Surface modified titanium stent with magnetic hydrogel coating and preparation method and application of surface modified titanium stent

Abstract

The invention discloses a surface modified titanium stent with a magnetic hydrogel coating and a preparation method and application of the surface modified titanium stent, and belongs to the technical field of biomedical materials. The surface modified titanium scaffold with the magnetic hydrogel coating comprises a titanium substrate, a polydopamine coating and a hydrogel precursor solution coating, wherein the polydopamine coating and the hydrogel precursor solution coating are compounded on the titanium substrate; the polydopamine coating is positioned between the titanium substrate and the hydrogel precursor solution coating; the hydrogel precursor solution is prepared from freeze-dried GelMA, Fe3O4 nanoparticles and ammonium persulfate. The prepared surface modified titanium scaffold with the magnetic hydrogel coating has excellent mechanical properties and stability, and can improve the ALP activity level of cells, improve the collagen secretion level and promote mineralization in the stem cell osteogenic differentiation process through an external magnetic field with certain strength, so that the bone regeneration process is accelerated, and the osteogenic differentiation effect is improved. The method has a wide application prospect in preparation of bone graft materials and bone repair materials.

Description

Technical Field

[0001] The present application belongs to the technical field of biomedical materials, and in particular relates to a surface-modified titanium stent with a magnetic hydrogel coating, and a preparation method and application thereof. Background Art

[0002] Bone tissue engineering is a new approach to treating bone defects. It can repair or reconstruct bone defects by combining biomaterials, cells, and signaling factors, providing a new approach to solving bone defect problems. Titanium, as an implantable scaffold, has excellent mechanical and processing properties and good biocompatibility. Although titanium materials are widely used as implantable scaffolds, they still have some limitations in clinical applications, such as being prone to stress shielding and poor bone integration, which is not conducive to the direct attachment, growth, and osteogenic differentiation of bone marrow mesenchymal stem cells, thus affecting their clinical application. Surface coating modification is currently an important method for improving the performance of titanium implantable scaffolds. Attaching magnetic hydrogels to the modified titanium surface is a method to improve the biological properties of titanium and promote the osteogenic differentiation of bone marrow mesenchymal stem cells. Therefore, it is necessary to develop a material with excellent comprehensive properties to achieve effective and safe treatment for bone tissue engineering.

[0003] The prior art application with publication number CN 119424731 A discloses a surface-modified Ti implant with an anisotropic magnetic hydrogel coating. Fe3O4 nanoparticles are deposited on carboxylated cellulose nanofibers by a coprecipitation method, and the surface is modified with polydopamine to prepare magnetic cellulose nanoparticles. The magnetic cellulose nanoparticles are then incorporated into a GelMA hydrogel solution together with calcium phosphate oligomers. The solution is then coated onto a polydopamine-modified Ti implant (Ti-PDA). After forming an anisotropic structure in a magnetic field, photocrosslinking is performed to obtain a Ti implant with an anisotropic hydrogel coating.

[0004] However, existing Ti implants have the following problems: First, ferroferric oxide is deposited on carboxylated cellulose nanofibers and then mixed with GelMA hydrogel. Due to the large specific surface area and surface energy of carboxylated cellulose nanofibers, they are prone to agglomeration during the dispersion process, which leads to uneven distribution of ferroferric oxide nanoparticles in the formed GelMA hydrogel coating, which may affect the mechanical properties of the hydrogel; second, the introduction of calcium phosphate oligomers improves the mechanical properties of the hydrogel coating, but makes the degradation rate difficult to control. Summary of the Invention

[0005] The present application discloses a surface-modified titanium stent with a magnetic hydrogel coating, a preparation method thereof, and an application thereof, aiming to solve the technical problems of poor mechanical properties and difficult-to-control degradation rate of Ti implants.

