Mesoporous bioactive glass hybrid materials, methods of making and using the same

Abstract

The application provides a mesoporous bioactive glass hybrid material and a preparation method and application thereof, relates to the technical field of biomedical materials, and prepares a mesoporous bioactive glass hybrid material with macrophage targeting, immunoregulation and bone regeneration functions through a multi-step reaction method by using a silane coupling agent and epsilon-polylysine as connecting molecules. The hybrid material can target macrophages, regulate the polarization state of the macrophages to transform from a pro-inflammatory phenotype to an anti-inflammatory phenotype, enhance the bone immunoregulation capacity, and exhibits excellent osteogenic induction performance in an in-vitro experiment, thereby providing a new idea for the research and development of bone repair materials. The method adopted in the application is simple and easy to implement, and the prepared mesoporous bioactive glass hybrid material has good application potential in bone immunoregulation and bone tissue repair.

Description

Technical Field

[0001] This invention relates to the field of functional biomedical materials technology, and in particular to a mesoporous bioactive glass hybrid material, its preparation method, and its application. Background Technology

[0002] Bone defect repair is a common clinical problem. Traditional bone repair methods, including autologous and allogeneic bone grafts, suffer from drawbacks such as donor shortages and immune rejection. Therefore, many biomaterials have become alternatives to traditional bone repair materials. Studies have shown that biomaterial implants induce immune responses in macrophages. Macrophage phenotypic polarization and the cytokines they secrete directly affect the bone regeneration microenvironment. Macrophage M1 phenotypic polarization can secrete pro-inflammatory factors and clear pathogens, while macrophage M2 phenotypic polarization can secrete anti-inflammatory factors and promote tissue repair. However, after biomaterial implantation, macrophage phenotypic transformation is often delayed, leading to the persistence of a pro-inflammatory microenvironment and ultimately affecting bone regeneration. Therefore, ideal bone regeneration materials should possess both osteoinductive and immunomodulatory functions. Thus, the preparation of osteogenic biomaterials with immunomodulatory functions 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 nano-bioceramic material with a mesoporous structure. Due to its osteoconductivity and osteoinductive properties, it is widely used in bone tissue engineering. However, traditional MBG lacks bone immunomodulation capabilities and is difficult to precisely 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, its preparation method, and its application to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a mesoporous bioactive glass hybrid material, its preparation method, and its application.

[0006] To achieve the above-mentioned objectives, 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. The MBG obtained in step S1 is silanized using a silane coupling agent to obtain epoxy-functionalized MBG.

[0009] S3. The epoxy-functionalized MBG obtained in step S2 is surface-functionalized using a polymer solution to obtain polymer-functionalized MBG, wherein the polymer contains amino and / or amine groups.

[0010] S4. The polymer-functionalized MBG obtained in step S3 is subjected to a secondary surface functionalization treatment 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 from at least one of γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropyltriethoxysilane, β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, and 3-epoxypropoxypropylmethyldiethoxysilane.

[0012] Preferably, in step S2, the silane coupling agent is a solution of γ-glycidoxypropyltrimethoxysilane, and the mass ratio of MBG to the γ-glycidoxypropyltrimethoxysilane in the 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, polyacrylamide, and polyamide-amine dendritic molecules.

[0014] Preferably, in step S3, the polymer solution is an aqueous solution of ε-polylysine with a concentration of 5-50 mg / mL; the mass ratio of the epoxy-functionalized MBG to the ε-polylysine in the aqueous solution of ε-polylysine 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 hours, 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 polymerically functionalized MBG to the mannose in the mannose solution is 1:1, and the reaction time is 6 hours.

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

[0019] The beneficial effects of this invention are:

[0020] 1. The method for preparing mesoporous bioactive glass hybrid materials provided by the present invention involves grafting mannose onto the surface of mesoporous bioactive glass using a multi-step reaction method. This combines the osteogenic properties of the mesoporous bioactive glass with the macrophage-targeting and macrophage-phenotype-regulating properties of mannose, thus preparing mannose-modified mesoporous bioactive glass. This yields a mesoporous bioactive glass hybrid material with macrophage-targeting, immunomodulatory, and bone regeneration functions. This hybrid material exhibits good biocompatibility, macrophage-targeting ability, immunomodulatory ability, and bone regeneration activity, showing promising application prospects in biomedical fields such as bone tissue repair materials or immunomodulation.

