Mesoporous bioactive glass hybrid material as well as preparation method and application thereof

Abstract

The invention provides a mesoporous bioactive glass hybrid material and a preparation method and application thereof, and relates to the technical field of biomedical materials.According to the method, through a multi-step reaction method, a silane coupling agent and epsilon-polylysine are used as connecting molecules, and the mesoporous bioactive glass hybrid material is prepared. A mesoporous bioactive glass hybrid material with macrophage targeting, immunoregulation and bone regeneration functions is prepared, and the hybrid material can target macrophages, regulate and control the polarization state of the macrophages to be converted from a proinflammatory phenotype to an anti-inflammatory phenotype, enhance the bone immunoregulation ability and improve the bone regeneration effect. And the material shows excellent osteogenic induction performance in an in-vitro experiment, and a new thought is provided for research and development of bone repair materials. The method adopted by the invention is simple and easy to implement, and the prepared mesoporous bioglass hybrid material has good application potential in bone immunoregulation and bone tissue repair.

Description

Technical Field

[0001] The present invention relates to the technical field of functional biomedical materials, and in particular to a mesoporous bioactive glass hybrid material and a preparation method and application thereof. Background Art

[0002] Bone defect repair is a common clinical problem. Traditional bone repair methods include autologous and allogeneic bone transplantation, which have defects such as donor shortage and immune rejection. Therefore, many biomaterials have become substitutes for traditional bone repair materials. Studies have shown that biomaterial implants can cause an immune response of macrophages in the body. The phenotypic polarization of macrophages and the cytokines they secrete will directly affect the bone regeneration microenvironment. The M1 phenotypic polarization of macrophages can secrete proinflammatory factors and eliminate pathogens, while the M2 phenotypic polarization of macrophages can secrete anti-inflammatory factors and promote tissue repair. However, after the implantation of biomaterials, macrophage phenotypic transformation is often delayed, resulting in the persistence of a proinflammatory microenvironment, which ultimately affects the effect of bone regeneration. Therefore, the ideal bone regeneration material should have both bone induction ability and bone immune regulation function. Therefore, the preparation of osteogenic biomaterials with immunoregulatory function is of great significance in the field of bone repair.

[0003] Mesoporous bioactive glass (MBG) is a silicate glass composed of inorganic components such as SiO2, CaO, and P2O5. It is a type of nanobioceramic material with a mesoporous structure. Due to its osteoconductivity and osteoinductivity, it is widely used in bone tissue engineering. However, traditional MBG lacks bone immune regulation ability and is difficult to accurately regulate the polarization state of macrophages, which will greatly limit the further application of mesoporous bioactive glass in the field of bone repair.

[0004] In view of this, it is necessary to design an improved mesoporous bioactive glass hybrid material and its preparation method and application to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to provide a mesoporous bioactive glass hybrid material and a preparation method and application thereof.

[0006] To achieve the above-mentioned object of the invention, on the one hand, the present invention provides a method for preparing a mesoporous bioactive glass hybrid material, comprising the following steps:

[0007] S1. Preparation of mesoporous bioactive glass MBG;

[0008] S2, silanizing the MBG obtained in step S1 using a silane coupling agent to obtain epoxy-functionalized MBG;

[0009] S3, performing surface functionalization treatment on the epoxy-functionalized MBG obtained in step S2 using a polymer solution to obtain polymer-functionalized MBG, wherein the polymer contains amino groups and / or amine groups;

[0010] S4. Performing secondary surface functionalization treatment on the polymer-functionalized MBG obtained in step S3 using a sugar solution to obtain a mesoporous bioactive glass hybrid material.

[0011] Preferably, in step S2, the silane coupling agent is a solution formed by at least one of γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropyltriethoxysilane, β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, and 3-glycidoxypropylmethyldiethoxysilane.

[0012] Preferably, in step S2, the silane coupling agent is a γ-glycidyloxypropyltrimethoxysilane solution, and the mass ratio of MBG to the γ-glycidyloxypropyltrimethoxysilane in the γ-glycidyloxypropyltrimethoxysilane solution is 1:(0.05-0.5).

