Glucose responsive composite material as well as preparation method and application thereof

Abstract

The invention discloses a glucose responsive composite material as well as a preparation method and application thereof. The composite material sequentially comprises porous metal, a polydopamine layer and a mesoporous silicon dioxide coating from inside to outside, the porous metal is coated with the polydopamine layer, the polydopamine layer is coated with the mesoporous silicon dioxide coating, and preparation raw materials of the mesoporous silicon dioxide coating comprise aminated mesoporous silicon dioxide, aspirin, 4-carboxyphenylboronic acid and sodium alginate. The composite material disclosed by the invention has blood sugar concentration dual-mode response capability, aspirin is suddenly released in a hyperglycemia environment, up-regulation expression of anti-inflammatory factors is remarkably promoted, and an anti-inflammatory effect is dominated; in a normal blood glucose environment, aspirin is slowly released, and the expression of osteogenic factors is remarkably up-regulated, so that osteogenic differentiation is promoted as a leading effect; the dual-mode response realizes accurate regulation and control of ASP dosage, organically combines bone formation promotion and anti-inflammatory functions, and provides a brand new solution for patients with bone defects, especially patients with bone defects caused by diabetes mellitus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a glucose-responsive composite material, its preparation method, and its uses. Background Technology

[0002] Diabetes mellitus, a globally prevalent metabolic disease, causes significant oral complications, with periodontitis prevalence reaching 80%-90%. These patients experience rapid disease progression and severe tissue damage, manifesting as intense periodontal inflammation, rapid alveolar bone resorption, periodontal ligament fibrosis, and significantly impaired repair capacity. Regarding bone health, diabetes severely disrupts osseointegration, leading to insufficient peri-implant bone formation and delayed mineralization, resulting in a significantly lower implant success rate compared to healthy individuals. The underlying mechanisms of this pathological process involve multiple aspects: a persistently high-glycemic microenvironment not only inhibits the expression of osteogenic growth factors but also promotes the abnormal accumulation of advanced glycation end products (AGEs), thereby impairing bone formation capacity and mechanical properties; simultaneously, the chronic low-grade inflammation characteristic of diabetes is continuously activated through the inflammasome pathway, further disrupting the implant-bone interface microenvironment homeostasis and creating a vicious cycle hindering osseointegration. However, currently used clinical implant materials struggle to cope with this complex pathological environment, resulting in a persistently high implant failure rate in diabetic patients with bone infection.

[0003] In the selection of implant materials, while traditional titanium implants possess excellent mechanical strength, their elastic modulus of 110-117 GPa differs significantly from that of bone tissue (cortical bone 12-18 GPa, cancellous bone 0.1-0.5 GPa). This mismatch in mechanical properties can easily lead to stress shielding effects, resulting in clinical problems such as bone resorption and implant loosening. In contrast, the bioinert metal tantalum (Ta) shows better application prospects. Its biocompatibility is superior to titanium, and it has stronger corrosion resistance and tissue affinity, and has been widely used in orthopedics, such as in artificial joints and bone defect filling. However, due to the high processing difficulty and cost limitations, the application of tantalum implants in dentistry is still in the exploratory stage. Current research on surface modification of porous tantalum mainly focuses on three dimensions: At the macroscopic scale, cell behavior is regulated by optimizing pore parameters, and the elastic modulus can be precisely controlled through advanced 3D printing technology to achieve a high degree of matching with the mechanical properties of natural bone tissue. This not only provides an ideal space for bone tissue ingrowth but also significantly improves the integration effect of the bone-implant interface; At the microscopic scale, surface nano-processing techniques such as sandblasting and acid etching are used to increase surface roughness, improve cell adhesion energy, and promote the migration, proliferation, and differentiation of osteoblasts; At the molecular scale, functional modification is achieved through the loading of bioactive molecules, all working together to improve the osteoconductivity, osteoinductive properties, and antibacterial properties of the implant.

[0004] However, even though porous tantalum performs excellently in the early stages of osseointegration, its long-term performance under pathological conditions such as diabetes still faces severe challenges. Oxidative stress, inflammatory responses, and impaired bone formation caused by high-glucose environments are particularly prominent, and simple surface structure optimization is insufficient to meet these complex needs. Therefore, researchers are dedicated to developing intelligent responsive surface modification technologies, among which glucose-responsive nanomedicine delivery systems have shown unique advantages. Currently, these systems are mainly divided into three categories: glucose oxidase (GODx), concanavalin A (ConA), and phenylboronic acid (PBA). Although GODx and ConA have attracted much attention in the field of glucose response, the former is sensitive to environmental pH, and the latter is biocompatible; both suffer from drawbacks such as biotoxicity, poor stability, and high cost. This characteristic makes the PBA system a promising candidate to overcome the limitations of GODx and ConA, pointing to a new research direction for the development of glucose-responsive nanomedicine delivery systems.

[0005] Therefore, there is a need to develop a safer and more effective glucose-responsive composite material to address the technical challenges of treating and repairing bone defects in diabetic patients. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a glucose-responsive composite material, its preparation method, and its uses.

[0007] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0008] The first aspect of the present invention protects a glucose-responsive composite material, comprising, from the inside out, a porous metal, a polydopamine layer coated on the porous metal, and a mesoporous silica coating coated on the polydopamine layer, wherein the raw materials for preparing the mesoporous silica coating include aminated mesoporous silica, aspirin, 4-carboxyphenylboronic acid, and sodium alginate.

[0009] The mesoporous silica coating of this application consists of core-shell structured particles. The core layer is 4-carboxyphenylboronic acid-modified mesoporous silica loaded with aspirin. It is obtained by loading aspirin onto aminated mesoporous silica as a framework after covalent modification with 4-carboxyphenylboronic acid. It can maintain the three-dimensional interconnected pore structure of aminated mesoporous silica. The shell layer is a sodium alginate coating layer, which is cross-linked to the surface of the core layer by sodium alginate through amide bonds. It covers and seals the mesoporous pores of the core layer to form a dense barrier.

[0010] This invention grafts 4-carboxyphenylboronic acid onto the surface of aminated mesoporous silica via an amidation reaction, while maintaining the mesoporous structure of the aminated mesoporous silica, ensuring efficient loading and controlled release of aspirin. An outer layer of sodium alginate forms a dense shell via amide bonds, sealing the mesoporous pores of the aminated mesoporous silica and acting as a "molecular valve." In the blood glucose microenvironment (blood glucose concentration of 5-25 mM), the dynamic competition between the o-dihydroxyl group of sodium alginate (SA) and the glucose molecule for the binding site of carboxyphenylboronic acid (CPBA) triggers shell swelling and pore opening, initiating the on-demand release of aspirin (burst release / sustained release). In other words, under normal blood glucose levels (corresponding to glucose ≤1 mg / mL in the release kinetics study or blood glucose concentration <11 mM in the in vivo osteogenic experiment), the valve closes and the orifice closes, allowing for the slow release of aspirin, preferentially activating osteogenic factors, and promoting osteogenic differentiation. Conversely, under hyperglycemic conditions (corresponding to glucose 2-5 mg / mL in the release kinetics study or a glucose concentration ≥11 mM as measured by a blood glucose meter), the valve opens and the orifice opens, releasing aspirin rapidly, quickly inhibiting inflammatory factors and upregulating the expression of anti-inflammatory factors, thus relieving the inhibition of cell proliferation and differentiation caused by high glucose. The composite material of this invention achieves the time-sequential synergistic release of anti-inflammatory factors (CD206, iNOS, and CD86) and osteogenic factors (ALP, RUNX-2, and OPN), significantly enhancing the bioactivity and integration efficacy of the composite material-bone interface.

[0011] Bone defects in diabetic patients present significant challenges in treatment and repair because new bone formation depends on the differentiation of mesenchymal stem cells into osteoblasts. Studies have shown that a high-glucose environment can inhibit not only the proliferation of bone marrow mesenchymal stem cells but also their osteogenic differentiation capacity. 4-Carboxyphenylboronic acid exists in both hydrophobic and charged hydrophilic forms, and can specifically bind to compounds with cis-diol units, exhibiting excellent glucose responsiveness. In the presence of glucose, the hydrophobic and uncharged PBA transforms into a charged hydrophilic form, responding to compounds with different diol structures through a competitive binding mechanism. Simultaneously, in conjunction with a sodium alginate coating, under hyperglycemic conditions (corresponding to glucose 2-5 mg / mL in release kinetics studies or a glucose concentration ≥11 mM as measured by a glucometer), phenylboronic acid-sodium alginate swells, opening pores and releasing aspirin rapidly. Aspirin rapidly downregulates the M1 macrophage markers iNOS and CD86, and upregulates the M2 marker CD206, "cooling down" the hyperglycemic-induced chronic inflammatory microenvironment and relieving the inhibition of cell proliferation by inflammation. Under normal blood glucose levels (corresponding to glucose ≤1 mg / mL in release kinetics studies or a blood glucose concentration less than 11 mM in in vivo osteogenic experiments), it promotes the expression of ALP, RUNX-2, and OPN, thus promoting osteogenic differentiation.