[0021] 2. The preparation method provided by this invention targets the changes in the immune microenvironment during bone defects and tissue regeneration. It uses carbohydrate molecules with macrophage targeting and immunomodulatory functions to modify the surface of mesoporous bioactive glass, combining immunomodulation 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 this invention is simple to operate, environmentally friendly, and has high reaction efficiency. The raw materials used are inexpensive, readily available, and have no toxic side effects. It is also applicable to the functionalization strategies of other materials. Attached Figure Description

[0023] Figure 1 This is a mechanism diagram of the preparation method of the mesoporous bioactive glass hybrid material proposed in this 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 The figures show the cell viability results of RAW264.7 cells and MC3T3-E1 cells after culturing 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 The experimental results of cultured macrophages with MBG-g-PL and MBG-g-Man prepared in Example 1 of the present invention and MBG prepared in Comparative Example 1 are shown.

[0027] Figure 5 The results are as follows: MBG-g-PL and MBG-g-Man prepared in Example 1 of this invention and MBG prepared in Comparative Example 1 were co-cultured with RAW264.7 cells.

[0028] Figure 6 The results are as follows: MBG-g-PL and MBG-g-Man prepared in Example 1 of this invention and MBG prepared in Comparative Example 1 were co-cultured with RAW264.7 cells.

[0029] Figure 7 The gene expression levels 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 were co-cultured with MC3T3-E1 cells for 7 days.

[0030] Figure 8 The results of co-culturing MC3T3-E1 cells for 7 days with macrophage conditioned medium prepared from MBG-g-PL and MBG-g-Man obtained in Example 1 and MBG obtained in Comparative Example 1.

[0031] Figure 9 The results of culturing MC3T3-E1 cells with macrophage conditioned medium prepared from MBG-g-PL and MBG-g-Man obtained in Example 1 and MBG obtained in Comparative Example 1 are presented. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

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

[0034] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0035] Please see 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. The MBG obtained in step S1 is silanized using a silane coupling agent to obtain epoxy-functionalized MBG.

[0038] S3. The epoxy-functionalized MBG obtained in step S2 is subjected to surface functionalization treatment using a polymer solution containing amino / amine groups to obtain polymer-functionalized MBG.

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

[0040] In some embodiments, in step S2, the silanization process is carried out 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 a silane coupling agent in an alcohol solution, with a volume concentration of 10-100%, preferably 80%, and the alcohol solution (methanol or ethanol) has a mass percentage of 80%. The stirring temperature is 10-35°C, and the stirring time is 0.1-12 h, preferably 3 h.

[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 can be adjusted as needed, as long as the aforementioned purpose can be achieved, and are not limited to this.

[0042] In some embodiments, in step S2, the silane coupling agent is a solution formed from at least one of γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropyltriethoxysilane, β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, and 3-epoxypropylmethyldiethoxysilane. 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, which is at least one of ε-polylysine, α-polylysine, linear polylysine, dendritic polylysine, hyperbranched polylysine, RADA16 peptide, chitosan, polyethyleneimine, polyacrylamide, and polyamide-amine dendritic molecules. When ε-polylysine is used as the amino / amine-containing polymer, its concentration is 5-50 mg / mL. 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. 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 to ε-polylysine within a specific range, it is possible to ensure that ε-polylysine is evenly distributed on the surface of MBG. Under the premise of maximizing the loading of ε-polylysine, the influence of ε-polylysine on the pore structure of MBG itself during the loading process is reduced, thus ensuring that the performance of MBG in the final hybrid material 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 porous structure of MBG, limiting some of MBG's functions (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 leverage the performance advantages of ε-polylysine, reducing its utilization rate. If the mass ratio is too small, the loading of ε-polylysine is too low, and the active sites on the MBG surface are not fully utilized, affecting the overall bioactivity and cell affinity of the material. At the same time, a low loading of ε-polylysine will also affect the subsequent coupling efficiency of mannose, impacting the material's targeting ability.