[0013] Preferably, in step S3, the solute of the polymer solution is at least one of ε-polylysine, α-polylysine, linear polylysine, dendritic polylysine, hyperbranched polylysine, RADA16 peptide, chitosan, polyethyleneimine, polyacrylamine, and polyamide-amine dendrimer molecules.

[0014] Preferably, in step S3, the polymer solution is an ε-polylysine aqueous solution with a concentration of 5-50 mg / mL; the mass ratio of the epoxy-functionalized MBG to the ε-polylysine in the ε-polylysine aqueous solution is 1:(0.5-5), the reaction time is 1-24 h, and the reaction temperature is 20-80°C.

[0015] Preferably, in step S3, the mass ratio of the epoxy-functionalized MBG to the ε-polylysine in the ε-polylysine aqueous solution is 1:1, the reaction time is 6 h, and the reaction temperature is 60°C.

[0016] Preferably, in step S4, the sugar solution is a mannose solution with a concentration of 5-50 mg / mL, the mass ratio of the polymer-functionalized MBG to the mannose in the mannose solution is 1:(0.5-5), and the reaction time is 1-24 h.

[0017] Preferably, in step S4, the mass ratio of the polymer-functionalized MBG to the mannose in the mannose solution is 1:1, and the reaction time is 6 h.

[0018] On the other hand, the present invention also provides applications of mesoporous bioactive glass hybrid materials, including applications in targeting macrophages, regulating macrophage polarization, improving bone immune microenvironment, and promoting bone regeneration.

[0019] The beneficial effects of the present invention are:

[0020] 1. The present invention provides a method for preparing a mesoporous bioactive glass hybrid material. By using a multi-step reaction method to graft mannose on the surface of a mesoporous bioactive glass, the osteogenic properties of the mesoporous bioactive glass are combined with the properties of mannose targeting macrophages and regulating macrophage phenotypes, thereby preparing a mannose-modified mesoporous bioactive glass. The obtained mesoporous bioactive glass hybrid material has macrophage targeting, immunomodulation and bone regeneration functions. The hybrid material has good biocompatibility, macrophage targeting ability, immunomodulation ability and bone regeneration activity, and has good application prospects in biomedical fields such as bone tissue repair materials or immunomodulation.

[0021] 2. The preparation method provided by the present invention targets the changes in the immune microenvironment during bone defects and tissue regeneration, and uses carbohydrate molecules with macrophage targeting and immune regulation functions to modify the surface of 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.

[0022] 3. The preparation method provided by the present invention is simple to operate, environmentally friendly, has high reaction efficiency, and the raw materials used are cheap, easily available, and have no toxic side effects. It is also applicable to the functionalization strategy of other materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a mechanism diagram of the preparation method of the mesoporous bioactive glass hybrid material proposed in the present invention;

[0024] Figure 2 TEM images of MBG-g-PL and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1;

[0025] Figure 3 Figure 2 is a graph showing the cell viability results of RAW264.7 cells and MC3T3-E1 cells cultured with MBG@KH560, MBG-g-PL, and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1;

[0026] Figure 4 These are the experimental results of culturing macrophages with MBG-g-PL and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1;

[0027] Figure 5 These are the experimental results of co-culturing MBG-g-PL and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1 with RAW264.7 cells;

[0028] Figure 6 These are the experimental results of co-culturing MBG-g-PL and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1 with RAW264.7 cells;

[0029] Figure 7 The gene expression results of macrophage conditioned medium prepared from MBG-g-PL and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1 and co-cultured with MC3T3-E1 cells for 7 days;

[0030] Figure 8 The experimental results of co-culturing macrophage conditioned medium prepared with MBG-g-PL and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1 with MC3T3-E1 cells for 7 days;

[0031] Figure 9 These are the experimental results of culturing MC3T3-E1 cells with macrophage conditioned medium prepared with MBG-g-PL and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1. DETAILED DESCRIPTION

[0032] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] It should also be noted here that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the solutions of the present invention are shown in the drawings, while other details that are not closely related to the present invention are omitted.

