A mesoporous bioactive glass hybrid material with active oxygen scavenging, immune regulation and bone regeneration functions, and its preparation method and application

Abstract

The present invention provides a method for preparing a mesoporous bioactive glass hybrid material, comprising the steps of: (1) performing an amination treatment on the mesoporous bioactive glass using a silane coupling agent to prepare an amino-functionalized mesoporous bioactive glass; (2) modifying a polymer on the amino-functionalized mesoporous bioactive glass by an amidation reaction to prepare a polymer-modified mesoporous bioactive glass; the polymer is a carboxyl-rich macromolecule; (3) modifying L-arginine on the polymer-modified mesoporous bioactive glass by an amidation reaction to obtain the mesoporous bioactive glass hybrid material. The mesoporous bioactive glass hybrid material prepared by modifying the surface of the mesoporous bioactive glass with L-arginine by reaction design can be used to remove excess ROS in a bone defect area, regulate the bone immune microenvironment, and promote bone tissue regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a bone tissue repair material, and more particularly to a mesoporous bioactive glass hybrid material with the functions of removing active oxygen, immunomodulation and bone regeneration, and a preparation method and application thereof. Background Art

[0002] Bone defects and nonunions caused by trauma, bone diseases, tumors, and congenital diseases are common problems in clinical practice. In the early stages of bone tissue healing and reconstruction, the immune system will be activated and recruit a large number of immune cells to the defect area, causing the rapid release of a large number of cytokines and inflammatory mediators such as tumor necrosis factor (TNF-a), interleukin-1 (IL-1), and interleukin-6 (IL-6), leading to increased tissue oxygen consumption and respiratory burst, and generating a large amount of reactive oxygen species (ROS) in a short period of time to effectively remove damaged cells and tissues. However, in many pathological conditions, local inflammation cannot subside in time, resulting in excessive local ROS levels, which will directly or indirectly damage normal tissues and hinder the regeneration of bone tissue. Excessive ROS can not only act as signal molecules to upregulate multiple inflammatory-related signaling pathways and aggravate inflammation in the defect area; at the same time, ROS can directly damage normal cells through various pathways, even leading to the loss of cell stemness, affecting the osteogenic differentiation of cells, and preventing bone tissue regeneration. Therefore, removing excessive ROS, regulating inflammation, and building a good regenerative microenvironment is an important strategy to promote bone repair.

[0003] Mesoporous bioactive glass (MBG) is a porous material mainly composed of silicon, calcium, and phosphorus oxides. It has good biocompatibility, degradability, osteoconductivity, and osteoinductivity, and is a commonly used material for bone tissue engineering. However, a single mesoporous bioactive glass has a single function and cannot meet the needs of bone regeneration in terms of regulating ROS levels in bone defect areas and regulating inflammation, which will greatly limit the application of mesoporous bioactive glass in the field of bone regeneration.

[0004] L-arginine (L-Arg) is a natural amino acid that can be used as a nitric oxide donor with good biocompatibility. Studies have shown that L-arginine can consume ROS and release nitric oxide and citrulline that are beneficial to tissue regeneration. It is still a challenge to combine mesoporous bioactive glass with L-arginine by a simple method to prepare multifunctional mesoporous bioactive glass hybrid materials, which is of great significance to promote the application of mesoporous bioactive glass in the biomedical field.

[0005] Based on the above reasons, this application is hereby filed. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a mesoporous bioactive glass hybrid material with the functions of scavenging reactive oxygen species, immunomodulation and bone regeneration, and a preparation method and application thereof. By modifying L-arginine to the surface of the mesoporous bioactive glass through reaction design, a mesoporous bioactive glass hybrid material with the functions of scavenging reactive oxygen species, immunomodulation and bone regeneration can be obtained, and the hybrid material can be used to remove excess ROS in the bone defect area, regulate the bone immune microenvironment, and promote bone tissue regeneration.