[0006] In order to achieve the above objectives, the technical solution of this application is:

[0007] A first aspect of the present application provides a surface-modified titanium stent with a magnetic hydrogel coating, comprising: a titanium substrate, and a polydopamine coating and a hydrogel precursor solution coating composited on the titanium substrate;

[0008] The polydopamine coating is located between the titanium substrate and the hydrogel precursor solution coating;

[0009] The hydrogel precursor solution is prepared from freeze-dried GelMA, Fe3O4 nanoparticles and ammonium persulfate.

[0010] In combination with the first aspect, preferably, the amount of the lyophilized GelMA added is 10-20% (w / v) of the hydrogel precursor solution.

[0011] In combination with the first aspect, preferably, the particle size of the Fe3O4 nanoparticles is 100-200 nm.

[0012] In combination with the first aspect, preferably, the concentration of the Fe3O4 nanoparticles is 0.1-0.2 mol / L.

[0013] The second aspect of the present application provides a method for preparing the surface-modified titanium stent with a magnetic hydrogel coating according to the first aspect, the method comprising:

[0014] Providing a polydopamine-modified titanium substrate;

[0015] Freeze-dried GelMA and Fe3O4 nanoparticles were added to PBS solution, mixed evenly, and then ammonium persulfate was added to react to obtain a hydrogel precursor solution;

[0016] The hydrogel precursor solution is dropped onto the surface of the polydopamine-modified titanium substrate to obtain the surface-modified titanium stent with the magnetic hydrogel coating.

[0017] In combination with the second aspect, preferably, when freeze-dried GelMA and Fe3O4 nanoparticles are added to a PBS solution for reaction, the reaction temperature is 40-50°C and the reaction time is 30-60 min.

[0018] In combination with the second aspect, preferably, when ammonium persulfate is added for reaction, the reaction temperature is 40-50° C. and the reaction time is 30-60 min.

[0019] In combination with the second aspect, preferably, when the hydrogel precursor solution is added dropwise to the surface of the polydopamine-modified titanium substrate, the reaction temperature is 50-60° C. and the reaction time is 3-4 h.

[0020] The third aspect of the present application provides the use of the surface-modified titanium stent with a magnetic hydrogel coating as described in the first aspect or the surface-modified titanium stent with a magnetic hydrogel coating prepared by the preparation method as described in the second aspect in the preparation of bone transplant materials and bone repair materials.

[0021] Compared with the prior art, the advantages or beneficial effects of the embodiments of the present application include at least:

[0022] The magnetic hydrogel coating provided in this application comprises a surface-modified titanium scaffold comprising freeze-dried GelMA, Fe3O4 nanoparticles, and ammonium persulfate to form a hydrogel precursor solution, which is then composited onto a polydopamine-modified titanium substrate. On the one hand, the synergistic effect of freeze-dried GelMA and ferroferric oxide nanoparticles significantly improves the mechanical properties of the hydrogel network without affecting the structure and biocompatibility of the hydrogel. On the other hand, the hydrogel coating maintains a strong bond with the titanium substrate, maintaining overall structural stability during implantation while also maintaining excellent hydrophilicity. Thirdly, during the osteogenic differentiation of stem cells, an external magnetic field of a certain intensity can increase the cell's ALP activity, increase collagen secretion, and promote mineralization, thereby accelerating bone regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the technical solutions of the embodiments of this application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some of the embodiments described in this application. Those skilled in the art can derive other drawings based on these drawings without inventive effort.

[0024] Figure 1 The scanning electron microscope image and EDS image of the hydrogel prepared in the examples of the present application;

[0025] Figure 2 This is a stress-strain diagram of the hydrogel prepared in the examples of the present application;

[0026] Figure 3 This is a magnetic characteristic diagram of the hydrogel prepared in the embodiment of the present application;

[0027] Figure 4 The swelling curve and degradation curve of the hydrogel prepared in the examples of the present application are shown;

[0028] Figure 5 AFM images of the titanium substrate before and after polydopamine modification prepared in the examples of the present application;

[0029] Figure 6 This is a graph showing the bonding strength before and after modification of a titanium substrate with polydopamine prepared in an embodiment of the present application;