[0034] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0035] See also Figure 1 As shown, the present invention provides a method for preparing a mesoporous bioactive glass hybrid material, comprising:

[0036] S1. Preparation of mesoporous bioactive glass MBG;

[0037] S2, silanizing the MBG obtained in step S1 using a silane coupling agent to obtain epoxy-functionalized MBG;

[0038] S3, performing surface functionalization treatment on the epoxy-functionalized MBG obtained in step S2 using a polymer solution containing amino groups / amine groups to obtain polymer-functionalized MBG;

[0039] S4. Performing secondary surface functionalization treatment on the polymer-functionalized MBG obtained in step S3 using a sugar solution to obtain a mesoporous bioactive glass hybrid material.

[0040] In some embodiments, in step S2, the silanization treatment is performed as follows: MBG is first dispersed in water, then added to a silane coupling agent solution. After stirring, the product is collected and washed, and then dried to obtain silanized MBG. The silane coupling agent solution is obtained by dispersing the silane coupling agent in an alcohol solution, wherein the volume concentration is 10-100%, preferably 80%, and the mass percentage of the alcohol solution (methanol or ethanol) is 80%. The stirring process temperature is 10-35°C, and the stirring time is 0.1-12 hours, preferably 3 hours.

[0041] In the above steps, the purpose of stirring is to promote full contact between the silane coupling agent and MBG, thereby improving the silanization efficiency. The stirring method and stirring rate of the stirring process can be adjusted as needed as long as the above purpose can be achieved, and this is not limited here.

[0042] In some embodiments, in step S2, the silane coupling agent is a solution formed by at least one of γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropyltriethoxysilane, β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, and 3-glycidoxypropylmethyldiethoxysilane. When γ-glycidoxypropyltrimethoxysilane is used as the silane coupling agent, the mass ratio of MBG to γ-glycidoxypropyltrimethoxysilane in the γ-glycidoxypropyltrimethoxysilane solution is 1:(0.05-0.5), preferably 1:0.1.

[0043] In some embodiments, in step S3, the amino / amine-containing polymer solution is obtained by dispersing an amino / amine-containing polymer in water, and the amino / amine-containing polymer is at least one of ε-polylysine, α-polylysine, linear polylysine, dendritic polylysine, hyperbranched polylysine, RADA16 peptide, chitosan, polyethyleneimine, polyacrylamine, and polyamide-amine dendrimers. When ε-polylysine is used as the amino / amine-containing polymer, its concentration is 5-50 mg / mL, and the mass ratio of epoxy-functionalized MBG to ε-polylysine in the ε-polylysine solution is 1:(0.5-5), preferably 1:1; the reaction time of epoxy-functionalized MBG in the ε-polylysine solution is 1-24 h, preferably 6 h; and the reaction temperature is 20-80° C., preferably 60° C.

[0044] In the above technical solution, by controlling the mass ratio of epoxy-functionalized MBG and ε-polylysine within a specific range, it is possible to ensure that ε-polylysine is evenly distributed on the surface of MBG, and while maximizing the loading amount of ε-polylysine, the influence of ε-polylysine on the pore structure of MBG itself during the loading process is reduced, thereby ensuring that the performance of MBG in the hybrid material finally obtained is fully utilized. If the mass ratio of epoxy-functionalized MBG to ε-polylysine exceeds the above range, excessive ε-polylysine molecules may cover the pore structure of MBG, limiting some functions of MBG (such as drug loading capacity or ion release rate). Furthermore, excessive ε-polylysine molecules are prone to self-aggregation, which not only affects the overall uniformity of the material, but also makes it difficult to exert the performance advantages of ε-polylysine, thereby reducing the utilization rate of ε-polylysine. If the mass ratio of the two is too small, the loading amount of ε-polylysine is too low, and the active sites on the MBG surface are not fully utilized, affecting the overall biological activity and cell affinity of the material. At the same time, the loading amount of ε-polylysine is too low, which will also affect the subsequent coupling efficiency of mannose and affect the targeting of the material.

[0045] In some embodiments, in step S4, the sugar solution is a mannose solution with a concentration of 5-50 mg / mL, preferably 10 mg / mL, and the mass ratio of the polymer-functionalized MBG to the mannose in the mannose solution is 1:(0.5-5), preferably 1:1; the reaction time of the polymer-functionalized MBG in the mannose solution is 1-24 h, preferably 6 h, and the temperature is 10-35°C.

