCs cell migration are as follows:

[0074] The effect of irisin / Ce-MBGNs on the migration of DPCs was evaluated using a scratch wound assay. First, DPCs were evenly seeded in a 6-well plate. When the cell density reached 80%, cells were scraped off using a sterile, disposable 10 μL pipette tip, creating two to three horizontal linear scratches. After rinsing with PBS until no suspended cells were evident, serum-free medium containing irisin, CH, Ce-MBGNs, and irisin / Ce-MBGNs was added and incubated. Images were captured at 4× magnification using an inverted microscope after 0, 12, and 24 hours of incubation. Migration rates were calculated using Image J software.

[0075] The scratch test results are as follows Figure 6 As shown in the figure, compared with the blank control group and Ce-MBGNs group, the scratch area of ​​Irisin / Ce-MBGNs was reduced after 12 hours and 24 hours, and the migration rate of DPCs was significantly accelerated.

[0076] Example 5

[0077] The effects of Irisin / Ce-MBGNs prepared in Example 1 on the expression of genes related to osteogenesis / odontogenesis are as follows:

[0078] DPCs were co-cultured with each group of materials for 3 days. The expression levels of osteogenic / odontogenic factors in each group of DPCs were compared by RT-qPCR. Compared with Ce-MBGN and Irisin stimulation alone, the mRNA expression levels of odontogenic differentiation markers (DSPP, DMP-1, and BMP-2) in the Irisin / Ce-MBGNs stimulation group on the third day were significantly higher overall. Compared with the CH group, the expression levels of osteogenic differentiation-related factors (ALP, OCN, Osterix, RUNX2, and COL1A1, etc.) were significantly upregulated in the Irisin / Ce-MBGNs group ( Figure 7 ). From the above results, it can be seen that Irisin / Ce-MBGNs have a stable and long-lasting ability to promote dentin regeneration.

[0079] Example 6

[0080] The ALP staining results after culturing DPCs with Irisin / Ce-MBGNs prepared in Example 1 are as follows:

[0081] DPCs were seeded in six-well plates and then replaced with irisin / Ce-MBGNs. After 7 days of osteogenic induction, the medium was aspirated, the cells were washed three times with ultrapure water, and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were then rinsed with ultrapure water and stained with BICP / NBT working solution for 30–60 minutes at room temperature in the dark. Images were taken using a scanner and recorded under an inverted microscope.

[0082] The experimental results showed that Irisin / Ce-MBGNs had a much better mineralization-inducing effect on DPCs compared with the CH group ( Figure 8 Quantitative analysis of ALP activity also showed that the irisin / ce-mbgns group had the strongest ALP activity, surpassing that of the osteogenic induction solution alone and any other treatment groups. Irisin / ce-mbgns can enhance the effects of the osteogenic induction solution.

[0083] Example 7

[0084] The effects of Irisin / Ce-MBGNs prepared in Example 1 on the expression of osteogenesis / odontoblastogenesis-related proteins are as follows:

[0085] After 7 days of co-culture of DPCs with Ce-MBGNs, Irisin / Ce-MBGNs, and CH, total protein was extracted from each group to detect the expression of osteogenic / odontogenic differentiation-related proteins RUNX2, Osterix, and OCN. After washing with PBS at 4°C, cells were added to RIPA lysis buffer supplemented with PMSF (1:100) for 10 minutes and further lysed using an ultrasonic disruptor. Centrifuge at 4°C, 12,000 rpm for 10 minutes, and the supernatant was collected. After adding 5× protein loading buffer (1:4), the mixture was mixed and boiled at 100°C for 5 minutes. After cooling, the cells can be frozen at -80°C.

[0086] Protein was extracted using Thermo Scientific M-PER Mammalian Protein Extraction Reagent, and protein concentration was determined using the BCA protein assay.

[0087] The results showed that at the protein level, Irisin / Ce-MBGNs significantly increased the expression levels of several important osteogenic markers (Runx2, Osterix, and OCN). Figure 9 ).

[0088] Example 8

[0089] The Micro-CT results of the Irisin / Ce-MBGNs prepared in Example 1 after treating pulpitis model rats are as follows:

[0090] Forty SPF male rats were randomly assigned to experimental groups. After anesthesia, the maxillary first molars were drilled intermittently with water cooling until a pink tint appeared. After 4 hours of LPS stimulation, the cavities were cleaned with saline. The perforations were then covered with CH, Ce-MBGNs, and Irisin / Ce-MBGNs, respectively: (a) negative control group (sterile PBS), (b) positive control group (CH), (c) Ce-MBGNs group, and (d) Irisin / Ce-MBGNs. Glass ionomer filling was then used for restoration. At 4 and 8 weeks postoperatively, the rats were sacrificed, and the maxillae were isolated and fixed in vitro with 4% paraformaldehyde. Micro-CT analysis was performed to analyze the pulp status and neodentine in each group.