[0012] In some embodiments, the mass ratio of the aminated mesoporous silica to 4-carboxyphenylboronic acid is 1:(5-7).

[0013] In some embodiments, the mass ratio of sodium alginate to 4-carboxyphenylboronic acid is 1:(1-10).

[0014] In some embodiments, the porous metal has a pore size of 400-600 μm and a porosity of 70-80%. Preferably, the pore size is 400 μm and the porosity is 70%.

[0015] In some embodiments, the metal in the porous metal is selected from tantalum, titanium, or alloys thereof. Preferably, it is tantalum. The applicant has previously conducted research on metallic materials and found that porous tantalum exhibits superior cell adhesion, proliferation, and migration properties compared to non-porous tantalum and titanium in cell proliferation experiments. Furthermore, after modification with polydopamine, porous tantalum demonstrates better adsorption of AMS and adhesion than non-porous tantalum and titanium.

[0016] In some embodiments, the porous metal is a cylinder with a diameter of 3-15 mm and a height of 1-5 mm. Preferably, it is a cylinder with a diameter of 3 mm and a height of 3 mm; or a cylinder with a diameter of 10 mm and a height of 3 mm.

[0017] In some embodiments, the polydopamine-modified porous metal is prepared by reacting the porous metal and dopamine hydrochloride in a solvent to obtain the polydopamine-modified porous metal.

[0018] In some embodiments, the reaction temperature is 30-44°C. Preferably, it is 37°C.

[0019] In some embodiments, the reaction time is 20-30 hours. Preferably, it is 24 hours.

[0020] In some embodiments, the solvent is an 8-12 mmol / L Tris-HCl buffer. Preferably, it is a 10 mmol / L Tris-HCl buffer.

[0021] In one specific embodiment, porous tantalum was immersed in a Tris-HCl buffer solution with a pH of 8.5 and a concentration of 2 mg / mL dopamine hydrochloride, and reacted at 37°C for 24 h to obtain polydopamine-modified porous tantalum (labeled as DT).

[0022] In some embodiments, the thickness of the mesoporous silica coating is 200-600 nm.

[0023] In some embodiments, the aminated mesoporous silica has a pore size of 6-8 nm and a particle size of 200-300 nm.

[0024] In some embodiments, the method for preparing the mesoporous silica coating is as follows:

[0025] 1) Amino-modified mesoporous silica and 4-carboxyphenylboronic acid react in an organic solvent under the action of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to obtain phenylboronic acid-modified mesoporous silica.

[0026] 2) The phenylboronic acid-modified mesoporous silica and aspirin are mixed in the first solvent to obtain aspirin-loaded phenylboronic acid-modified mesoporous silica;

[0027] 3) The aspirin-loaded phenylboronic acid-modified mesoporous silica and sodium alginate are reacted in a second solvent under the action of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC to obtain a mesoporous silica coating.

[0028] In this application, the mesoporous silica modified with phenylboronic acid retains the channel-like pore morphology of mesoporous silica, while after being coated with sodium alginate, the channel-like pores of the mesoporous silica are closed, forming a dense surface.

[0029] In some specific embodiments, in 1), the mass ratio of 4-carboxyphenylboronic acid, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is 1:(1-3):(2-8). Preferably, it is 1:2.2:4.1.

[0030] In some specific embodiments, the mass ratio of the aminated mesoporous silica to 4-carboxyphenylboronic acid is (0.1-10):1. Preferably, it is 1.5:1.

[0031] In some specific embodiments, in 1), the organic solvent is selected from DMSO.

[0032] In some specific embodiments, in 1), the temperature of the reaction is room temperature.

[0033] In some specific embodiments, in step 1), the reaction time is 20-30 hours. Preferably, it is 24 hours.

[0034] In some specific embodiments, in step 1), the reaction is further followed by centrifugation and washing, wherein the washing agent is one or more of DMSO, water, and methanol. Preferably, DMSO, water, and methanol are used for washing sequentially.

[0035] In one specific embodiment, 67 mg of 4-carboxyphenylboronic acid (CPBA), 83.3 mg of NHS and 276.2 mg of EDC·HCl were mixed in DMSO, and then 5 mL of 20 mg / mL aminated mesoporous silica nanoparticles (MSN-NH2) were added dropwise. After reacting for 24 hours, the mixture was centrifuged and washed sequentially with DMSO, water and methanol to obtain phenylboronic acid-modified mesoporous silica.

[0036] In this application, 4-carboxyphenylboronic acid and aminated mesoporous silica nanoparticles were directly reacted, and it was found that phenylboronic acid could not be grafted onto the outer surface of mesoporous silica.

[0037] In some specific embodiments, in 2), the first solvent is selected from ethanol. Preferably, it is anhydrous ethanol.

[0038] In some specific embodiments, in step 2), the mass ratio of aspirin to phenylboronic acid-modified mesoporous silica is 1:(2-6). Preferably, it is 1:1.5.

[0039] In some specific embodiments, in step 2), the mixing time is 20-30 hours. Preferably, it is 24 hours.

[0040] In some specific implementations, in 2), the mixing condition is light protection.

[0041] In some specific embodiments, step 2) further includes centrifugation, washing, and drying after mixing. The washing agent used is ethanol. The drying temperature is 60°C.

[0042] In one specific embodiment, 60 mg of CPBA-MSN particles were placed in 10 mL of a 4 mg / mL aspirin ethanol solution and stirred for 24 hours at room temperature in the dark. The precipitate was then collected by centrifugation and washed with ethanol. The cleaned precipitate was then vacuum-dried at 60°C for 10 hours to obtain aspirin-loaded phenylboronic acid-modified mesoporous silica.

[0043] In some specific embodiments, in 3), the second solvent is selected from a phosphate buffer solution.

[0044] In some specific embodiments, in step 3), the mass ratio of sodium alginate, NHS, and EDC is 1:(1-4):(3-8). Preferably, it is 1:2.5:5.

[0045] In some specific embodiments, in step 3), the mass ratio of sodium alginate to aspirin-loaded phenylboronic acid-modified mesoporous silica is 1:(1-4). Preferably, it is 1:3.

[0046] In some specific embodiments, in 3), the temperature of the reaction is room temperature.

[0047] In some specific implementations, in 3), the reaction time is 3 hours.

[0048] In some specific embodiments, in step 3), centrifugation is further included after the reaction.

[0049] In a specific embodiment, 10 mg of sodium alginate (SA) was dissolved in a phosphate buffer solution, and 25 mg of NHS and 50 mg of EDC·HCl were added as catalysts. The mixture was stirred for 30 min to obtain an SA solution. 30 mg of phenylboronic acid-modified mesoporous silica loaded with aspirin was added to the SA solution and stirred for 3 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 5 min, and the precipitate was retained. The precipitate was a mesoporous silica coating.

[0050] The second aspect of this invention protects a method for preparing the composite material described above, comprising the following:

[0051] A porous metal is coated with a polydopamine layer to obtain a polydopamine-modified porous metal.

[0052] The composite material is obtained by mixing and incubating the polydopamine-modified porous metal and mesoporous silica coating.

[0053] In some embodiments, the mixing incubation is carried out in a phosphate buffer solution, preferably by mixing the mesoporous silica coating and the phosphate buffer solution to form a solution with a concentration of 10 mg / ml, and then drop-coating it onto the polydopamine-modified porous metal.

[0054] In some embodiments, the incubation time is 0.5-2 hours, preferably 1 hour.

[0055] In some embodiments, the incubation temperature is 30-42°C. Preferably, it is 37°C.

[0056] A third aspect of this invention protects the use of the aforementioned composite material in the preparation of bone defect repair products or osteogenesis-promoting products.