[0046] In the above technical solution, by controlling the mass ratio of the polymer-functionalized MBG to mannose within a specific range, the comprehensive performance of the resulting hybrid material can be ensured to be optimal. This is because: if the mass ratio of the two is too small, the amount of mannose is too low, resulting in insufficient reaction of the surface amino groups of the polymer-functionalized MBG, a low mannose grafting rate, and weakening its targeted binding ability to specific receptors (such as the mannose receptor CD206 on the surface of macrophages). Secondly, the incomplete reaction of the amino groups may cause the surface positive charge of the hybrid material to be too high, causing nonspecific cell adsorption or toxicity. When the mass ratio of the two is too large, the excess mannose may lead to intermolecular competition reaction, which in turn reduces the coupling efficiency of individual mannose and amino groups and hinders effective grafting reaction. Secondly, mannose may be adsorbed in the MBG pores through hydrogen bonds or van der Waals forces, blocking the mesopores and affecting some functions of MBG (such as drug loading capacity or ion release rate). Thirdly, unreacted free mannose needs to be removed additionally, increasing the preparation cost.

[0047] In particular, the mesoporous bioactive glass hybrid material prepared by the preparation method proposed in the present invention can be used as a biomedical material or further made into a functional biomedical material, such as a macrophage targeting material, a bone immune microenvironment regulating material, a bone tissue regeneration material, etc., or used to target macrophages, regulate macrophage polarization, improve the bone immune microenvironment, and promote bone regeneration.

[0048] The following is a further description of the mesoporous bioactive glass hybrid material, its preparation method and application proposed by the present invention in conjunction with specific embodiments:

[0049] Example 1

[0050] In this embodiment, a mesoporous bioactive glass hybrid material is prepared, and the preparation method thereof comprises the following steps:

[0051] S1. Mesoporous bioactive glass MBG was prepared by sol-gel method. The specific preparation method is as follows: 1.4 g of hexadecyltrimethylammonium bromide (CTAB) was dissolved in 66 mL of water, ultrasonically dispersed, and then continuously stirred at 30 ° C. When CTAB was completely dissolved, 20 mL of ethyl acetate was added to the mixed solution, and the mixture was stirred for 30 min to form microemulsion droplets. Then, 14 mL of 1 M ammonia water (analytical grade) was added to the mixed solution, and the mixed solution was stirred for 15 min. After the stirring was completed, 7.2 mL of Ethyl orthosilicate (analytical grade) was added, and after 30 minutes, 0.72 mL of triethyl phosphate (analytical grade) was added. After another 30 minutes, 4.554 g of calcium nitrate tetrahydrate was added. The resulting solution was stirred for 4 hours until the solution became turbid to obtain a white suspension. The white suspension was centrifuged, and the white precipitate therein was collected, washed three times with ethanol, and then washed three times with water. The resulting product was freeze-dried and calcined in a muffle furnace at 700°C for 6 hours to obtain MBG with a particle size of 100 nm, a pore size distribution range of 7.5-17.5 nm, and a specific surface area of 248.65 m 2 / g, pore volume 1.14cm 3 / g; wherein, the heating rate of the calcination process from 25°C to 700°C is 2°C / min; those skilled in the art should understand that in other embodiments, other methods can also be used to prepare mesoporous bioactive glass MBG;

[0052] S2. Disperse 0.2 g of MBG obtained in step S1 in 10 mL of water to obtain an MBG solution; add the MBG solution dropwise to 10 mL of γ-glycidyloxypropyltrimethoxysilane solution and magnetically stir at 25° C. for 3 h; after stirring, separate the precipitate by centrifugation, wash it three times with water, and freeze-dry it to obtain the silanized mesoporous bioactive glass MBG@KH-560; wherein the γ-glycidyloxypropyltrimethoxysilane solution is obtained by dissolving 0.02 g of γ-glycidyloxypropyltrimethoxysilane in 10 mL of 80% by mass methanol and magnetically stirring it at 25° C. for 30 min;