[0007] The present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a mesoporous bioactive glass hybrid material having the functions of removing active oxygen, immunomodulation and bone regeneration, comprising the following steps:

[0009] (1) using a silane coupling agent to perform amino treatment on the mesoporous bioactive glass to prepare amino-functionalized mesoporous bioactive glass;

[0010] (2) modifying a polymer on an amino-functionalized mesoporous bioactive glass by an amidation reaction to prepare a polymer-modified mesoporous bioactive glass; the polymer is a carboxyl-rich macromolecule;

[0011] (3) L-arginine is modified on the polymer-modified mesoporous bioactive glass by an amidation reaction to obtain the mesoporous bioactive glass hybrid material.

[0012] The present invention uses mesoporous bioactive glass as a matrix, and a silane coupling agent is dehydrated and condensed on the surface of the matrix to provide amino groups on the surface of the mesoporous bioactive glass. Then, the amino groups on the surface and the carboxyl groups on the polymer can be used to modify the polymer to the surface of the mesoporous bioactive glass through an amidation reaction, providing a large number of reaction sites for the introduction of L-arginine. The polymer acts as a connecting bridge and undergoes an amidation reaction with L-arginine again, so that L-arginine is grafted onto the molecular chain of the polymer, thereby modifying the surface of the mesoporous bioactive glass.

[0013] Through amination treatment and amidation reaction, L-arginine and mesoporous bioactive glass can be successfully combined using carboxyl-rich macromolecules as connecting bridges. The modified L-arginine can consume excess reactive oxygen species (ROS) in the microenvironment and release nitric oxide that is beneficial to tissue regeneration, giving mesoporous bioactive glass the ability to scavenge reactive oxygen species and regulate immunity, playing an important role in regulating inflammation and promoting bone tissue regeneration.

[0014] Furthermore, the silane coupling agent is an aminosilane coupling agent, including but not limited to γ-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, anilinomethyltriethoxysilane, and N-β (aminoethyl)-γ-aminopropylmethyldiethoxysilane.

[0015] Furthermore, in step (1), the mass ratio of the mesoporous bioactive glass to the silane coupling agent is 1:1-3, and the reaction conditions are 80°C for 24 hours.

[0016] Among them, the mass ratio of mesoporous bioactive glass to silane coupling agent is preferably 1:2.

[0017] Furthermore, a carboxyl activating agent is added in both the amidation reactions of step (2) and step (3).

[0018] Furthermore, the carboxyl activator comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:0.5-2.

[0019] The molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is preferably 1:1.

[0020] Furthermore, the polymer includes but is not limited to hyaluronic acid, alginate, gelatin, and carboxymethyl cellulose.

[0021] Furthermore, in step (2), the mass ratio of amino-functionalized mesoporous bioactive glass to polymer is 1:1-3, and the reaction conditions are room temperature for 24 hours.

[0022] Among them, the mass ratio of amino-functionalized mesoporous bioactive glass to polymer is preferably 1:2.

[0023] Furthermore, in step (3), the mass ratio of the polymer-modified mesoporous bioactive glass to L-arginine is 1:0.5-2, and the reaction conditions are room temperature for 24 hours.

[0024] Wherein, the mass ratio of polymer-modified mesoporous bioactive glass to L-arginine is preferably 1:1.

[0025] Based on the same inventive concept, the present invention also provides a mesoporous bioactive glass hybrid material prepared by the above preparation method and having the functions of removing active oxygen, immunomodulation and bone regeneration.

[0026] Based on the same inventive concept, the present invention also provides applications of mesoporous bioactive glass hybrid materials with the functions of scavenging reactive oxygen species, immunomodulation and bone regeneration, including 1) applications of scavenging reactive oxygen species and improving cell microenvironment, 2) applications of regulating the polarization of macrophages to anti-inflammatory macrophages and eliminating inflammation, and 3) applications of promoting osteogenic differentiation of cells and accelerating bone tissue regeneration.

[0027] The present invention also provides the use of the mesoporous bioactive glass hybrid material with the functions of removing active oxygen, immunomodulation and bone regeneration in bone tissue repair materials or immunomodulation materials.