[0057] In some embodiments, the bone defect is a diabetic bone defect.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] 1) The composite nanomaterial of this invention utilizes sodium alginate (SA) and 4-carboxyphenylboronic acid (CPBA) modified MSN-NH2 to construct a dynamically reversible borate ester bond and load aspirin (ASP), giving the composite nanomaterial a glucose concentration-dependent "intelligent drug release" characteristic. At normal blood glucose levels (corresponding to glucose ≤1 mg / mL in release kinetic studies or blood glucose concentration <11 mM in in vivo osteogenic experiments), ASP can be continuously and slowly released (cumulative release over 7 days reaches 28.14%), directionally promoting osteogenic differentiation and regulating bone metabolism. However, in a hyperglycemic environment (corresponding to glucose 2-5 mg / mL in release kinetic studies or glucose concentration ≥11 mM measured by a blood glucose meter), glucose competitively binds to CPBA, triggering rapid ASP release, promptly alleviating inflammation and inhibiting bone resorption. This "dual-mode blood glucose concentration response" mechanism enables precise control of ASP dosage, organically combining bone-promoting and anti-inflammatory functions, providing a novel solution for patients with bone defects, especially those with diabetes-related bone defects.

[0060] 2) In in vitro osteogenic experiments, the composite nanomaterials of this invention were found to not only promote the activity of alkaline phosphatase, an early osteogenic marker, but also promote the deposition of late-stage mineralized nodules. Furthermore, the composite material of this invention promotes cell proliferation and increases cell density, and is safe for cells and exhibits good blood compatibility.

[0061] 3) The composite nanomaterial of the present invention has a glucose threshold-dependent bimodal behavior reprogramming capability. That is, at normal blood glucose levels (corresponding to glucose <1 mg / mL in the release kinetic study or blood glucose concentration <11 mM in the in vivo osteogenic experiment), it significantly upregulates the expression of osteogenic factors (ALP, RUNX-2 and RUNX-2), thereby promoting osteogenic differentiation. At high blood glucose levels (corresponding to glucose 2-5 mg / mL in the release kinetic study or blood glucose concentration ≥11 mM as measured by a blood glucose meter), the phenylboronic acid of the composite material forms a reversible glycol ester with glucose in the environment, triggering shell swelling and opening the "molecular valve" to rapidly release high doses of aspirin, significantly inhibiting iNOS and CD86 expression and promoting CD206 upregulation, thus becoming dominated by anti-inflammatory effects.

[0062] 4) When the composite nanomaterial of the present invention was implanted in a type I diabetic rat femoral defect model for 8 weeks, Micro-CT quantitatively showed the bone volume fraction and the new bone in the defect area was completely bridged, confirming that it can still effectively promote bone regeneration and rapidly fill bone defects in a high-glucose microenvironment.

[0063] 5) The elastic modulus of the composite material of the present invention is 0.923 MPa, which is highly matched with the mechanical properties of human cancellous bone (0.1-2 GPa), showing excellent biomechanical compatibility. Attached Figure Description

[0064] Figure 1 These are transmission electron microscopy images of aminated mesoporous silica nanoparticles (MSN), phenylboronic acid-modified mesoporous silica (CPBA-MSN), and sodium alginate-coated CPBA-MSN@ASP (AMS) from Example 3 of this invention.

[0065] Figure 2 This is the Fourier transform infrared spectrum of the AMS synthesized in Example 3 of this invention.

[0066] Figure 3 This invention relates to the particle size analysis and zeta potential analysis of aminated mesoporous silica nanoparticles (MSN), phenylboronic acid modified mesoporous silica (CPBA / MSN), and sodium alginate-coated CPBA-MSN@ASP (AMS) in Example 3 of this invention.

[0067] Figure 4 These are photographs and scanning electron microscope images of the composite material in Example 3 of this invention.

[0068] Figure 5 The images (magnification: 3K) and EDS analysis of porous tantalum, polydopamine-modified tantalum (DT), and polydopamine-modified tantalum with MSN coating (DT-MSN) and AMS coating (DT-AMS) coating are from Example 3 of this invention.

[0069] Figure 6 This is a graph showing the release results of the composite material DT-AMS in simulated body fluids with different glucose concentrations in Example 4 of the present invention.

[0070] Figure 7 This is a fluorescence curve of the response test of CPBA-MSN under different glucose concentrations in Example 4 of the present invention.

[0071] Figure 8 This is a schematic diagram of the hemolysis test results of different groups in Example 5 of the present invention.

[0072] Figure 9 This is a schematic diagram of the test results of cell proliferation in different groups in Example 5 of the present invention.

[0073] Figure 10 These are photographs of osteogenic induction test results (Alizarin Red ARS staining) from different groups in Example 6 of this invention.

[0074] Figure 11 These are photographs of osteogenic induction test results (alkaline phosphatase ALP staining) from different groups in Example 6 of this invention.

[0075] Figure 12These are staining results of DCFH-DA fluorescent probes in different groups in Example 7 of this invention.

[0076] Figure 13 This is a graph showing the test results of osteogenic and anti-inflammatory gene expression in different groups in Example 7 of the present invention.

[0077] Figure 14-1 These are photographs showing the protein expression test results of osteogenic factors in different groups in Example 7 of this invention.

[0078] Figure 14-2 These are photographs showing the protein expression test results of anti-inflammatory genes in different groups in Example 7 of this invention.

[0079] Figure 15 This is a photograph of the composite material implanted in the femur in Embodiment 8 of the present invention.

[0080] Figure 16 These are micro-CT images of rat femurs made of different composite materials in Example 8 of this invention.

[0081] Figure 17 This refers to the Micro-CT 3D reconstruction of the femur after 8 and 12 weeks of implantation treatment with different groups of composite materials in Example 8 of the present invention.

[0082] Figure 18 This is a statistical analysis chart of the bone volume fraction of the femur after 8 and 12 weeks of implantation treatment with different groups of composite materials in Example 8 of the present invention.

[0083] Figure 19 The figure shows the CCK8 experimental results of the non-porous tantalum, porous tantalum and non-porous titanium groups in Comparative Example 1 of this invention.

[0084] Figure 20 The figures show the results of live and dead staining and migration experiments of the non-porous tantalum, porous tantalum and non-porous titanium groups in Comparative Example 1 of this invention.

[0085] Figure 21 This is a comparison chart showing the adhesion performance test results of the non-porous titanium surface modified and unmodified polydopamine layers in Comparative Example 1 of the present invention.

[0086] Figure 22 This is a comparison chart showing the adhesion performance test results of the porous tantalum surface modified and unmodified polydopamine layers in Comparative Example 1 of the present invention.

[0087] Figure 23 This is a comparison chart showing the adhesion performance test results of the non-porous tantalum surface modified and unmodified polydopamine layers in Comparative Example 1 of the present invention. Detailed Implementation

[0088] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0089] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0090] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0091] In the following embodiments of this application, aminated mesoporous silica is labeled MSN; aspirin is labeled ASP; 4-carboxyphenylboronic acid is labeled CPBA; sodium alginate is labeled SA; 4-carboxyphenylboronic acid-grafted MSN is labeled CPBA-MSN; CPBA-MSN loaded with aspirin is labeled CPBA-MSN@ASP; and CPBA-MSN@ASP surface coated with sodium alginate is labeled AMS.

[0092] In the following embodiments of this application, the in vitro experiments used porous tantalum (Ta) cylinders with a diameter of 10 mm and a height of 3 mm. The pore size was 400 μm, and the porosity was 70%.

[0093] In vivo experiments used porous tantalum (Ta) cylinders with a diameter of 3 mm and a height of 6 mm. The pore size was 400 μm, and the porosity was 70%.

[0094] Unless otherwise specified, the reactions in the following embodiments of this application are carried out at room temperature, i.e., 20-35°C.

[0095] Example 1

[0096] This embodiment provides a glucose-responsive composite material and its preparation method, including the following steps:

[0097] 1. Preparation of polydopamine-modified porous tantalum (labeled DT)

[0098] A 10×3 mm (diameter×height) porous tantalum (T) was immersed in a dopamine hydrochloride solution and reacted in a shaker at 37°C in the dark for 24 hours. Afterward, the tantalum was removed and repeatedly rinsed with deionized water to remove any unadhered polydopamine, yielding polydopamine-modified porous tantalum (labeled DT). The dopamine hydrochloride solution was prepared by adding dopamine hydrochloride (PDA) to 10 mmol / L Tris-HCl buffer to obtain a dopamine hydrochloride solution with a concentration of 2 mg / ml and a pH of 8.5.