[0091] result( Figure 10 A) shows that after 4 weeks, the mineralized bridge formed in the Irisin / Ce-MBGNs group was clearer and thicker than that in the other groups, and the bone in the root apex was intact. After 8 weeks, a thin layer of mineralized tissue was observed near the pulp foramen in the Ce-MBGNs group, and ectopic mineralization was observed in the pulp cavity. In addition, the reparative dentin structure in the Irisin / Ce-MBGNs group was clearer and stronger than that at 4 weeks, and no obvious bone destruction was observed in the root apex. Quantitative analysis also confirmed that the Irisin / Ce-MBGNs group formed more reparative dentin in the pulp cavity than the Ce-MBGNs group ( Figure 10 BC).

[0092] Example 9

[0093] Effects of Irisin / Ce-MBGNs prepared in Example 1 on inflammatory macrophages in the dental pulp of rats with pulpitis model:

[0094] After the above experimental rats were collected, the maxillary teeth and bones were fixed with 4% paraformaldehyde, decalcified, embedded, and sliced. Immunohistochemistry was performed using anti-CD86 antibody to evaluate macrophage infiltration. Figure 11 ), pulp necrosis occurred in the PBS and CH groups, and CD86 was positive In contrast, the Irisin / Ce-MBGNs group had a significantly increased number of CD86 positive cells. The area occupied is significantly reduced.

[0095] In summary, Irisin / Ce-MBGNs can influence macrophage polarization and suppress intracellular ROS production, thereby alleviating the dental pulp immune inflammatory response and promoting the odontoblastic differentiation of DPCs. An in vitro rat pulpitis model further validated the anti-inflammatory and dentin regenerative properties of Irisin / Ce-MBGNs. This invention provides a theoretical basis for the development of novel drugs for vital pulp preservation treatments, focusing on immunomodulation and dentin regeneration.

[0096] Example 10

[0097] A method for preparing an irisin-based marrow preservation agent comprises the following steps:

[0098] (1) MBGNs were synthesized by the sol-gel method: 3.5 g of ammonium bromide was dissolved in 132 mL of 35°C deionized water, 60 mL of ethyl acetate was added, the temperature was adjusted to 25°C, and the mixture was stirred for 30 min. 30 mL of ammonia water was added and stirred for 15 min. Then, 15.6 mL of ethyl orthosilicate and 10.24 g of calcium nitrate tetrahydrate were added to the mixture in sequence over 30 min and stirred for 4 h. The resulting white suspension was collected by centrifugation and washed twice with deionized water and once with 96% ethanol. The collected sediment was dried at 60°C overnight and then calcined at 700°C for 3 h at a heating rate of 2°C / min to obtain dry MBGNs.

[0099] (2) Synthesis of Ce-MBGNs: 5 g of MBGNs were immersed in 60 mL of cerium nitrate ethanol solution, stirred continuously at room temperature for 24 h, washed twice with ethanol, dried at 60 °C overnight, and finally calcined at 680 °C for 2 h to stabilize the loaded Ce ions;

[0100] (3) Synthesis of Irisin / Ce-MBGNs: 700 mg of Ce-MBGNs powder was dissolved in 50 mL of PBS, and then 200 ng / mL of irisin was added. The mixture was shaken vigorously, vortexed for 20 seconds, and incubated at room temperature for 24 hours. After incubation, the mixture was frozen at -80°C overnight and freeze-dried to powder. The obtained irisin / Ce-MBGNs powder was stored at -20°C.