[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 polymerically functionalized MBG to the mannose in the mannose solution is 1:(0.5-5), preferably 1:1; the reaction time of the polymerically 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 polymerically functionalized MBG to mannose within a specific range, the optimal comprehensive performance of the resulting hybrid material can be ensured. This is because: if the mass ratio is too small, the amount of mannose is too low, resulting in insufficient reaction of the surface amino groups of the polymerically functionalized MBG, low mannose grafting rate, and weakened targeting binding ability to specific receptors (such as the mannose receptor CD206 on the surface of macrophages). Secondly, insufficiently reacted amino groups may lead to excessively high positive charge on the surface of the hybrid material, causing non-specific cell adsorption or toxicity. On the other hand, when the mass ratio is too large, excessive mannose may lead to intermolecular competitive reactions, which may reduce the coupling efficiency between individual mannose and amino groups, hindering effective grafting reactions. Furthermore, mannose may be adsorbed into the MBG pores through hydrogen bonds or van der Waals forces, blocking mesopores and affecting some functions of MBG (such as drug loading capacity or ion release rate). Moreover, unreacted free mannose needs to be removed, increasing the preparation cost.

[0047] In particular, the mesoporous bioactive glass hybrid material prepared by the preparation method proposed in this 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 regulation material, a bone tissue regeneration material, etc., or applied to target macrophages, regulate macrophage polarization, improve the bone immune microenvironment, and promote bone regeneration.

[0048] The following specific embodiments further illustrate the mesoporous bioactive glass hybrid material proposed in this invention, its preparation method, and its applications:

[0049] Example 1

[0050] This embodiment prepares a mesoporous bioactive glass hybrid material, and the preparation method includes the following steps:

[0051] S1. Mesoporous bioactive glass (MBG) was prepared using the 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. After CTAB was completely dissolved, 20 mL of ethyl acetate was added to the above mixed solution, and the mixture was stirred for 30 min to form microemulsion droplets. Then, 14 mL of 1 M ammonia solution (analytical grade) was added to the mixed solution, and the mixture was stirred for 15 min. After stirring, 7.2 mL of [unspecified solution] was added to the solution. Tetraethyl orthosilicate (analytical grade) was added, followed by 0.72 mL of triethyl phosphate (analytical grade) after 30 min. Then, 4.554 g of calcium nitrate tetrahydrate was added after another 30 min. The resulting solution was stirred for 4 h until it became cloudy, yielding a white suspension. The white suspension was centrifuged, and the white precipitate was collected. The precipitate was washed three times with ethanol and then three times with water. The product was freeze-dried and calcined in a muffle furnace at 700 °C for 6 h to obtain MBG. MBG had 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 m². 2 / g, pore volume is 1.14cm³ 3 / g; wherein, the heating rate of the calcination process from 25℃ to 700℃ is 2℃ / 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 0.2g of MBG obtained in step S1 in 10mL of water to obtain an MBG solution; add the MBG solution dropwise to 10mL of γ-glycidoxypropyltrimethoxysilane solution and stir magnetically at 25℃ for 3h; after stirring, centrifuge to separate the precipitate, wash with water three times, and freeze-dry to obtain silanized mesoporous bioactive glass MBG@KH-560; wherein, the γ-glycidoxypropyltrimethoxysilane solution is obtained by dissolving 0.02g of γ-glycidoxypropyltrimethoxysilane in 10mL of 80% methanol and stirring magnetically at 25℃ for 30min;

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

[0054] S4. Add 0.25g of mannose to 5mL of ultrapure water to prepare a mannose solution; disperse 0.25g of MBG-g-PL obtained in step S3 in 10mL of water and then add it dropwise to the mannose solution. Stir and react at 25℃ for 6h. After the reaction is complete, collect the precipitate by centrifugation, wash it three times with water, and freeze-dry the obtained product to obtain the mesoporous bioactive glass hybrid material MBG-g-Man, with a particle size of about 110nm, a pore size distribution range of 5-20nm, and a specific surface area of ​​428.81m². 2 / g, pore volume is 1.19cm³ 3 / g. It should be noted that, unless otherwise specified, the reagents and raw materials used in the embodiments of the present invention can be obtained by purchasing them from the market; in addition, the purpose of the stirring operation in steps S1-S4 is to achieve mixing, and the stirring method can be selected according to actual needs, which will not be described in detail here.