[0099] 2. Preparation of mesoporous silica coating (labeled AMS)

[0100] 2.1 Phenylboronic acid-modified mesoporous silica (labeled as CPBA-MSN particles)

[0101] The synthesis process of MSN-NH2 and CPBA was optimized.

[0102] First, 67 mg of 4-carboxyphenylboronic acid (CPBA), 83.3 mg of NHS, and 276.2 mg of EDC·HCl were dissolved in DMSO and stirred at room temperature for 30 minutes to obtain a DMSO mixture. Then, 5 mL of pre-purified aminated mesoporous silica nanoparticles (MSN-NH2) were slowly added dropwise to the DMSO mixture at a concentration of 20 mg / mL. After stirring at room temperature for 24 hours, centrifugation was performed, followed by washing with DMSO, water, and methanol sequentially to obtain phenylboronic acid-modified mesoporous silica (i.e., CPBA-MSN particles).

[0103] 2.2 CPBA-MSN nanoparticles loaded with aspirin (ASP) (labeled as CPBA-MSN@ASP particles)

[0104] The 60 mg of CPBA-MSN particles obtained in step 2.1 were placed in 10 mL of a 4 mg / mL aspirin ethanol solution and stirred for 24 hours at room temperature in the dark. Afterwards, the precipitate was collected by centrifugation and gently washed with ethanol. The cleaned precipitate was then vacuum-dried at 60 °C for 10 hours to obtain ASP-loaded CPBA-MSN particles (labeled as CPBA-MSN@ASP particles).

[0105] 2.3 Sodium alginate-coated CPBA-MSN@ASP particles (labeled as AMS particles)

[0106] Dissolve 10 mg of sodium alginate (SA) in 5 mL of phosphate buffer solution, add 25 mg of NHS and 50 mg of EDC·HCl as catalysts, stir for 30 min to obtain SA solution; add 30 mg of CPBA-MSN@ASP particles obtained in step 2.2 to SA solution and stir for 3 h. Centrifuge at 10000 rpm for 5 min to retain the precipitate, which is sodium alginate coated CPBA-MSN@ASP particles (labeled AMS), and store at 4 °C for further use.

[0107] 3. AMS modifies DT

[0108] The AMS precipitate obtained in step 2.3 was dissolved in phosphate buffer with a pH of 6.5 to form an AMS solution with a concentration of 10 mg / ml. The AMS solution was then slowly drop-coated onto the polydopamine-modified porous tantalum DT obtained in step 1 after freeze-drying, and incubated at 37°C for 1 h to attach AMS to the dopamine coating, thereby obtaining a glucose-responsive composite material (denoted as DT-AMS).

[0109] The applicant also followed the reference (Hou L et al. Self-Regulated Carboxyphenylboronic Acid-Modified Mesoporous Silica Nanoparticles with "TouchSwitch" Releasing Property for Insulin delivery. ACS Appl Mater Interfaces. 2018 Jul 5; 10(26):21927-21938. doi:10.1021 / acsami.8b06998.Epub2018Jun 22.PMId:29932320), that is, without using NHS and EDC·HCl to prepare phenylboronic acid-modified mesoporous silica, and the rest were the same as in step 2.1, to obtain the product. The obtained product was characterized by Fourier transform infrared spectroscopy (FTIR), see Figure 2 B.

[0110] from Figure 2 As can be seen from B, without the use of NHS and EDC·HCl, 1300cm -1 At this point, the peak is relatively flat and no peak appears, indicating that the target chemical bond B–O was not effectively formed, and the surface grafting reaction did not occur successfully; or, although the grafting reaction occurred, the product concentration did not reach the detection limit of the infrared spectrum due to the low grafting efficiency.

[0111] Example 2: ASP Load

[0112] 1) Preparation of the standard curve: Aspirin was dissolved in anhydrous ethanol to a concentration of 1 mg / ml. The solution was diluted to 100, 80, 64, 51.2, 40.96, and 32.768 μg / L (all 8-fold dilutions). The diluted solutions were then measured at OD0.05. 280 The absorbance was measured at nm. The standard curve of aspirin in anhydrous ethanol was calculated by using the absorbance and concentration.

[0113] 2) Calculation of ASP loading: The total ASP is calculated using the 4 mg / ml aspirin ethanol solution from step 2.2 of Example 1. After the reaction is complete, the solution is centrifuged, the supernatant is collected, and the OD value (280 nm) is measured. This represents the free ASP. The drug loading, or loading efficiency, is calculated using the following formula.

[0114] Loading efficiency=(total amount ofASP-[free ASP]) / (total amountofCPBA-MSN)×100%

[0115] In the formula, "total amount of ASP" refers to the amount of ASP added, expressed in mg.

[0116] Free ASP refers to the free ASP in the supernatant after centrifugation, and the unit is mg.

[0117] Then, the drug loading is divided by the mass of CPBA-MSN to obtain the ASP loading.

[0118] Based on the total mass of CPBA-MSN, the aspirin loading is 20%.

[0119] Example 3: Performance Study of Composite Materials

[0120] In this embodiment, the composite material obtained in Example 1 is characterized, including mechanical properties, energy dispersive X-ray spectroscopy, and other tests.

[0121] 3.1 Morphological Analysis

[0122] The morphologies of three groups of materials—aminated mesoporous silica nanoparticles (MSN) from Example 1, phenylboronic acid-modified mesoporous silica (CPBA-MSN) from step 2.1 of Example 1, and sodium alginate-coated CPBA-MSN@ASP (AMS) from step 2.2 of Example 1—were observed using transmission electron microscopy (TEM, Hitachi, HT7700, Japan). The morphological analysis results are shown below. Figure 1 .

[0123] from Figure 1 It was observed that all three materials—MSN (aminated mesoporous silica nanoparticles), CPBA-MSN (phenylboronic acid-modified mesoporous silica), and AMS (sodium alginate-coated CPBA-MSN@ASP)—maintained a highly regular spherical structure. The raw MSN sample exhibited typical mesoporous characteristics, with a clear channel-like pore structure on its surface and a uniform particle size distribution (215.6 ± 12.3 nm, n = 3). Notably, the CPBA modification process did not significantly alter the mesoporous structure of MSN; CPBA-MSN retained its intact channel-like pore morphology. More importantly, AMS formed after SA coating exhibited a unique core-shell structure. TEM observation revealed a uniformly thick (15-20 nm) transparent coating layer (i.e., a sodium alginate coating layer) on the surface of the CPBA-MSN@ASP particles, which effectively sealed most of the mesoporous entrances on the MSN.

[0124] Fourier transform infrared (FTIR) spectrometer (Nicolet iS50, Thermo Fisher Scientific, USA) with a spectral range of 500 to 4000 cm⁻¹ -1 Chemical groups were identified in 4-carboxyphenylboronic acid (CPBA), amino-modified mesoporous silica nanoparticles (MSN), phenylboronic acid-modified mesoporous silica (CPBA / MSN, also known as CPBA-MSN), and sodium alginate-coated CPBA-MSN@ASP (AMS) samples. Results are shown in […]. Figure 2 .

[0125] from Figure 2 As shown in section A, in the infrared spectrum of phenylboronic acid-modified mesoporous silica (CPBA / MSN), 1636.71 cm⁻¹ -1 The stretching vibration peak of the CO bond at 1321.23 cm⁻¹ -1 The characteristic peak of the BO bond at [location] confirms the successful grafting of CPBA. However, the complete disappearance of the characteristic peak of the BO bond in the AMS infrared spectrum (1321.23 cm⁻¹) further confirms this. -1 This confirms that a dynamic and reversible covalent connection has been formed between CPBA and SA.

[0126] The particle size distributions of amino-modified mesoporous silica nanoparticles (MSN), phenylboronic acid-modified mesoporous silica (CPBA-MSN), and sodium alginate-coated CPBA-MSN@ASP (AMS) nanoparticles were precisely measured using dynamic light scattering (DLS) technology, and the surface charge state of the particles was evaluated using Zeta potential analysis. Results are shown below. Figure 3 .

[0127] from Figure 3It was found that the average hydrodynamic diameter of AMS significantly increased to 329.17±18.5 nm (n=3), which was significantly higher than that of CPBA-MSN (224.58±5.67 nm). Surface potential analysis revealed that the electrochemical properties of the material surface changed significantly with the modification process: MSN (+8.92 mV) → CPBA-MSN (-24.6 mV) → AMS (-41.6 mV). This result provides direct evidence for the mechanism of SA as a highly efficient "pore-sealing agent" for mesoporous silica surfaces.