[0028] For example, a material for scavenging active oxygen can be prepared, which includes the above-mentioned mesoporous bioactive glass hybrid material with the functions of scavenging active oxygen, immunomodulation and bone regeneration; an immunomodulatory material can be prepared, which includes the above-mentioned mesoporous bioactive glass hybrid material with the functions of scavenging active oxygen, immunomodulation and bone regeneration; and a bone tissue repair material can also be prepared, which includes the above-mentioned mesoporous bioactive glass hybrid material with the functions of scavenging active oxygen, immunomodulation and bone regeneration.

[0029] The present invention has the following beneficial effects:

[0030] 1. The present invention uses a multi-step reaction method to graft L-arginine on the surface of mesoporous bioactive glass, combines the osteogenic property of mesoporous bioactive glass with the characteristics of L-arginine in scavenging ROS and releasing NO, prepares L-arginine-modified mesoporous bioactive glass, and obtains a mesoporous bioactive glass hybrid material with the functions of scavenging reactive oxygen species, immunomodulation and bone regeneration. The hybrid material has good biocompatibility, ROS scavenging ability, immunomodulation ability and bone regeneration performance, and has good application prospects in biomedical fields such as bone tissue repair materials or immunomodulation.

[0031] 2. The preparation method of the present invention is simple, environmentally friendly, has high reaction efficiency, and the raw materials used are cheap, easily available and have no toxic side effects.

[0032] 3. The present invention targets the changes in the local microenvironment during bone defects and tissue regeneration, and uses the natural amino acid L-arginine to modify the mesoporous bioactive glass, combining immune regulation with bone regeneration function to create a good bone immune microenvironment to meet the needs of bone defect repair. It is also applicable to other tissue regeneration fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 This is a process flow chart of preparing a mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration in one embodiment of the present invention;

[0035] Figure 2Transmission electron micrographs of MBG prepared in comparative example 1 of the present invention, MBG@HA prepared in comparative example 2, and MBG@L-Arg prepared in example 1;

[0036] Figure 3 The results of the test of the in vitro ROS scavenging ability and NO generating ability of MBG@L-Arg prepared in Example 1 of the present invention and MBG in Comparative Example 1;

[0037] Figure 4 The results of the MBG@L-Arg prepared in Example 1 of the present invention and the MBG in Comparative Example 1 in clearing ROS in macrophages (RAW264.7) and mouse embryonic osteoblast precursor cells (MC3T3-E1) are shown;

[0038] Figure 5 The gene expression levels of M1 macrophage-related factors and M2 macrophage-related factors after co-culturing the MBG@L-Arg prepared in Example 1 of the present invention and the MBG in Comparative Example 1 with RAW264.7 cells stimulated with lipopolysaccharide (LPS);

[0039] Figure 6 The protein bands after co-culture of MBG@L-Arg prepared in Example 1 of the present invention and MBG in Comparative Example 1 with RAW264.7 cells stimulated with lipopolysaccharide (LPS) and quantitative analysis of related protein expression;

[0040] Figure 7 The expression of osteogenic-related genes of MBG@L-Arg prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1 after co-culture with cells for 7 days under the condition of oxidative damage of MC3T3-E1 cells;

[0041] Figure 8 This is a characterization diagram of the osteogenic performance of MBG@L-Arg prepared in Example 1 of the present invention and MBG in Comparative Example 1. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The embodiment of the present invention provides a method for preparing a mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration, comprising the following steps:

[0044] (1) using a silane coupling agent to perform amino treatment on the mesoporous bioactive glass to prepare amino-functionalized mesoporous bioactive glass;

[0045] (2) modifying a polymer on an amino-functionalized mesoporous bioactive glass by an amidation reaction to prepare a polymer-modified mesoporous bioactive glass; the polymer is a carboxyl-rich macromolecule;

[0046] (3) L-arginine is modified on the polymer-modified mesoporous bioactive glass by an amidation reaction to obtain the mesoporous bioactive glass hybrid material.

[0047] See also Figure 1 In this embodiment, the polymer is selected from hyaluronic acid, but the polymer of the present invention is not limited to hyaluronic acid. Macromolecules rich in carboxyl groups can be used as connecting bridges to modify the mesoporous bioactive glass, such as alginate, gelatin, carboxymethyl cellulose, etc.