[0030] Figure 7Live / dead cell staining image of A1-pTi-PDA-GelMA@Fe3O4 prepared in the examples of this application;

[0031] Figure 8 Laser confocal microscopy images of bone marrow mesenchymal stem cells seeded on A1-pTi-PDA-GelMA@Fe3O4 under magnetic and non-magnetic conditions prepared in the examples of this application;

[0032] Figure 9 ALP staining and quantitative statistical graphs of bone marrow mesenchymal stem cells seeded on the Al-pTi-PDA-GelMA@Fe3O4 surface under different magnetic field conditions prepared in the examples of this application;

[0033] Figure 10 Collagen secretion staining and quantitative statistical graphs of bone marrow mesenchymal stem cells seeded on the Al-pTi-PDA-GelMA@Fe3O4 surface under different magnetic field conditions prepared in the examples of this application;

[0034] Figure 11 These are the mineralization staining images and quantitative statistical graphs of bone marrow mesenchymal stem cells seeded on the Al-pTi-PDA-GelMA@Fe3O4 surface under different magnetic field conditions prepared in the examples of this application. DETAILED DESCRIPTION

[0035] The following will be combined with the accompanying drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0036] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, B exists alone, and both A and B exist. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0037] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0038] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0039] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0040] It should be noted that all raw materials and reagents in the examples of the present application were purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0041] In a first aspect, an embodiment of the present application provides a surface-modified titanium stent with a magnetic hydrogel coating, comprising: a titanium substrate, and a polydopamine coating and a hydrogel precursor solution coating composited on the titanium substrate;

[0042] The polydopamine coating is located between the titanium substrate and the hydrogel precursor solution coating;

[0043] The hydrogel precursor solution is prepared from freeze-dried GelMA, Fe3O4 nanoparticles and ammonium persulfate.

[0044] Among them, on the one hand, the synergistic effect of GelMA freeze-drying and ferroferric oxide nanoparticles can significantly improve the mechanical properties of the hydrogel network without affecting the structure and biocompatibility of the hydrogel; on the other hand, it can maintain a strong bonding between the hydrogel coating and the titanium substrate, maintain the stability of the overall structure during the implantation process, and at the same time maintain excellent hydrophilicity; thirdly, during the osteogenic differentiation of stem cells, an external magnetic field of a certain intensity can increase the ALP activity level of the cells, increase the level of collagen secretion, and promote mineralization, thereby accelerating the bone regeneration process.

[0045] In the examples of the present application, the lyophilized GelMA accounts for 10-20% (w / v) of the hydrogel precursor solution. The specific source of the lyophilized GelMA used in the present application is not particularly limited and can be synthesized by commercial routes or methods known in the art. For example, the preparation method used in the examples of the present application is:

[0046] Weigh 10g of gelatin and dissolve it in 100ml of phosphate buffered saline (PBS, pH=7.4). Heat and stir in a 50℃ water bath. After the gelatin particles are completely dissolved, wrap them in tin foil and slowly add 6ml of methacrylic anhydride (MA). Continue to react at 50℃ in the dark for 3h before stopping stirring. Preheat 3 times the reaction volume of PBS solution and add it to the solution to terminate the reaction. Dialyze in a 50℃ water bath for 3-5 days to remove unreacted MA impurities. The dialysis bag should have a molecular weight cutoff of 8-14kDa. After dialysis, freeze-dry to obtain freeze-dried GelMA.

[0047] In the examples of the present application, the particle size of the Fe3O4 nanoparticles is preferably 100-200 nm, and the concentration in the solution is preferably 0.1-0.2 mol / L. The specific source of the Fe3O4 nanoparticles used in the present application is not particularly limited, and can be synthesized by commercial routes or methods known in the art. For example, the preparation method used in the examples of the present application is:

[0048] 0.1 mol / L FeCl3·6H2O and 1 mol / L ammonium acetate (NH4Ac) were added to ethylene glycol (EG). The mixture was mixed thoroughly under a magnetic stirrer and then transferred to an autoclave for reaction at 200°C for 8 hours. After the reaction was completed, the mixture was cooled to room temperature, subjected to ultrasonic centrifugation in ethanol, and dried at 60°C to obtain Fe3O4 nanoparticles.