[0053] S3. Disperse 0.2 g of MBG@KH-560 prepared in step S2 in 10 mL of water, add the solution dropwise to the ε-polylysine solution, and react at 60° C. for 6 h. After the reaction, collect the precipitate by centrifugation, wash three times with water, and freeze-dry the resulting product to obtain the ε-polylysine surface-functionalized mesoporous bioactive glass MBG-g-PL; wherein the ε-polylysine solution is obtained by dissolving 0.2 g of ε-polylysine with a concentration of 10 mg / mL in water;

[0054] S4, 0.25 g of mannose was added to 5 mL of ultrapure water to prepare a mannose solution; 0.25 g of MBG-g-PL prepared in step S3 was dispersed in 10 mL of water and then added dropwise to the mannose solution. The mixture was stirred at 25°C for 6 h. After the reaction was completed, the precipitate was collected by centrifugation and washed three times with water. The obtained product was freeze-dried to obtain a mesoporous bioactive glass hybrid material MBG-g-Man, which had a particle size of about 110 nm, a pore size distribution range of 5-20 nm, and a specific surface area of 428.81 m 2 / g, pore volume 1.19cm 3 It should be noted that, unless otherwise specified, the reagents and raw materials used in the embodiments of the present invention can be obtained from the market. In addition, in steps S1-S4, the purpose of the stirring operation is to achieve mixing. The stirring method can be selected according to actual needs and will not be described in detail here.

[0055] In the above technical scheme, the preparation mechanism of MBG-g-Man is as follows: first, γ-glycidyloxypropyltrimethoxysilane (silane coupling agent) is used to introduce abundant epoxy groups into MBG to facilitate the surface chemical modification of MBG. At the same time, silanization can reduce the surface energy of MBG and prevent MBG particles from agglomerating and affecting the subsequent surface chemical modification. Then, ε-polylysine and mannose are used to modify the surface of MBG in sequence. The epoxy groups on the surface of the alkylated MBG and the ε-polylysine react with each other to form a bond. Through electrostatic adsorption, ε-polylysine is grafted on the surface of MBG. ε-polylysine acts as a connecting bridge to add more action sites for MBG to react with mannose. The aldehyde group of mannose reacts with the amino group of ε-polylysine to form a Schiff base reaction, thereby modifying mannose on the surface of MBG. The surface-modified mannose can specifically recognize the mannose receptor on the surface of macrophage membranes, improve the uptake efficiency, induce the M2 polarization of macrophages, and then secrete anti-inflammatory factors to create a favorable microenvironment, thereby synergistically promoting bone regeneration with mesoporous bioactive glass.

[0056] Example 2

[0057] The only difference between this embodiment and embodiment 1 is that in step S2, the amount of γ-glycidyloxypropyltrimethoxysilane added to the γ-glycidyloxypropyltrimethoxysilane solution is 0.01 g, and the other experimental parameters are the same as those in embodiment 1 and are not repeated here.

[0058] Example 3

[0059] The only difference between this embodiment and embodiment 1 is that in step S2, the amount of γ-glycidyloxypropyltrimethoxysilane added to the γ-glycidyloxypropyltrimethoxysilane solution is 0.1 g, and the other experimental parameters are the same as those in embodiment 1 and are not repeated here.

[0060] Example 4

[0061] The only difference between this embodiment and embodiment 1 is that in step S2, the amount of ε-polylysine added to the ε-polylysine solution is 0.1 g, and the other experimental parameters are the same as those in embodiment 1 and will not be repeated here.

[0062] Example 5

[0063] The only difference between this embodiment and embodiment 1 is that in step S3, the amount of ε-polylysine added to the ε-polylysine solution is 1 g, and the other experimental parameters are the same as those in embodiment 1 and will not be repeated here.

[0064] Example 6

[0065] The only difference between this embodiment and embodiment 1 is that in step S3, the reaction time of MBG@KH-560 and ε-polylysine solution is 1 hour. The other experimental parameters are the same as those in embodiment 1 and are not described again here.

[0066] Example 7

[0067] The only difference between this embodiment and embodiment 1 is that in step S3, the reaction time of MBG@KH-560 and ε-polylysine solution is 24 h. The other experimental parameters are the same as those in embodiment 1 and are not described again here.

[0068] Example 8

[0069] The only difference between this embodiment and embodiment 1 is that in step S4, the mass of mannose in the mannose solution is 0.125 g. The remaining experimental parameters are the same as those in embodiment 1 and are not described again here.