[0048] Mesoporous bioactive glass is used as a matrix. Silane coupling agents (γ-aminopropyltriethoxysilane, etc.) can dehydrate and condense on the surface of various substrates to provide amino groups on the surface of mesoporous bioactive glass, which is conducive to the further functionalization of mesoporous bioactive glass. Hyaluronic acid is rich in carboxyl groups and can be modified to the surface of mesoporous bioactive glass through amidation reaction and provide abundant reaction sites for L-arginine. Modified L-arginine can remove excess ROS and regulate the host immune response, thereby synergistically promoting bone regeneration with mesoporous bioactive glass.

[0049] Specifically, step (1) disperses the mesoporous bioglass in ethanol, adds a silane coupling agent, refluxes at 80° C. for 24 hours, collects the precipitate by centrifugation, washes, and freeze-dries to obtain the amino-functionalized mesoporous bioactive glass;

[0050] Step (2) may be: dispersing the amino-functionalized mesoporous bioactive glass in water to obtain a dispersion, dissolving hyaluronic acid in water, adding a carboxyl activator after 24 hours, and continuing to add the amino-functionalized mesoporous bioactive glass dispersion after 2 hours, stirring for 24 hours, collecting the precipitate by centrifugation, washing, and freeze-drying to obtain the hyaluronic acid-modified mesoporous bioactive glass;

[0051] Step (3) may be: dispersing the hyaluronic acid-modified mesoporous bioactive glass in water, adding a carboxyl activator and stirring for 2 hours, adding L-arginine, continuing stirring for 24 hours, collecting the precipitate by centrifugation, washing, and freeze-drying to obtain the mesoporous bioglass hybrid material.

[0052] In a specific embodiment, the silane coupling agent is an aminosilane coupling agent, including but not limited to γ-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, anilinomethyltriethoxysilane, N-β (aminoethyl)-γ-aminopropylmethyldiethoxysilane, and the like.

[0053] In a specific embodiment, the mass ratio of the mesoporous bioactive glass to the silane coupling agent in step (1) is 1:1-3, preferably 1:2.

[0054] In a specific embodiment, a carboxyl activator is added to the amidation reaction of step (2) and step (3). The carboxyl activator includes 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride to N-hydroxysuccinimide is 1:0.5-2, preferably 1:1.

[0055] In a specific embodiment, the mass ratio of the amino-functionalized mesoporous bioactive glass to the polymer in step (2) is 1:1-3, preferably 1:2.

[0056] In a specific embodiment, in step (3), the mass ratio of the polymer-modified mesoporous bioactive glass to L-arginine is 1:0.5-2, preferably 1:1.

[0057] The present invention will be further described below by means of specific embodiments:

[0058] The experimental methods used in the following examples are all conventional methods unless otherwise specified; the raw materials, reagents, etc. used are all raw materials and reagents that can be obtained from conventional commercial channels, etc. unless otherwise specified.

[0059] Example 1

[0060] The specific process of preparing the mesoporous bioactive glass hybrid material in this embodiment is as follows:

[0061] (1) Amino-functionalization treatment: Mesoporous bioactive glass (0.3 g) was dispersed in 100 mL of ethanol, 0.6 mL of γ-aminopropyltriethoxysilane was added, and then refluxed at 80 °C for 24 h. The precipitate was collected by centrifugation, washed three times with ultrapure water, and freeze-dried to obtain amino-functionalized mesoporous bioactive glass (MBG@NH2).

[0062] (2) Preparation of hyaluronic acid modified mesoporous bioactive glass by amidation reaction: MBG@NH2 (0.05 g) obtained in step (1) was dispersed in 2 mL of water; 0.1 g of hyaluronic acid was dissolved in 8 mL of water, and 0.1919 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.1151 g of N-hydroxysuccinimide were added after 24 hours, and MBG@NH2 dispersion was added after two hours, and stirring was continued for 24 hours. The precipitate was collected by centrifugation, washed three times with ultrapure water, and freeze-dried to obtain hyaluronic acid modified mesoporous bioactive glass (MBG@HA).