[0049] In a second aspect, the present invention further provides a method for preparing the surface-modified titanium stent with the magnetic hydrogel coating according to the first aspect, the method comprising:

[0050] Providing a polydopamine-modified titanium substrate;

[0051] Freeze-dried GelMA and Fe3O4 nanoparticles were added to phosphate buffer, mixed evenly, and then ammonium persulfate was added for reaction to obtain a hydrogel precursor solution;

[0052] The hydrogel precursor solution is dropped onto the surface of the polydopamine-modified titanium substrate to obtain the surface-modified titanium stent with the magnetic hydrogel coating.

[0053] It should be noted that the specific source of the polydopamine-modified titanium substrate provided in this application is not particularly limited and can be synthesized by commercial routes or methods known in the art. For example, the preparation method used in the examples of this application is:

[0054] First, pretreat the titanium substrate: cut the Ti sheet into 1cm×1cm size, polish it with sandpaper of 400#, 1000#, and 2000# grit in turn, then use triple-distilled water and anhydrous ethanol for ultrasonic polishing, cleaning, and drying, mix 66% sulfuric acid and 0.2% hydrofluoric acid in a ratio of 1:1 (v:v), preheat the mixed solution at 85°C for 15 minutes, then place the Ti sheet in the preheated solution and continue heating at 85°C for 15 minutes to stop the reaction, wash and dry it with triple-distilled water, and calcine it at 450°C in a muffle furnace for 120 minutes to obtain a Ti substrate with a rough surface, thereby obtaining a pretreated titanium substrate (p-Ti).

[0055] Polydopamine-modified titanium substrate: The pretreated titanium substrate (p-Ti) was shaken in a Tris-HCl (pH = 8.5) solution containing 2 mg / mL dopamine hydrochloride for 8 h in the dark. After the reaction was completed, it was ultrasonically cleaned with triple-distilled water to obtain a polydopamine-modified titanium substrate (pTi-PDA).

[0056] In the embodiment of the present application, when freeze-dried GelMA and Fe3O4 nanoparticles were added to PBS solution for reaction, the reaction temperature was 40-50°C and the reaction time was 30-60 min. 。 When adding ammonium persulfate to react, the reaction temperature is 40-50°C and the reaction time is 30-60 minutes. When the hydrogel precursor solution is dropwise added to the surface of the polydopamine-modified titanium substrate, the reaction temperature is 50-60°C and the reaction time is 3-4 hours. By controlling the temperature and time of each reaction, the lyophilized GelMA and Fe3O4 nanoparticles can be stably composited on the titanium substrate, maintaining excellent overall structural stability during implantation.

[0057] The third aspect of this application provides the use of a surface-modified titanium scaffold with a magnetic hydrogel coating as described in the first aspect, or a surface-modified titanium scaffold with a magnetic hydrogel coating prepared by the preparation method described in the second aspect, in the preparation of bone graft materials and bone repair materials. The surface-modified titanium scaffold with a magnetic hydrogel coating exhibits excellent stability and mechanical properties. During the osteogenic differentiation of stem cells, an applied magnetic field of a certain intensity can increase the cell's ALP activity, increase collagen secretion, and promote mineralization, thereby accelerating bone regeneration.

[0058] The technical solution of the present application will be further described below in conjunction with specific embodiments.