[0128] Scanning electron microscopy (SEM) images of the composite material were observed, and the results are shown below. Figure 4 In this image, A is the actual photograph, and B is a scanning electron microscope image at different magnifications.

[0129] from Figure 4 As can be seen from A, the diameter and thickness of porous tantalum increase slightly after forming a composite material.

[0130] from Figure 4 As shown in section B, the composite material has a uniformly distributed three-dimensional interconnected pore structure with controllable pore size.

[0131] In summary, the successful construction and structural characteristics of the composite material were systematically verified from multiple dimensions, including morphology, size, surface chemistry, and molecular structure. This gradient change in potential not only confirms the successful modification of SA, but also indicates a significant increase in the negative charge density on the composite material surface. This enhanced electronegativity will effectively improve the colloidal stability of the composite material, thereby extending its in vivo cycling time.

[0132] 3.2 Microstructure Characterization Results and Energy-Dispersive X-ray Spectroscopy (EDS)

[0133] The adhesion of porous tantalum, polydopamine-modified porous tantalum (DT), polydopamine-modified porous tantalum coated with mesoporous silica (DT-MSN), and polydopamine-modified porous tantalum coated with AMS (DT-AMS) coating were observed in detail using scanning electron microscopy (SEM, ZEISS, Germany). Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDS). The analytical results are shown below. Figure 5 In the image, A is a scanning electron microscope image, and B is an EDS analysis image.

[0134] The preparation method of polydopamine-modified porous tantalum coated with mesoporous silica coating (DT-MSN) is as follows: pre-purified MSN-NH2 is slowly added dropwise at a concentration of 2 mg / ml to the polydopamine coating coated with freeze-dried polydopamine-modified porous tantalum DT (prepared in the same way as step 1 in Example 1), and incubated at 37°C for 1 h to attach MSN to the polydopamine coating. The resulting product is denoted as DT-MSN.

[0135] The preparation method of polydopamine-modified porous tantalum surface coated with AMS coating (DT-AMS) is as follows: The AMS solution obtained in step 2.3 of Example 1 is slowly added dropwise at a concentration of 2 mg / ml to the polydopamine coating of freeze-dried polydopamine-modified tantalum DT (prepared in the same way as step 1 in Example 1), and incubated at 37°C for 1 h to attach AMS to the polydopamine coating. The resulting product is denoted as DT-AMS.

[0136] from Figure 5 As shown in Figure A, at a magnification of 3000x, it can be clearly observed that MSN and AMS nanoparticles in DT-MSN and DT-AMS are uniformly distributed on the porous tantalum surface, forming a complete and dense coating.

[0137] from Figure 5 As shown in Figure B, porous tantalum only detected Ta element signals; polydopamine-modified porous tantalum (DT) detected significant C (12.3±0.5%) and O (15.7±0.3%) element signals, confirming the successful modification of polydopamine; the MSN-coated polydopamine-modified porous tantalum group (DT-MSN) detected characteristic Si element signals (18.8±0.2%); particularly noteworthy is the detection of Na element signals (10.0±0.1%) in the AMS-coated polydopamine-modified porous tantalum group (DT-AMS), which directly confirms the successful modification of sodium alginate (SA). These characterization results, from macroscopic to microscopic dimensions, systematically verify the successful preparation of the composite material and its excellent physicochemical properties, laying a solid foundation for subsequent biomedical applications.

[0138] Example 4: Drug Release Performance Study

[0139] 4.1 Release kinetics of DT-AMS

[0140] To evaluate the ASP release behavior of the composite material (DT-AMS) obtained in Example 1 under four different glucose concentrations (0 mg / ml, 1 mg / ml, 2 mg / ml, 5 mg / ml), the experiment was conducted as follows:

[0141] First, glucose was dissolved in SBF simulated body fluid at concentrations of 0 mg / ml, 1 mg / ml, 2 mg / ml and 5 mg / ml, respectively, to prepare a series of glucose concentration solutions.

[0142] Subsequently, DT-AMS was added to these solutions to simulate the in vivo environment for drug release experiments.

[0143] Then, every 24 hours, 1 mL of the supernatant was taken out and the release amount of ASP was measured using a microplate reader at a wavelength of 280 nm. After the test was completed, the taken supernatant was put back into the original release system to maintain the consistency of experimental conditions.

[0144] The analysis results are shown below. Figure 6 .

[0145] from Figure 6 It was found that the DT-AMS system exhibits a significant concentration-dependent release behavior. In the DT-AMS + 0 mg / mL glucose group, aspirin (ASP) followed a basal osmotic diffusion pattern, with a cumulative release of 28.14% over 7 days. When the glucose concentration increased to physiological levels (1 mg / mL), the release kinetics changed significantly, with a cumulative release of 41.82% over 7 days and 77.85% over 17 days, a 2.8-fold increase compared to the DT-AMS + 0 mg / mL glucose group (28.14%). Notably, the release behavior under high glucose conditions exhibited a typical concentration gradient response: the release rate in the 2 mg / mL glucose solution was 56.12% over 7 days (an increase of 11.78% compared to the 1 mg / mL group), further increasing to 68.49% at 5 mg / mL (an increase of 12.37% compared to the 2 mg / mL group). This intelligent response mechanism stems from the dynamic competition between the o-dihydroxy group of sodium alginate (SA) and the binding site of glucose molecule on carboxyphenylboronic acid (CPBA)—as glucose concentration increases, the SA-CPBA bond breaks more rapidly, leading to an increased drug release rate. Kinetic analysis shows that the release curve exhibits a non-linear increase in the 0-1 mg / mL range, while transitioning to a linear increase in the 1-5 mg / mL range, providing an important theoretical basis for personalized drug administration in diabetic patients.

[0146] 4.2 Response and release characteristics of the CPBA-MSN system under different glucose concentrations

[0147] To verify glucose responsiveness, the phenylboronic acid-modified mesoporous silica (CPBA-MSN) produced in step 2.1 of Example 1 was investigated for its release characteristics under different glucose concentrations. The investigation included the following:

[0148] First, aqueous solutions of Alizarin Red (ARS) and CPBA-MSN were prepared independently, both using deionized water as the solvent. For the CPBA-MSN solution, uniform dispersion was ensured by sonication, and stirring was performed under light-protected conditions. While continuously stirring, the Alizarin Red aqueous solution was slowly added dropwise to the CPBA-MSN solution to promote the reaction between the two. This reaction process lasted for 3 hours.

[0149] The reaction mixture was then centrifuged at 10,000 rpm to precipitate the complex. It was washed three times with deionized water and dried under vacuum at 50°C to obtain pure CPBA-MSN-ARS.

[0150] Then, CPBA-MSN-ARS was placed in glucose solutions of different concentrations (0, 1, 2, 5 mg / ml) (prepared from SBF simulated body fluids) and the reaction was carried out using a fluorescence spectrophotometer.

[0151] The fluorescence scanning results are shown in Figure 7 .

[0152] from Figure 7 It was observed that as the glucose concentration gradually increased from 0 mg / mL to 5 mg / mL, the fluorescence intensity of the CPBA-MSN group showed a significant decreasing trend, indicating that the CPBA-MSN is glucose-sensitive and the released drug amount is directly proportional to the glucose concentration. Within the 1-2 mg / mL range (the borderline value for diabetes), the decrease in fluorescence intensity was relatively gradual, indicating that the binding of Alizarin Red (ARS) to CPBA was relatively stable. When the glucose concentration exceeded 2 mg / mL, the fluorescence intensity decreased significantly; and under the condition of 5 mg / mL (hyperglycemic state), the fluorescence intensity further decreased. This concentration-dependent change in fluorescence intensity is highly consistent with the release behavior of ARS, confirming the sensitivity of CPBA-MSN to changes in glucose concentration.

[0153] Example 5 Cell Experiment

[0154] 5.1 Hemolysis test

[0155] Fresh rat blood was collected and centrifuged at 1500 rpm for 10 min to collect red blood cells. These red blood cells were then resuspended in an equal volume of PBS, and this washing process was repeated three times until the supernatant became almost colorless. Next, the red blood cells were diluted with PBS to a 4% red blood cell suspension.