[0070] Example 9

[0071] The only difference between this embodiment and embodiment 1 is that in step S4, the mass of mannose in the mannose solution is 1.25 g. The remaining experimental parameters are the same as those in embodiment 1 and are not described again here.

[0072] Example 10

[0073] The only difference between this embodiment and embodiment 1 is that in step S4, the reaction time of the mannose solution and MBG-g-PL is adjusted to 1 h. The other experimental parameters are the same as those in embodiment 1 and are not described again here.

[0074] Example 11

[0075] The only difference between this embodiment and embodiment 1 is that in step S4, the reaction time of the mannose solution and MBG-g-PL is adjusted to 24 h. The other experimental parameters are the same as those in embodiment 1 and are not described again here.

[0076] The experimental results show that Examples 1 to 11 can all produce mesoporous bioactive glass hybrid materials with excellent performance.

[0077] Comparative Example 1

[0078] The only difference between Comparative Example 1 and Example 1 is that MBG is not subjected to surface chemical modification, but is directly used as a mesoporous bioactive glass hybrid material. The preparation method of MBG is the same as that of Example 1 and will not be repeated here.

[0079] Comparative Example 2

[0080] The only difference between Comparative Example 2 and Example 1 is that mannose is not modified on the surface of MBG-g-PL, but MBG-g-PL is directly used as a mesoporous bioactive glass hybrid material. The preparation method of MBG-g-PL is the same as that of Example 1 and is not repeated here.

[0081] The TEM images of MBG-g-PL and MBG-g-Man prepared in Example 1 and MBG prepared in Comparative Example 1 are as follows: Figure 2 As shown in the figure, it can be seen that the overall morphologies of MBG-g-PL, MBG-g-Man and MBG are not much different, indicating that surface modification will not have a great impact on the morphology of MBG.

[0082] Furthermore, during the experiment, the application of MBG-g-PL and MBG-g-Man prepared in Example 1 and MBG prepared in Comparative Example 1 in promoting tissue regeneration and immune regulation was explored, and the test results of biocompatibility, macrophage targeting ability, immune regulation ability, and in vitro osteogenesis ability were used to illustrate the application. The specific test process and test results are as follows:

[0083] (1) Biocompatibility test:

[0084] MBG prepared in Comparative Example 1 and MBG@KH-560, MBG-g-PL, and MBG-g-Man prepared in Example 1 were dispersed in cell culture medium to prepare suspensions of 200, 100, 50, and 25 μg / mL, respectively. Cells were cultured at a rate of 2×10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate. After incubation in a cell culture incubator, cell culture media prepared with different concentrations of the materials were added and co-cultured with RAW264.7 cells and MC3T3-E1 cells for 24 hours and 72 hours. The biocompatibility of the materials was evaluated using the Cell Counting Kit-8 (CCK-8) method. The cell viability results are shown in the figure. Figure 3 As shown, Figure 3 Figures AB in the figure are the cell viability results of MC3T3-E1 cells treated for 24 hours and 72 hours respectively. Figure 3Figures CD are the cell viability results of RAW264.7 cells treated for 24 hours and 72 hours, respectively. The results show that the cell activity after treatment with the four materials is higher than 90%, and the number of RAW264.7 cells and MC3T3-E1 cells treated with MBG-g-Man is greater than that of MBG, MBG@KH-560, and MBG-g-PL, indicating that MBG has low toxicity to cells before and after functional modification, and that the modification of MBG with ε-polylysine and mannose can reduce its toxicity to cells to a certain extent. The results show that the mesoporous bioactive glass hybrid material prepared by the method proposed in the present invention has good biocompatibility.

[0085] (2) Macrophage targeting ability test:

[0086] MBG prepared in Comparative Example 1 and MBG-g-PL and MBG-g-Man prepared in Example 1 were dispersed in cell culture medium to prepare a 100 μg / mL suspension, which was used as cell culture medium. Figure 4 The cell transwell device shown in Figure A is used to culture macrophages. The device includes a culture dish and two chambers arranged vertically relative to each other in the culture dish. A porous membrane is provided between the two chambers. The two chambers and the semipermeable membrane together constitute an upper chamber for placing macrophages. The space inside the culture dish excluding the upper chamber is a lower chamber for holding cell culture medium.