[0063] (3) Preparation of L-arginine-modified mesoporous bioactive glass by amidation reaction: The MBG@HA (0.065 g) obtained in step (2) was dispersed in deionized water, and 0.0959 g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.0576 g N-hydroxysuccinimide were added. After two hours, 0.065 g L-arginine was added, and stirring was continued for 24 hours. The precipitate was collected by centrifugation, washed three times with deionized water, and freeze-dried to obtain L-arginine-functionalized mesoporous bioactive glass (MBG@L-Arg).

[0064] Example 2

[0065] The steps and operations of this embodiment are basically the same as those of embodiment 1, except that the silane coupling agent used is γ-aminopropyltrimethoxysilane.

[0066] Example 3

[0067] The steps of this embodiment are basically the same as those of embodiment 1, except that the selected carboxyl-containing polymer is alginic acid.

[0068] Example 4

[0069] The steps and operations of this embodiment are basically the same as those of embodiment 1, the only difference being that the carboxyl-containing polymer selected is gelatin.

[0070] Example 5

[0071] The steps of this embodiment are basically the same as those of embodiment 1, except that the amount of γ-aminopropyltriethoxysilane is 0.9 mL.

[0072] Example 6

[0073] The steps of this embodiment are basically the same as those of embodiment 1, except that the amount of hyaluronic acid is 0.05 g.

[0074] Example 7

[0075] The steps of this embodiment are basically the same as those of embodiment 1, except that the amount of hyaluronic acid is 0.15 g.

[0076] Example 8

[0077] The steps of this embodiment are basically the same as those of embodiment 1, except that the amount of L-arginine is 0.0325 g.

[0078] Example 9

[0079] The steps of this embodiment are basically the same as those of embodiment 1, except that the amount of L-arginine is 0.13 g.

[0080] Comparative Example 1

[0081] The mesoporous bioactive glass without any treatment is used. The mesoporous bioactive glass can be prepared by a sol-gel method or purchased commercially.

[0082] Comparative Example 2

[0083] The mesoporous bioactive glass modified with hyaluronic acid (MBG@HA) was prepared by adopting steps (1)-(2) in Example 1. In this comparative example, the L-arginine modification in step (3) was not performed.

[0084] The products obtained in Example 1, Comparative Example 1 and Comparative Example 2 were analyzed by transmission electron microscopy. Figure 2 , the morphology of the mesoporous bioactive glass did not change significantly.

[0085] Performance Testing

[0086] (1) In vitro ROS scavenging and NO generation capabilities testing

[0087] The MBG@L-Arg and MBG prepared in Example 1 and Comparative Example 1 were respectively dispersed in deionized water to prepare suspensions of 1000, 500, 250, 125, and 62.5 μg / mL. The antioxidant capacity of MBG@L-Arg prepared in Example 1 and MBG in Comparative Example 1 at different concentrations was detected by a total antioxidant capacity test kit (ABTS rapid method).

[0088] The MBG@L-Arg prepared in Example 1 and the MBG prepared in Comparative Example 1 were dispersed in deionized water to prepare suspensions of 1000 and 500 μg / mL, respectively. A nitric oxide detection kit was used to detect the ability of MBG@L-Arg and MBG prepared in Example 1 and Comparative Example 1 at different concentrations to generate NO in 100 μmol / L hydrogen peroxide (H2O2).

[0089] Figure 3(A) is the test result of ROS scavenging ability of MBG@L-Arg prepared in Example 1 of the present invention and MBG of Comparative Example 1, and (B) is the test result of the ability of MBG@L-Arg prepared in Example 1 of the present invention to generate NO in the presence of 100 μmol / LH2O2; the results show that MBG@L-Arg prepared in Example 1 can effectively scavenge ROS and generate NO by utilizing ROS, and the ability to scavenge ROS and generate NO is related to the concentration of mesoporous bioactive glass hybrid material.