[0156] In the experimental group, red blood cell suspension and extraction solution were mixed at a volume ratio of 1:1 and then incubated at 37°C for 4 hours. After incubation, the mixture was centrifuged again, and the absorbance (OD value) of the supernatant was measured at a wavelength of 545 nm. The hemolysis ratio (%) was calculated according to the following formula, and the average value was recorded for analysis. The extraction solution refers to the supernatant obtained by soaking porous tantalum, polydopamine-modified porous tantalum (DT), and amino-mesoporous silica-coated DT (DT-MSN) in DMEM medium for 48 hours, and then collecting the supernatant. Furthermore, the amino-mesoporous silica-coated DT+ microenvironment with 1 mg / ml glucose (DT-MSN(0)) refers to the supernatant of DT(DT-MSN) coated with amino-mesoporous silica after soaking in DMEM medium with 1 mg / ml glucose for 48 h; the amino-mesoporous silica-coated DT+ microenvironment with 2 mg / ml glucose (DT-MSN(2)) refers to the supernatant of DT(DT-MSN) coated with amino-mesoporous silica after soaking in DMEM medium with 2 mg / ml glucose for 48 h; the amino-mesoporous silica-coated DT+ microenvironment with 5 mg / ml glucose (DT-MSN(5)) refers to the supernatant of DT(DT-MSN) coated with amino-mesoporous silica after soaking in DMEM medium with 5 mg / ml glucose for 48 h.

[0157] Porous tantalum, polydopamine-modified porous tantalum (DT), amino-mesoporous silica-coated DT (DT-MSN), amino-mesoporous silica-coated DT+ normal blood glucose (DT-MSN(0)), amino-mesoporous silica-coated DT+ microenvironment 2 mg / ml glucose (DT-MSN(2)), and amino-mesoporous silica-coated DT+ microenvironment 5 mg / ml glucose (DT-MSN(5)) were placed in 4% red blood cell suspension to evaluate their hemolytic effects.

[0158] Meanwhile, distilled water was set up as a positive control and PBS as a negative control.

[0159]

[0160] Among them, OD Sample These are the absorbance values ​​(OD values) of the extracts from each experimental group after incubation with red blood cells. Positive This is the absorbance value of the positive control group, OD. Negative This is the absorbance value of the negative control group.

[0161] See results Figure 8 .

[0162] from Figure 8It was found that no obvious hemolysis occurred after co-incubation with blood samples in any group, and the hemolysis rate of each group was less than 5%. There was no significant difference, indicating that all materials have good blood compatibility.

[0163] 5.2 Cell proliferation assay and AO / EB staining assay

[0164] The effects of different materials on the viability, proliferation, and apoptosis-necrosis status of BMSCs and RAW264.7 cells were systematically evaluated using CCK-8 assay and AO / EB staining techniques. Details are as follows:

[0165] CCK-8 assay: Bone marrow mesenchymal stem cells (BMSCs) and macrophages (RAW264.7) were administered at doses of 2×10⁻⁶. 4 Seeds were seeded at a density of [number] cells / well in 24-well plates. The corresponding extraction solution was added to each experimental group's wells, and the plates were co-cultured for 1, 3, 5, and 7 days. After replacing the medium with fresh DMEM, 10 v / v% CCK8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 2 hours. Then, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 450 nm using a UV spectrophotometer. Results are shown below. Figure 9 In the image, A and B are shown. A represents bone marrow mesenchymal stem cells (BMSCs), and B represents macrophages (RAW264.7).

[0166] AO / EB staining: Bone marrow mesenchymal stem cells (BMSCs) and macrophages (RAW264.7) were stained at a concentration of 2 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 24-well plates. The appropriate extraction solution was added to each experimental group's wells, and after co-culturing for 1 and 3 days, cells were stained using an AO / EB staining kit. Cell viability was observed using a fluorescence microscope, and cell numbers were counted. Results are shown below. Figure 9 C and D in the middle.

[0167] The corresponding extraction solution refers to the extraction solution prepared from the following 6 groups of materials according to step 5.1. 500 μL of the extraction solution is added to each well of the 24-well plate.

[0168] The samples were divided into 6 groups: polydopamine-modified porous tantalum (DT), amino-mesoporous silica-modified DT (DT-MSN), AMS-coated DT+ normal blood glucose concentration (labeled as DT-AMS(0)), AMS-coated DT+ microenvironment 2 mg / ml glucose (labeled as DT-AMS(2)), AMS-coated DT+ microenvironment 5 mg / ml glucose (labeled as DT-AMS(5)) and porous tantalum.

[0169] from Figure 9As shown in the cck8 statistical graph in A, in the BMSCs cell proliferation experiment (left column of A), the cell viability of all experimental groups (DT, DT-MSN, DT-AMS(0), DT-AMS(2), DT-AMS(5)) was better than that of porous tantalum, and the proliferation peak appeared on days 3-5 of culture. Among them, the proliferation-promoting effect of the DT-AMS(2) group was the most significant, and the cell viability was significantly higher than that of the porous tantalum group DT, DT-MSN and other experimental groups (p<0.05). In the RAW264.7 cell proliferation experiment (right column of A), the proliferation-promoting effect of the DT-AMS(5) group was the best, significantly higher than that of the other groups (p<0.5).

[0170] from Figure 9 As shown in Figure B, the upper layer of the BMSCs cell group shows AO / EB staining results from day 1, and the lower layer shows AO / EB staining results from day 3; similarly, the upper layer of the RAW264.7 cell group shows AO / EB staining results from day 1, and the lower layer shows AO / EB staining results from day 3. In summary, the cells in all groups exhibited good morphology and membrane integrity, with no significant apoptosis or necrosis.

[0171] from Figure 9 As shown in C (BMSCs cells) and D (RAW264.7 cells), quantitative analysis revealed that the cell density in the experimental group was significantly higher than that in the porous tantalum group (p<0.05).

[0172] In summary, DT, DT-MSN, and DT-AMS(0), DT-AMS(2), and DT-AMS(5) groups all exhibited good biocompatibility and no cytotoxicity. Among them, DT-AMS(2) and DT-AMS(5) showed the best proliferative effects on BMSCs and RAW264.7 cells, respectively.

[0173] Example 6 In vitro osteogenic experiment

[0174] In this embodiment, alkaline phosphatase (ALP) activity, an early osteogenic marker, and Alizarin Red S (ARS) staining of late mineralized nodules in BMSCs were performed in vitro to evaluate the effect of composite materials on the osteogenic activity of BMSCs.

[0175] Alkaline phosphatase (ALP) activity: BMSCs were added at 2 × 10⁻⁶ per well. 4 Cells were seeded at a density of [number] cells per well in 24-well plates and co-cultured with extracts from each group for 3 and 7 days, respectively. The culture medium was then removed, and the cells were washed three times with PBS buffer. Subsequently, the cells were fixed with 4% paraformaldehyde solution for 30 min and washed three more times with PBS. Cells were stained according to the alkaline phosphatase staining kit instructions, and PBS was added to terminate the reaction after staining. Finally, the alkaline phosphatase staining results for each group were observed and recorded using an inverted microscope. Figure 11 .

[0176] Alizarin Red S (ARS) staining: BMSCs were stained with 2 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 24-well plates and co-cultured with extracts from each group for 14 and 21 days, respectively. The culture medium was then aspirated, and the cells were washed three times with PBS buffer. Subsequently, the cells were fixed with 4% cell fixative for 30 min, washed three more times with PBS, and 1% (w / v) Alizarin Red S (pH 4.2) staining solution was added to the wells. Staining was performed at room temperature for 20 min, followed by gentle washing twice with deionized water. The staining results for each group were observed and recorded using an inverted microscope. See [link to table]. Figure 10 .

[0177] The extraction solutions for each group are the same as in step 5.2 of Example 5, with an addition amount of 500 μL.

[0178] The osteogenic differentiation process of BMSCs was evaluated using alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining system. Results are shown in [Figure number missing]. Figure 11 and Figure 10 .

[0179] from Figure 11 As can be seen, ALP detection showed that the alkaline phosphatase activity of each experimental group (the experimental groups refer to DT, DT-MSN, DT-AMS(0), DT-AMS(2), and DT-AMS(5)) was significantly higher than that of the porous tantalum group on days 3-7 of culture, with the DT-AMS(2) group showing the most outstanding performance.

[0180] from Figure 10 As can be seen from the ARS staining results, all experimental groups showed obvious calcified nodule deposition on day 14, and the mineralization degree of the DT-AMS(2) group was significantly better than that of the other groups; by day 21, the mineralization of each group was further enhanced (p<0.05), and the DT-AMS(2) group formed characteristic large calcified nodules.