[0087] Add 500uL of material suspension to the lower chamber at a concentration of 1.5×10 4 Macrophages (RAW264.7) were seeded into the chamber at a density of cells / , and then the macrophages and materials were co-cultured in a cell culture incubator for 6 hours and 12 hours respectively. Cells were counted using crystal violet staining, and 1% crystal violet solution was prepared to stain the cells. The cells were observed and photographed using a stereomicroscope. The results are shown in Figure 2. Figure 4 As shown in Figure B, it can be seen from the figure that the number of cells in the experimental group with the addition of MBG-g-Man is significantly greater than that in the other groups, indicating that MBG-g-Man has a certain ability to recruit macrophages. After taking the picture, the crystal violet dye on each group of membranes was dissolved with an equal amount of 33% acetic acid, and then its wavelength was measured at an OD value of 570nm. The results are shown in Figure B. Figure 4 As shown in Figure C, the results indicate that MBG-g-Man can effectively recognize and capture macrophages.

[0088] (3) Immunomodulatory ability test:

[0089] MBG prepared in Comparative Example 1 and MBG-g-PL and MBG-g-Man prepared in Example 1 were dispersed in cell culture medium to prepare a 100 μg / mL suspension. First, RAW264.7 cells were cultured at a density of 2×105 The density of cells / well was seeded in 6-well plates and incubated in cell culture medium containing the materials for 1 day and 3 days respectively. Then, the total RNA and total protein of macrophages were extracted. The results of total RNA of macrophages were as follows: Figure 5 As shown, Figure 5 Figure A shows the gene expression levels of M1 macrophage-related factors. Figure 5 Figure B shows the gene expression levels of M2 macrophage-related factors. Real-time fluorescence quantitative PCR (RT-qPCR) and western blotting (WB) experiments were used to detect the expression of macrophage polarization-related genes and proteins. The results showed that MBG-g-Man can dynamically regulate the polarization state of macrophages and effectively promote the transformation of macrophages from M1 (pro-inflammatory) to M2 (anti-inflammatory) in the later stage of co-culture, thereby improving the bone immune microenvironment.

[0090] Figure 6 Figure A shows the protein bands related to inflammation after MBG prepared in Comparative Example 1 and MBG-g-PL and MBG-g-Man prepared in Example 1 were co-cultured with RAW264.7 cells. Figure 6 Figure B is a quantitative analysis of iNOS and CD206 expression. The results show that MBG-g-Man can promote cell polarization to M2 type on the third day of co-culture with macrophages, which is beneficial to immune regulation and tissue regeneration.

[0091] (4) In vitro osteogenic capacity test:

[0092] MBG prepared in Comparative Example 1 and MBG, MBG-g-PL, and MBG-g-Man prepared in Example 1 were co-cultured with macrophages for three days to obtain macrophage-conditioned medium co-cultured with the MBG, MBG-g-PL, and MBG-g-Man materials. This medium was mixed with complete osteogenic induction medium at a ratio of 1:2 to prepare macrophage-conditioned osteogenic medium. The potential of macrophage-conditioned osteogenic medium to stimulate the osteogenic differentiation of mouse embryonic osteoblast precursor cells (MC3T3-E1) was investigated using real-time fluorescence quantitative PCR (RT-qPCR), western blotting (WB), alkaline phosphatase (ALP) staining, and Alizarin red staining (ARS).

[0093] MC3T3-E1 cells were cultured at 1.5 × 10 4 The cells were seeded at a density of 100 cells / well in a 6-well plate and cultured with macrophage-conditioned osteogenic medium prepared from different groups. After 7 days of osteogenic induction, total RNA and total protein were collected from MC3T3-E1 cells. The gene expression levels of OCN, OPN, and Runx-2 were shown in Figure 2. Figure 7The results showed that the gene expression levels of macrophages cultured with MBG-g-Man were significantly higher than those in other groups, indicating that MBG-g-Man can promote the osteogenic differentiation of cells and is beneficial to bone tissue regeneration; the osteogenesis-related protein bands and quantitative analysis of protein bands after co-culture of macrophage conditioned medium with MC3T3-E1 cells for 7 days are shown in Figure 2. Figure 8 Figure A and Figure 8 As shown in Figure B, the results show that MBG-g-Man can promote the expression of cell osteogenesis-related proteins, which is beneficial to bone tissue regeneration.