[0090] (2) Test for the ability to remove intracellular reactive oxygen species

[0091] 1 mL of RAW 264.7 cell suspension was diluted with 3 × 10 4 cell / mL was inoculated in a 24-well plate, and 1 μg / mL of LPS was added after 10 hours and cultured for another 24 hours. The old culture medium was discarded and replaced with a culture medium containing MBG@L-Arg (250 μg / mL) prepared in Example 1 and MBG (250 μg / mL) of Comparative Example 1 for co-culture. After 24 hours, the newly prepared DCFH-DA fluorescent probe was added and incubated in the dark for 30 minutes. After washing with PBS three times, the reactive oxygen positive cells were observed under a fluorescence microscope.

[0092] 1 mL of MC3T3-E1 cell suspension was diluted with 3 × 10 4 cell / mL was inoculated in a 24-well plate, and 100 μmol / L H2O2 was added after 10 hours and cultured for another 24 hours. The old culture medium was discarded and replaced with a culture medium containing MBG@L-Arg (250 μg / mL) prepared in Example 1 and MBG (250 μg / mL) of Comparative Example 1 for co-culture. After 24 hours, the newly prepared DCFH-DA fluorescent probe was added and incubated in the dark for 30 minutes. After washing with PBS three times, the reactive oxygen positive cells were observed under a fluorescence microscope.

[0093] Figure 4 (A) and (B) are the detection results of MBG@L-Arg prepared in Example 1 of the present invention and MBG in Comparative Example 1 for clearing ROS in macrophages (RAW264.7) and mouse embryonic osteoblast precursor cells (MC3T3-E1), respectively; the experimental results show that MBG@L-Arg prepared in Example 1 can effectively clear intracellular ROS and protect cells from oxidative stress.

[0094] (3) Test of immune regulation ability

[0095] 1 mL of macrophage (RAW 264.7) suspension was added at 3×10 4cell / mL was inoculated in a 24-well plate, and 1 μg / mL of lipopolysaccharide (LPS) was added after 10 hours and cultured for 12 hours. The old culture medium was discarded and replaced with a culture medium containing MBG@L-Arg (250 μg / mL) prepared in Example 1 and MBG (250 μg / mL) of Comparative Example 1 for co-culture. Total RNA and total protein were extracted after 48 hours to detect the expression of genes and proteins related to pro-inflammatory factors and anti-inflammatory factors.

[0096] Figure 5 (A) is the gene expression level of M1 macrophage-related factors after co-culture of MBG@L-Arg prepared in Example 1 of the present invention and MBG of comparative example 1 with RAW264.7 cells stimulated by LPS, and (B) is the gene expression level of M2 macrophage-related factors after co-culture of MBG@L-Arg prepared in Example 1 of the present invention and MBG of comparative example 1 with RAW264.7 cells stimulated by LPS; the experimental results show that MBG@L-Arg prepared in Example 1 can inhibit the expression of pro-inflammatory factors under inflammatory conditions, and promote cell polarization to M2 type, which is beneficial to tissue repair.

[0097] Figure 6 Quantitative analysis of protein bands and related protein expressions after co-culture of MBG@L-Arg prepared in Example 1 of the present invention and MBG in Comparative Example 1 with RAW264.7 cells stimulated by LPS; the results show that MBG@L-Arg prepared in Example 1 can inhibit cell polarization to M1 type and promote cell polarization to anti-M2 type under inflammatory conditions, which is beneficial to immune regulation and tissue regeneration.

[0098] (4) Osteogenesis performance test

[0099] 1 mL of mouse embryonic osteoblast precursor cell suspension (MC3T3-E1) was added at 1×10 4 cell / mL was inoculated in a 24-well plate, and 100 μmol / L H2O2 was added after 10 hours and cultured for 12 hours. The old culture medium was discarded and replaced with a culture medium containing MBG (250 μg / mL) of Comparative Example 1 and MBG@L-Arg (250 μg / mL) prepared in Example 1 for co-culture. Total RNA and total protein were extracted after 7 days of co-culture, and the expression of osteogenesis-related genes and proteins was detected. Alkaline phosphatase activity was detected by an alkaline phosphatase detection kit. After 14 days of co-culture, the formation of mineralized nodules was detected by Alizarin red staining.