[0055] In the above technical solution, the preparation mechanism of MBG-g-Man is as follows: First, γ-glycidyl etheroxypropyltrimethoxysilane (silane coupling agent) is used to introduce abundant epoxy groups into MBG to facilitate 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 alkylated MBG and ε-polylysine... Electrostatic adsorption allows for the grafting of ε-polylysine onto the surface of MBG. ε-polylysine acts as a connecting bridge, providing MBG with more sites for mannose reactions. The aldehyde group of mannose reacts with the amino group of ε-polylysine via a Schiff base reaction, thus modifying the MBG surface with mannose. The surface-modified mannose can specifically recognize mannose receptors on the macrophage membrane, improving uptake efficiency, inducing macrophage M2 polarization, and subsequently secreting anti-inflammatory factors to create a favorable microenvironment. This, in turn, synergistically promotes bone regeneration with mesoporous bioactive glass.

[0056] Example 2

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

[0058] Example 3

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

[0060] Example 4

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

[0064] Example 6

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

[0066] Example 7

[0067] The only difference between this embodiment and Example 1 is that in step S3, the reaction time of MBG@KH-560 with ε-polylysine solution is 24h. The other experimental parameters are the same as in Example 1 and will not be repeated 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 0.125g. The other experimental parameters are the same as in Embodiment 1 and will not be repeated 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 1.25g. The other experimental parameters are the same as in Embodiment 1 and will not be repeated here.

[0072] Example 10

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

[0074] Example 11

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

[0076] Experimental results show that high-performance mesoporous bioactive glass hybrid materials can be obtained from Examples 1 to 11.

[0077] Comparative Example 1

[0078] The only difference between Comparative Example 1 and Example 1 is that MBG is not chemically modified on the surface, 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 will not be repeated here.

[0081] TEM images of MBG-g-PL and MBG-g-Man prepared in Example 1 and MBG prepared in Comparative Example 1 are shown below. Figure 2 As shown in the figure, the overall morphology of MBG-g-PL, MBG-g-Man and MBG is not much different, indicating that surface modification does not have a significant impact on the morphology of MBG.

[0082] Furthermore, the experiments also explored the applications 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. The results of tests on biocompatibility, macrophage targeting ability, immunomodulatory ability, and in vitro osteogenic capacity were used to illustrate these applications. The specific test procedures and results are as follows:

[0083] (1) Biocompatibility testing:

[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 then cultured at a concentration of 2 × 10⁻⁶ μg / mL. 3 Cells were seeded at different density per well in 96-well plates and incubated in a cell culture incubator until adherence. Different concentrations of culture medium prepared with the material were then added, and RAW264.7 and MC3T3-E1 cells were co-cultured for 24 h and 72 h, respectively. The biocompatibility of the material was assessed using the Cell Count Kit-8 (CCK-8) assay. Cell viability results are shown in the figure below. Figure 3 As shown, where, Figure 3 Figures A and B in the figure show the cell viability results of MC3T3-E1 cells after 24 hours and 72 hours of treatment, respectively. Figure 3Figures C and D in the figure show the cell viability results of RAW264.7 cells after 24 h and 72 h of treatment, respectively. The results show that the cell viability 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. This indicates that MBG before and after functionalization has low cytotoxicity. Furthermore, the simultaneous modification of MBG with ε-polylysine and mannose can reduce its cytotoxicity to a certain extent. The results show that the mesoporous bioactive glass hybrid material prepared by the method proposed in this invention has good biocompatibility.

[0085] (2) Macrophage targeting ability test:

[0086] MBG obtained in Comparative Example 1 and MBG-g-PL and MBG-g-Man obtained in Example 1 were dispersed in cell culture medium to prepare suspensions of 100 μg / mL, which were then used as cell culture media. Figure 4 The cell transporation device shown in Figure A is used to culture macrophages. The device includes a culture dish and two chambers arranged vertically in the culture dish. A porous membrane is provided between the two chambers. The two chambers and the semipermeable membrane together form an upper chamber for placing macrophages. The space inside the culture dish excluding the upper chamber is the lower chamber, which is used to hold the cell culture medium.