[0101] (4) Synthesis of methacryloylated gelatin (GelMA)

[0102] Type a gel (porcine skin type a gel (300 bloom)) 10% w / v was dissolved in phosphate-buffered saline (PBS) and stirred continuously on a hot plate at 50°C; then, 8 ml of methacrylate was slowly added; after 2 hours, 8 ml of PBS was added to the solution to interrupt the methacrylate reaction; then, a dialysis membrane (12-14 kDa) was filled with the solution and soaked in deionized water (DI) at 45°C for one week, with the water changed twice a day; after the dialysis process, the solution was filtered, placed in a 50 ml falcon tube, frozen at -80°C overnight, and lyophilized for 7 days. The final GelMA foam was stored at -20°C; 15% GelMA solution was prepared using 450 mg of GelMA foam and 3 ml of PBS; the two components were mixed and placed on a hot plate at a temperature of 50±5°C and a rotation speed of 300 rpm;

[0103] (5) 10% of Irisin / Ce-MBGNs powder was then added to the GelMA solution and mixed under the same conditions. After the Irisin / Ce-MBGNs powder was appropriately dispersed in the GelMA, a silicon mold (6 × 2 mm) was filled with the solution and cross-linked using light curing (Bluephase Style, Ivoclar, Amherst, NY, USA) to obtain the Irisin / Ce-MBGNs vital bone marrow preservative.

[0104] Example 11

[0105] A method for preparing an irisin-based marrow preservation agent comprises the following steps:

[0106] (1) MBGNs were synthesized by the sol-gel method: 4.2 g of ammonium bromide was dissolved in 132 mL of 35°C deionized water, 80 / mL of ethyl acetate was added, the temperature was adjusted to 25°C, and the mixture was stirred for 30 min. 32 mL of ammonia water was added and stirred for 15 min. Then, 16.7 mL of ethyl orthosilicate and 11.5 g of calcium nitrate tetrahydrate were added to the mixture in sequence over 30 min and stirred for 4 h. The resulting white suspension was collected by centrifugation and washed twice with deionized water and once with 96% ethanol. The collected sediment was dried at 60°C overnight and then calcined at 700°C for 3 h at a heating rate of 2°C / min to obtain dry MBGNs.

[0107] (2) Synthesis of Ce-MBGNs: 9 g of MBGNs were immersed in 60 mL of cerium nitrate ethanol solution, stirred continuously at room temperature for 24 h, washed twice with ethanol, dried at 60 °C overnight, and finally calcined at 680 °C for 2 h to stabilize the loaded Ce ions.

[0108] (3) Synthesis of Irisin / Ce-MBGNs: 900 mg of Ce-MBGNs powder was dissolved in 50 mL of PBS, and then 400 ng / mL of irisin was added. The mixture was shaken vigorously, vortexed for 20 seconds, and incubated at room temperature for 24 hours. After incubation, the mixture was frozen at −80°C overnight and freeze-dried to a powder. The obtained irisin / Ce-MBGNs powder was stored at −20°C.

[0109] (4) Synthesis of glycopeptide hydrogel

[0110] (4.1) Synthesis of oxidized dextran (OD): Dextran (MW 70,000) was oxidized with NaIO4 in the dark for 3 h, and impurities were removed using dialysis tubing (MW = 3500). OD was then freeze-dried to obtain the product.

[0111] (4.2) Preparation of OD-PLL hydrogel: Polylysine (PLL) solution (0.32 g / mL, 1 mL) was added to 80 mL of 0.06 g / mL OD solution, and then adjusted to neutrality by adding NaOH solution. The mixture was reacted at 50°C for 10 min to obtain OD-PL hydrogel. Dextran (MW 70,000 Da) and polylysine (PLL) were purchased from Shanghai McLean Biochemical Co., Ltd.

[0112] (5) Irisin / Ce-MBGNs powder and OD-PLL hydrogel were mixed and stirred at 4°C to prepare Irisin / Ce-MBGNs vital pulp preservation agent.

[0113] Example 12

[0114] The irisin-based pulp preservation agent prepared by the present invention can be used to prepare drugs for preventing or treating pulpitis, for example, for direct pulp capping treatment and indirect pulp capping treatment after the removal of decayed teeth, as well as for pulpotomy treatment of mature permanent teeth and young permanent teeth diagnosed with reversible pulpitis or even with symptoms of irreversible pulpitis.

[0115] The pulp preservation agent of the present invention has stable physicochemical properties and exhibits excellent irisin sustained-release and enrichment capabilities. In vitro cell and animal experiments have confirmed that the Irisin / Ce-MBGNs pulp preservation agent can significantly alleviate dental pulp inflammatory responses by targeted reduction of overexpressed ROS and regulation of macrophage polarization, and has the effect of positively promoting the odontoblast differentiation of dental pulp cells. Compared with traditional pulp preservation agents, the present invention overcomes the shortcomings of traditional pulp preservation agents, such as unstable anti-inflammatory properties and poor dentin repair ability. It provides new ideas and research foundations for the development of new pulp preservation preparations and further improves the efficacy of traditional pulp preservation agents, and has great clinical application prospects.