[0100] Figure 7The expression of osteogenic-related genes of MBG@L-Arg prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1 after co-culture with MC3T3-E1 cells for 7 days under the condition of oxidative damage; the results show that MBG@L-Arg prepared in Example 1 can restore the differentiation ability of cells, promote the osteogenic differentiation of cells, and is beneficial to bone tissue regeneration.

[0101] Figure 8 (A) is the osteogenesis-related protein bands of MBG@L-Arg prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1 after co-culture with cells for 7 days under the condition of oxidative damage to MC3T3-E1 cells, (B) is the quantitative analysis of the protein bands, (C) and (D) are the alkaline phosphatase activity detection results of MBG prepared in Comparative Example 1 of the present invention and MBG@L-Arg prepared in Example 1 after co-culture with cells for 7 days under the condition of oxidative damage to MC3T3-E1 cells, and the calcified nodule detection results after co-culture for 14 days, respectively; the results show that MBG@L-Arg prepared in Example 1 can restore the differentiation ability of cells, promote the expression of cell osteogenesis-related proteins and alkaline phosphatase, accelerate the formation of calcified nodules, and is beneficial to bone tissue regeneration.

[0102] In summary, the present invention uses a multi-step reaction method to graft L-arginine on the surface of mesoporous bioactive glass, combines the osteogenic property of mesoporous bioactive glass with the characteristics of L-arginine in scavenging ROS and releasing NO, prepares L-arginine-modified mesoporous bioactive glass, and obtains a mesoporous bioactive glass hybrid material with the functions of scavenging reactive oxygen species, immunomodulation and bone regeneration; the hybrid material has good biocompatibility, ROS scavenging ability, immunomodulation ability and bone regeneration performance, and has good application prospects in biomedical fields such as bone tissue repair materials or immunomodulation.

[0103] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a mesoporous bioactive glass hybrid material with active oxygen scavenging, immune regulation and bone regeneration functions, characterized in that: The following steps are involved: (1) using a silane coupling agent to perform an amination treatment on the mesoporous bioactive glass to prepare an amino-functionalized mesoporous bioactive glass, wherein the mass ratio of the mesoporous bioactive glass to the silane coupling agent is 1:1-3; (2) modifying a polymer on an amino-functionalized mesoporous bioactive glass by an amidation reaction to prepare a polymer-modified mesoporous bioactive glass; the polymer is a carboxyl-rich macromolecule, and the mass ratio of the amino-functionalized mesoporous bioactive glass to the polymer is 1:1-3; (3) L-arginine is modified on the polymer-modified mesoporous bioactive glass by an amidation reaction to obtain the mesoporous bioactive glass hybrid material, wherein the mass ratio of the polymer-modified mesoporous bioactive glass to L-arginine is 1:0.5-2.

2. The method for preparing the mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration according to claim 1, characterized in that: The silane coupling agent in step (1) is an aminosilane coupling agent, including but not limited to γ-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, anilinomethyltriethoxysilane, and N-β (aminoethyl)-γ-aminopropylmethyldiethoxysilane.

3. The method for preparing the mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration according to claim 1, characterized in that: In step (1), the mass ratio of the mesoporous bioactive glass to the silane coupling agent is 1:

2.

4. The method for preparing the mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration according to claim 1, characterized in that: A carboxyl activating agent is added in both the amidation reactions of step (2) and step (3).

5. The method for preparing the mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration according to claim 4, characterized in that: The carboxyl activator comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:0.5-2.

6. The method for preparing the mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration according to claim 1, characterized in that: The polymer includes but is not limited to hyaluronic acid, alginate, gelatin, and carboxymethyl cellulose.

7. The method for preparing the mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration according to claim 1, characterized in that: The mass ratio of the amino-functionalized mesoporous bioactive glass to the polymer in step (2) is 1:

2.

8. The method for preparing the mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration according to claim 1, characterized in that: In step (3), the mass ratio of the polymer-modified mesoporous bioactive glass to L-arginine is 1:

1.

9. A mesoporous bioactive glass hybrid material having the functions of scavenging active oxygen, immunomodulation and bone regeneration, prepared by the preparation method according to any one of claims 1 to 8.

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