[0087] Add 500 μL of material suspension to the lower chamber at a concentration of 1.5 × 10⁻⁶. 4 Macrophages (RAW264.7) were seeded into chambers at a density of [number] cells / [number], and then co-cultured with the culture medium in a cell culture incubator for 6 h and 12 h, respectively. Cell counting was performed using crystal violet staining. A 1% crystal violet solution was prepared for staining the cells, and observation and photography were conducted using a stereomicroscope. The results are shown below. Figure 4 As shown in Figure B, the number of cells in the experimental group with added MBG-g-Man was significantly higher than that in other groups, indicating that MBG-g-Man has a certain recruitment ability for macrophages. After photography, the crystal violet dye on the membrane of each group was dissolved with an equal amount of 33% acetic acid, and then the wavelength was measured at an OD value of 570 nm. The results are as follows. Figure 4 As shown in Figure C, the results indicate that MBG-g-Man can effectively recognize and capture macrophages.

[0088] (3) Immune regulation capacity 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 suspensions of 100 μg / mL. First, RAW264.7 cells were cultured at 2 × 10⁻⁶ cells / mL.5 Cells were seeded at a density of 1 / well in 6-well plates and incubated in cell culture medium containing the material for 1 day and 3 days, respectively. Total RNA and total protein were then extracted from macrophages. The results of the total RNA from macrophages are shown below. Figure 5 As shown, where, 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. The expression of macrophage polarization-related genes and proteins was detected by real-time quantitative PCR (RT-qPCR) and Western blot (WB) assays. 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 results of inflammation-related protein bands after co-culturing MBG prepared in Comparative Example 1 and MBG-g-PL and MBG-g-Man prepared in Example 1 with RAW264.7 cells. Figure 6 Figure B shows the quantitative analysis of iNOS and CD206 expression. The results indicate that MBG-g-Man can promote cell polarization to the 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] The MBG prepared in Comparative Example 1, along with 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 MBG, MBG-g-PL, and MBG-g-Man materials, and osteogenic induction complete medium. These were then mixed at a 1:2 ratio to prepare macrophage conditioned osteogenic medium. The potential of macrophage conditioned osteogenic medium to stimulate osteogenic differentiation of mouse embryonic osteoblast precursor cells (MC3T3-E1) was investigated using real-time quantitative PCR (RT-qPCR), Western blotting (WB), alkaline phosphatase (ALP) staining, and Alizarin Red (ARS) staining.

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

[0094] MC3T3-E1 cells were loaded at 5×10 4 Cells / well were seeded at a density of [number] cells / well in 24-well plates. ALP staining was performed 14 days after osteogenic induction. The alkaline phosphatase activity assay results after 14 days of induction are as follows: Figure 9 As shown in Figure A; MC3T3-E1 cells were grown at a rate of 3 × 10⁻⁶. 4 Cells / well were seeded at a density of 1:1000 cells / well in 24-well plates. ARS staining was performed 21 days after osteogenic induction. The results of calcified nodules detection 21 days after induction are as follows: Figure 9 As shown in Figure B, the results indicate that MBG-g-Man can promote osteogenic differentiation and alkaline phosphatase expression in cells, accelerate the formation of calcified nodules, and is beneficial for bone tissue regeneration. It should be noted that... Figure 2-9 The “Control” in the text indicates that cells are cultured using a cell culture medium without any hybrid materials.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention 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, Includes the following steps: S1. Preparation of mesoporous bioactive glass MBG; S2. The MBG obtained in step S1 is silanized using a silane coupling agent to obtain epoxy-functionalized MBG. S3. The epoxy-functionalized MBG obtained in step S2 is subjected to surface functionalization treatment using a polymer solution to obtain polymer-functionalized MBG, wherein the polymer contains amino and / or amine groups; 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, polyacrylamide, and polyamide-amine dendritic molecules. S4. The polymer-functionalized MBG obtained in step S3 is subjected to a secondary surface functionalization treatment using a sugar solution to obtain a mesoporous bioactive glass hybrid material; the sugar solution is a mannose solution, and the mass ratio of the polymer-functionalized MBG to the mannose in the mannose solution is 1:(0.5-5).

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

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

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

5. The preparation method according to claim 4, 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.

6. The preparation method according to claim 1, characterized in that, In step S4, the concentration of the mannose solution is 5-50 mg / mL, and the reaction time of the polymer-functionalized MBG in the mannose solution is 1-24 h.

7. The preparation method according to claim 6, 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.

8. A mesoporous bioactive glass hybrid material prepared by any one of claims 1-7.

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