[0116] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A marrow preservation agent based on irisin, characterized in that: The invention comprises irisin, cerium-containing mesoporous bioactive glass nanoparticles and hydrogel. The irisin is loaded on the mesoporous structure of the cerium-containing mesoporous bioactive glass nanoparticles, and the hydrogel is filled between the cerium-containing mesoporous bioactive glass nanoparticles loaded with irisin.

2. The irisin-based marrow preservation agent according to claim 1, characterized in that: The hydrogel is a PNI / CS / GP hydrogel loaded with 5-FU.

3. A method for preparing the irisin-based marrow preservation agent according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Preparation of mesoporous bioactive glass nanoparticles by sol-gel method; (2) Ce ions were incorporated into mesoporous bioactive glass nanoparticles to obtain Ce-MBGNs powder; (3) Irisin was loaded into the mesoporous structure of Ce-MBGNs to obtain Irisin / Ce-MBGNs powder; (4) preparing a hydrogel; (5) The Irisin / Ce-MBGNs powder and the hydrogel are mixed and stirred to obtain a vital bone marrow preservation agent.

4. The method for preparing an irisin-based marrow preservation agent according to claim 3, characterized in that: In the step (1), ammonium bromide is dissolved in deionized water, ethyl acetate is added, and the mixture is stirred at room temperature. Ammonia water is added to adjust the pH value, and stirring is continued. Then, ethyl orthosilicate and calcium nitrate tetrahydrate are added in sequence, and stirring is continued to perform a sol-gel reaction. The precipitate was collected by centrifugation, washed, dried at low temperature and calcined at high temperature to obtain MBGNs powder with a mesoporous structure.

5. The method for preparing an irisin-based marrow preservation agent according to claim 3, characterized in that: In the step (2), the MBGNs powder is immersed in a cerium nitrate ethanol solution, stirred at room temperature, washed with ethanol, dried, and calcined at high temperature to obtain Ce-MBGNs powder.

6. The method for preparing an irisin-based marrow preservation agent according to claim 3, characterized in that: In the step (3), Ce-MBGNs powder is dissolved in PBS, Irisin is added, the mixture is shaken vigorously, vortexed, and incubated at room temperature. After the incubation, the mixture is frozen and lyophilized to obtain Irisin / Ce-MBGNs powder, which is stored at -20°C.

7. The method for preparing an irisin-based marrow preservation agent according to claim 3, characterized in that: In step (4), the hydrogel is a 5-FU-loaded PNI / CS / GP hydrogel, and the preparation steps are as follows: NIPAAm and IA were dissolved in THF, stirred under nitrogen atmosphere, AIBN was added, and polymerization reaction was carried out in an oil bath. After cooling to room temperature, the hydrogel precursor was purified and vacuum dried to obtain poly (N-isopropylacrylamide-co-IA), denoted as PNI. At room temperature, CS was dissolved in acetic acid solution to prepare CS solution, and then PNI was added and stirred to obtain PNI / CS solution; Prepare GP solution by magnetically stirring the PNI / CS solution and GP solution in an ice-water bath and adjust the pH to 7-8. Finally, heat the mixture to 37°C to form a PNI / CS / GP hydrogel. 5-FU was added to the PNI / CS / GP hydrogel and stirred until completely dissolved to obtain the 5-FU-loaded PNI / CS / GP hydrogel.

8. The method for preparing an irisin-based marrow preservation agent according to claim 7, characterized in that: 100 mg of CS was dissolved in 5 ml of 0.1 mol / L acetic acid solution to prepare CS solution, and 150 mg of PNI was added and stirred to mix thoroughly; After preparing a 40% GP w / v solution in distilled water, the PNI / CS solution was mixed with the GP solution in an ice-water bath at a ratio of 5:1 with continuous magnetic stirring; The 5-FU was added to the PNI / CS / GP hydrogel solution at a concentration of 3% w / w.

9. The method for preparing an irisin-based marrow preservation agent according to claim 3, characterized in that: In the step (5), the Irisin / Ce-MBGNs powder and the hydrogel are mixed and stirred at 4°C.

10. Use of the irisin-based pulp preservation agent according to any one of claims 1 to 2 or the irisin-based pulp preservation agent prepared by the preparation method according to any one of claims 3 to 9 in the preparation of a drug for preventing or treating pulpitis.

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