[0094] MC3T3-E1 cells were cultured at 5×10 4 Cells / well were seeded into 24-well plates, and ALP staining was performed 14 days after osteogenic induction. The results of alkaline phosphatase activity test after 14 days of induction were as follows: Figure 9 As shown in Figure A; MC3T3-E1 cells were grown at 3×10 4 Cells / well were seeded into 24-well plates, and ARS staining was performed 21 days after osteogenic induction. The results of calcified nodules detection after 21 days of induction were as follows: Figure 9 As shown in Figure B, the results show that MBG-g-Man can promote the osteogenic differentiation ability of cells and the expression of alkaline phosphatase, accelerate the formation of calcified nodules, and is beneficial to bone tissue regeneration. It should be noted that Figure 2-9 The “Control” in the table indicates that cells were cultured using cell culture medium without any hybrid material.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a mesoporous bioactive glass hybrid material, characterized in that: The steps include: S1. Preparation of mesoporous bioactive glass MBG; S2, silanizing the MBG obtained in step S1 using a silane coupling agent to obtain epoxy-functionalized MBG; S3, performing surface functionalization treatment on the epoxy-functionalized MBG obtained in step S2 using a polymer solution to obtain polymer-functionalized MBG, wherein the polymer contains amino groups and / or amine groups; S4. Performing secondary surface functionalization treatment on the polymer-functionalized MBG obtained in step S3 using a sugar solution to obtain a mesoporous bioactive glass hybrid material.

2. The preparation method according to claim 1, characterized in that In step S2, the silane coupling agent is a solution formed by at least one of γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropyltriethoxysilane, β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, and 3-glycidoxypropylmethyldiethoxysilane.

3. The preparation method according to claim 2, characterized in that In step S2, the silane coupling agent is a γ-glycidyloxypropyltrimethoxysilane solution, and the mass ratio of MBG to the γ-glycidyloxypropyltrimethoxysilane in the γ-glycidyloxypropyltrimethoxysilane solution is 1:(0.05-0.5).

4. The preparation method according to claim 1, characterized in that In step S3, the solute of the polymer solution is at least one of ε-polylysine, α-polylysine, linear polylysine, dendritic polylysine, hyperbranched polylysine, RADA16 peptide, chitosan, polyethyleneimine, polyacrylamine, and polyamidoamine dendrimers.

5. The preparation method according to claim 4, characterized in that In step S3, the polymer solution is an ε-polylysine aqueous solution with a concentration of 5-50 mg / mL; the mass ratio of the epoxy-functionalized MBG to the ε-polylysine in the ε-polylysine aqueous solution is 1:(0.5-5), the reaction time is 1-24 h, and the reaction temperature is 20-80°C.

6. The preparation method according to claim 5, characterized in that In step S3, the mass ratio of the epoxy-functionalized MBG to the ε-polylysine in the ε-polylysine aqueous solution is 1:1, the reaction time is 6 hours, and the reaction temperature is 60°C.

7. The preparation method according to claim 1, characterized in that In step S4, the sugar solution is a mannose solution with a concentration of 5-50 mg / mL, the mass ratio of the polymer-functionalized MBG to the mannose in the mannose solution is 1:(0.5-5), and the reaction time is 1-24 h.

8. The preparation method according to claim 7, characterized in that In step S4, the mass ratio of the polymer-functionalized MBG to the mannose in the mannose solution is 1:1, and the reaction time is 6 hours.

9. A mesoporous bioactive glass hybrid material prepared by the preparation method according to any one of claims 1 to 8.

10. A use of a mesoporous bioactive glass hybrid material prepared by the preparation method according to any one of claims 1 to 8 or the mesoporous bioactive glass hybrid material according to claim 9, characterized in that: It includes applications in targeting macrophages, regulating macrophage polarization, improving the bone immune microenvironment, and promoting bone regeneration.

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