[0181] The above results confirm that the DT-AMS(2) group has the best osteogenic induction ability.

[0182] Example 7: Combined Evaluation Experiment of Inflammation and Osteogenesis

[0183] This embodiment evaluated the effect of the composite material on the antioxidant properties of RAW264.7 cells in vitro, and studied its effects on anti-inflammatory and osteogenic factors in BMSCs cells.

[0184] 7.1 Observation under a fluorescence microscope

[0185] A reactive oxygen species (ROS) assay kit (DCFH-DA) was used to evaluate the ROS scavenging efficiency of RAW264.7 cells. RAW264.7 cells were cultured at 2 × 10⁶ cells per well.5 The tantalum substrate was seeded at a density suitable for 6-well plates, and LPS was added to each well to induce polarization. Then, 500 μL of the extract from each group was added to each well, and the plates were incubated at 37°C for 24 hours. The porous tantalum group (without LPS) served as a negative control. Subsequently, the DCFH-DA probe was added to each well. After stimulation at 37°C and 5% CO2 for 30 minutes, ROS levels were observed using an inverted fluorescence microscope. The extracts for each group were obtained in the same manner as in step 5.2 of Example 5.

[0186] To verify the antioxidant protective effect of DT-AMS, the intracellular reactive oxygen species (ROS) level in RAW264.7 cells was detected using the DCFH-DA fluorescent probe. Figure 12 ).

[0187] from Figure 12 It can be seen that the DT-AMS(5) group exhibited significant ROS scavenging ability and could effectively protect cells from oxidative stress damage.

[0188] 7.2 Observation using laser confocal microscopy

[0189] BMSCs (8×10 3 (pieces / hole) or RAW264.7 (1×10) 4 Cells (number per well) were seeded onto 24-well plates and cultured with 500 μL of different extracts. BMSCs were cultured in osteogenic induction medium, while RAW264.7 cells were stimulated with LPS. After reaching the designated time point, the culture medium was removed, and cells were fixed with 4% cell fixative for 30 min, followed by permeabilization with Triton-X100 for 5 min, and then blocked in 5 mg / mL BSA solution for 30 min. Primary antibody was then added and incubated overnight at 4°C. The next day, cell slides were incubated with Cy3 or 488-labeled secondary antibody at room temperature in the dark for 1 h. Cell nuclei were stained with DAPI for 1 hour. Finally, observation and imaging were performed using a laser confocal microscope. Figure 14-1 and Figure 14-2 The results of laser confocal microscopy were statistically analyzed, and the results are shown in [the table below]. Figure 13 * indicates a significant difference.

[0190] Osteogenic factors include: alkaline phosphatase (ALP), which is an early osteogenic marker and catalyzes the initiation of mineralization; osteogenic master transcription factor (RUNX-2); and osteopontin (OPN), which is a marker of late osteogenic differentiation and mediates mineralization maturation and matrix adhesion.

[0191] Anti-inflammatory factors include: mannose receptor (CD206), a marker of M2 macrophages, whose upregulated expression level indicates a good anti-inflammatory effect; INOS (inducible nitric oxide synthase) and CD86, which are markers of M1 macrophages, and whose downregulated expression level indicates a good anti-inflammatory effect.

[0192] from Figure 13 and Figure 14-1 , 14-2 The results collectively reveal the dual regulatory role of DT-AMS in cell behavior. Regarding osteogenic differentiation, the DT-AMS(2) group exhibited the most significant promoting effect, with significantly higher fluorescence intensities of ALP, OPN, and RUNX-2 compared to other groups (p<0.05). Figure 14-1 , 14-2 The mRNA expression levels of the corresponding genes in the DT-AMS(2) group were 2.2, 2.6, and 1.8 times higher than those in the porous tantalum group, respectively (p<0.05). Figure 13 In terms of immune regulation, the DT-AMS(5) group exhibited excellent anti-inflammatory properties, significantly inhibiting the expression of M1 macrophage markers (iNOS and CD86) (p<0.05), while promoting the expression of the M2 marker CD206 (p<0.05). Figure 13 These results indicate that DT-AMS exhibits a dose-dependent duality: it promotes osteogenic differentiation at a concentration of 2 mg / mL, while exhibiting optimal immunomodulatory effects at a concentration of 5 mg / mL.

[0193] Example 8 In vivo osteogenic experiment

[0194] The composite material used in the in vivo experiment differs from that in Example 1 in that it is prepared using tantalum metal with a diameter of 3 mm and a height of 6 mm; otherwise, it is the same as in Example 1.

[0195] This study used a type 1 diabetic rat femoral defect model to systematically evaluate the regulatory effects of different composite materials on bone regeneration capacity under diabetic pathological conditions. While strictly ensuring the biosafety of the coating materials in all experimental groups, the study focused on the differential effects of each composite material on bone regeneration in the femoral defect area of ​​male rats.

[0196] Male SD rats aged 6 to 8 weeks (250-280g) were selected and randomly assigned to six experimental groups: porous tantalum group, DT group, DT-MSN group, DT-AMS(0) group, DT-AMS(2) group, and DT-AMS(5) group. DT-AMS(0) group represented a rat system implanted with DT-AMS at normal blood glucose concentrations (blood glucose concentration ≤11mM as measured by a blood glucose meter); DT-AMS(2) group represented a rat system implanted with DT-AMS at medium blood glucose concentrations (blood glucose concentration 16-20mM as measured by a blood glucose meter); and DT-AMS(5) group represented a rat system implanted with DT-AMS at high blood glucose concentrations (blood glucose concentration ≥24mM as measured by a blood glucose meter). Drug release was achieved by adjusting for fluctuations in blood glucose concentration. These rats first underwent a three-day acclimatization period. During this period, rats in the DT-AMS(0) group maintained a regular diet, while rats in the other groups received a high-sugar, high-fat diet for four weeks. After a 28-day feeding period, rats received an intraperitoneal injection of streptozotocin (35 mg / kg), diluted with citrate buffer (pH 4.5). Blood samples were collected via tail vein 3 to 7 days post-injection, and non-fasting blood glucose levels (BGL) were measured using an electronic glucometer. A BGL value not lower than 16.7 mmol / L was considered a successful establishment of the diabetic model, suitable for subsequent experimental procedures.

[0197] The implants (composite materials, namely porous tantalum, DT, DT-MSN, DT-AMS(0), DT-AMS(2), DT-AMS(5)) were surgically and precisely implanted into specific locations in the rat femur. Figure 15 Femoral specimens were collected at 8 and 12 weeks post-surgery for Micro-CT scanning analysis to comprehensively assess the microstructural characteristics of the bone tissue. The Micro-CT scan results are shown below. Figure 16 .

[0198] from Figure 16 As can be seen from the sagittal plane, the newly formed bone tissue at the edge of the bone defect and the composite material formed a good bone integration interface. The defect in the porous tantalum group was the most obvious and did not spontaneously heal over time, indicating that the model construction was effective. As the glucose concentration increased, the defect was gradually filled, and the DT-AMS(5) group almost closed.

[0199] from Figure 17As can be seen, the cross-sectional images clearly show the spatial distribution characteristics of the trabeculae in each group. In the porous tantalum group, the defect area was completely vacuous at 8 and 12 weeks, with thin peripheral cortical bone and no new bone formation. Furthermore, with the increase of glucose concentration, the bone density of the DT-AMS(0), DT-AMS(2), and DT-AMS(5) groups increased, and also increased over time. At 12 weeks, the defect in the DT-AMS(2) group was almost completely filled.

[0200] Bone volume fraction was obtained using CTA software analysis of micro-CT scans; results are shown below. Figure 18 The left image shows the bone volume fraction at 8 weeks, and the right image shows the bone volume fraction at 12 weeks.

[0201] from Figure 18 It can be seen that the bone volume fraction of the DT-AMS(2) group was significantly higher than that of the DT-AMS(0), DT-AMS(5), DT, DT-MSN and porous tantalum groups.

[0202] The above results fully demonstrate that the composite material has superior bone regeneration potential under diabetic pathological conditions.

[0203] Comparative Example 1

[0204] This comparative study investigated the biocompatibility of composite materials formed from different metallic materials, and evaluated them using CCK-8 cell proliferation and tear tests.

[0205] 1) CCK-8 cell proliferation experiment

[0206] Composite material 1 and composite material 2 were obtained using tantalum and titanium, respectively, and prepared using the same method as in Example 1. Simultaneously, cell proliferation experiments were conducted on the composite material obtained in Example 1. Tantalum and titanium are non-porous materials.

[0207] The CCK-8 cell proliferation experiment was performed in step 5.2 of Example 5. The corresponding extraction solution consisted of composite material 1 and composite material 2, which were soaked in DMEM medium for 48 hours; 500 μL of extraction solution was added to each well of the 24-well plate. The absorbance at 450 nm was measured on days 1, 3, and 5 using a UV spectrophotometer.

[0208] The results of the CCK-8 cell proliferation experiment are as follows: Figure 19 As shown.

[0209] from Figure 19 It can be seen that on the 3rd and 5th days of culture, the absorbance (OD value) of the porous tantalum group was significantly higher than that of the non-porous tantalum group and the non-porous titanium group, indicating that porous tantalum is more conducive to cell proliferation.

[0210] from Figure 20It can be seen that, according to the live / dead staining and cell migration experiments, after 1 day and 3 days of culture, the cell survival rate and the number of cells that migrated were higher in the porous tantalum group than in the non-porous tantalum group and the non-porous titanium group.

[0211] The above data collectively indicate that porous tantalum performs best in promoting cell adhesion, proliferation and migration, and its biocompatibility is significantly better than that of traditional non-porous tantalum and non-porous titanium materials.

[0212] 2) Tear-off test

[0213] Using non-porous titanium, non-porous tantalum, and porous tantalum as implant materials, respectively, peel tests were conducted with or without polydopamine coating on their surfaces to investigate the adhesion performance between AMS and the implant.

[0214] I) Surface treatment: AMS solution was uniformly dropped onto the surface of each implant and the implant coated with polydopamine, and incubated at 37°C for 1 hour.

[0215] II) Cleaning process: Gently rinse three times with sterile phosphate buffered solution (PBS, pH=7.4) to remove unbound solution components;

[0216] III) Drying treatment: Place the cleaned implants in a 55℃ constant temperature drying oven until completely dry;

[0217] IV) Adhesion Test: Standard 3M tape (1cm wide) was vertically adhered to the implant surface, and a constant pressure (approximately 300g / cm) was applied. 2 After 10 seconds, quickly (within 1-2 seconds) tear it off vertically;

[0218] V) Surface characterization: Field emission scanning electron microscopy was used to observe the changes in the surface morphology of the implants before and after treatment and the presence of adhesive tape residue.

[0219] Scanning electron microscopy results of implants without or with polydopamine coating are shown below. Figure 21 , 22 And 23. Among them, the left column is before tearing, and the right column is after tearing.

[0220] from Figure 21 As shown in the left column, the surface hydrophilicity of the non-porous titanium group without polydopamine modification (top left column in the figure) is poor, which makes it difficult for AMS solution to be effectively fixed. SEM shows that the amount of AMS adsorbed on its surface is extremely low, which is the lowest among the three groups of non-porous titanium, non-porous tantalum and porous tantalum.

[0221] from Figure 21As shown in the right column, after peeling, compared with the unmodified polydopamine-modified non-porous titanium group (top right column in the figure), the amount of AMS residue on the surface of the polydopamine-modified non-porous titanium group (bottom right column in the figure) was not significantly different, indicating that the inherent binding ability of non-porous titanium to AMS is relatively weak. Although the amount of AMS adsorbed on the surface of non-porous titanium increased after polydopamine modification, large-scale detachment still occurred after peeling, with only a small amount of AMS remaining in the micron-level uneven areas of the surface.

[0222] from Figure 22 As shown in the middle left column, compared with the unmodified polydopamine porous tantalum group (top left column in the figure), the surface AMS adsorption capacity of the polydopamine-modified porous tantalum group (bottom left column in the figure) is significantly increased. This indicates that polydopamine modification further enhances the AMS loading capacity of the porous tantalum group.

[0223] from Figure 22 As shown in the right column, after peeling, compared with the unmodified polydopamine porous tantalum group (top right column in the figure), only a portion of the microspheres on the surface of the polydopamine-modified porous tantalum group (bottom right column in the figure) detached. This demonstrates that the introduction of polydopamine can significantly improve the interfacial bonding performance between AMS and the porous tantalum surface through its chemical coupling properties. Although some surface microspheres detached, a large amount of AMS remained inside the pores, indicating that the porous structure combined with dopamine chemical modification can achieve high drug loading and high retention rate of AMS.

[0224] from Figure 23 As shown in the left and middle columns, the surface of the non-porous tantalum group without polydopamine modification (top left column) is flat and lacks a porous structure. AMS microspheres can only be fixed through surface adsorption, resulting in a significantly lower loading capacity compared to the porous tantalum group. The polydopamine-modified non-porous tantalum group (bottom left column) can improve the adhesion density of AMS.

[0225] from Figure 23 As shown in the right column, after tearing, the AMS microsphere detachment rate of the non-porous tantalum group modified with polydopamine (bottom right column in the figure) was lower than that of the unmodified non-porous tantalum group (top right column in the figure), confirming that the chemical coupling effect of polydopamine can enhance the bonding strength between AMS and the surface of the non-porous tantalum group.

[0226] In summary, porous tantalum combined with polydopamine modification can synergistically improve the loading efficiency and adhesion stability of AMS. Its porous structure provides drug storage space, while the polydopamine coating enhances microsphere anchoring through chemical bonding, thereby significantly reducing the risk of high drug shedding during treatment. These results provide important evidence for optimizing composite materials.

[0227] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A glucose-responsive composite material, characterized in that, From the inside out, it sequentially includes a porous metal, a polydopamine layer covering the porous metal, and a mesoporous silica coating covering the polydopamine layer. The raw materials for preparing the mesoporous silica coating include aminated mesoporous silica, aspirin, 4-carboxyphenylboronic acid, and sodium alginate.

2. The composite material as described in claim 1, characterized in that, The mass ratio of the aminated mesoporous silica to 4-carboxyphenylboronic acid is 1:(5-7); And / or, the mass ratio of sodium alginate to 4-carboxyphenylboronic acid is 1:(1-10); And / or, the porous metal has a pore size of 400-600 μm and a porosity of 70-80%; And / or, the porous metal is selected from tantalum, titanium or alloys thereof; And / or, the thickness of the mesoporous silica coating is 200-600 nm.

3. The composite material as described in claim 1, characterized in that, The aminated mesoporous silica has a pore size of 6-8 nm and a particle size of 200-300 nm. And / or, the preparation method of polydopamine-coated porous metal is: porous metal and dopamine hydrochloride react in a solvent.

4. The composite material as described in claim 3, characterized in that, The reaction temperature is 20-35℃; And / or, the solvent is Tris-HCl buffer.

5. The composite material as described in claim 2, characterized in that, The method for preparing the mesoporous silica coating is as follows: 1) Aminated mesoporous silica, 4-carboxyphenylboronic acid, NHS and EDC are mixed in an organic solvent to obtain phenylboronic acid-modified mesoporous silica; 2) The phenylboronic acid-modified mesoporous silica and aspirin are mixed in the first solvent to obtain aspirin-loaded phenylboronic acid-modified mesoporous silica; 3) The aspirin-loaded phenylboronic acid-modified mesoporous silica and sodium alginate are reacted in a second solvent under the action of NHS and EDC to obtain a mesoporous silica coating.

6. The composite material as described in claim 5, characterized in that, In 1), the mass ratio of the aminated mesoporous silica to 4-carboxyphenylboronic acid is (0.1-10):1; And / or, in 1), the organic solvent is selected from DMSO; In and / or, in 2), the mass ratio of the aspirin to the phenylboronic acid-modified mesoporous silica is 1:(2-6); And / or, in 2), the first solvent is selected from ethanol; And / or, in 3), the second solvent is selected from a phosphate buffer solution; In and / or, 3), the mass ratio of sodium alginate to phenylboronic acid-modified mesoporous silica loaded with aspirin is 1:(1-4).

7. The method for preparing the composite material according to any one of claims 1-6, characterized in that, Including the following: A porous metal is coated with a polydopamine layer to obtain a polydopamine-modified porous metal. The composite material is obtained by mixing and incubating the polydopamine-modified porous metal and mesoporous silica coating.

8. The preparation method according to claim 7, characterized in that, The incubation time is 1-48 hours; And / or, the incubation temperature is 20-35°C.

9. Use of the composite material according to any one of claims 1-6 in the preparation of bone defect repair products or products that promote osteogenesis.

10. The use as described in claim 9, characterized in that, The bone defect is diabetic bone defect.

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