[0059] Example 1

[0060] This embodiment provides a method for preparing a surface-modified titanium stent with an Al-magnetic hydrogel coating, which specifically includes:

[0061] S101: 15% (w / v) freeze-dried GelMA was dissolved in a 50°C PBS solution, and ferrosoferric oxide magnetic nanoparticles were added. The solution was stirred evenly with an electric stirrer to form magnetic hydrogels GelMA@Fe3O4 with different concentrations of 0.15M. After stirring, ammonium persulfate thermal crosslinker was added at a ratio of 1:10 (w / v) to obtain A1-hydrogel precursor solution;

[0062] The mixed A1-hydrogel precursor solution was added to the mold and placed in a 60°C environment for 3 hours to form a gel to obtain the A1-hydrogel;

[0063] S102: Take 100 μl of A1-hydrogel precursor solution and add it dropwise on the prepared pTi-PDA surface, place it in a 60°C environment for 3 hours to form a gel, and thus obtain a surface-modified titanium scaffold with A1-magnetic hydrogel coating (denoted as A1-pTi-PDA-GelMA@Fe3O4).

[0064] In order to verify the appearance of the magnetic hydrogel coating prepared in the embodiment of the present application, the raw materials added in the embodiment were subjected to scanning electron microscopy and energy spectrum testing. The results were as follows: Figure 1 As shown, a) is the SEM image of A1-hydrogel; b) is the EDS image of A1-hydrogel.

[0065] according to Figure 1 It can be seen that Fe3O4 is evenly distributed in the hydrogel, indicating that the preparation of A1-hydrogel is successful.

[0066] Example 2

[0067] This example provides a method for preparing a hydrogel, and the component ratios, preparation operations, and process parameters are substantially the same as those in Example 1, except that in this example, ferroferric oxide magnetic nanoparticles are not added in the first step, and the mass volume ratios of freeze-dried GelMA are 10%, 15%, and 20%, respectively, to obtain B2-hydrogel (denoted as 10% GelMA), B3-hydrogel (denoted as 15% GelMA), and B4-hydrogel (denoted as 20% GelMA).

[0068] Example 3

[0069] This example provides a method for preparing a hydrogel. The component ratios, preparation operations, and process parameters are substantially the same as those in Example 1, except that the mass volume ratio of the freeze-dried GelMA added in the first step is 15%, and the concentrations of ferrosoferric oxide nanoparticles added are 0.1 M and 0.2 M, respectively, to obtain A2-hydrogel (denoted as 0.1 M Fe3O4) and A3-hydrogel (denoted as 0.2 M Fe3O4).

[0070] In order to verify the optimal addition amount of GelMA when preparing hydrogels in the examples of this application, the hydrogels prepared in Examples 2 and 3 were tested and the results were as follows: Figures 2 to 4 shown.

[0071] according to Figure 2 It can be seen that each group of hydrogels was prepared into a cylinder with a diameter of 8mm and a height of 5mm. The Young's modulus and stress relaxation properties of the hydrogels were tested on a universal mechanical testing machine equipped with a 30N load cell at a loading speed of 1mm / min. When the sample deformation reached 60%, the compression was stopped and the stress change curve of the hydrogel was recorded. The Young's modulus value range is 5%-15% of the slope of the stress-strain curve. Figure 2 From the stress-strain curves, it can be seen that the mechanical properties of the composite hydrogel group doped with ferroferric oxide nanoparticles are better than those of the pure GelMA group as a whole, indicating that the incorporation of ferroferric oxide magnetic nanoparticles into the hydrogel can effectively improve the mechanical properties of GelMA.

[0072] according to Figure 3 It can be seen that the washed and dried ferroferric oxide magnetic nanoparticles can be directly characterized by instruments. For each group of prepared hydrogel samples, the test was carried out after freeze-drying. Fe3O4 nanoparticles have superparamagnetism, and their saturation magnetic induction intensity is 77.6emu / g. The magnetism of the composite hydrogel increases with the increase of the concentration of magnetic particles in the hydrogel. The saturation magnetic induction intensities of the 0.1M, 0.15M, and 0.2M groups are 12emu / g, 21emu / g, and 29emu / g, respectively. Pure GelMA hydrogel has no magnetism.

[0073] according to Figure 4 It can be seen that each group of hydrogels prepared was freeze-dried and the dry weight (W d Subsequently, the hydrogels were immersed in PBS solution at 37°C, with three replicates per group. The hydrogels were taken out regularly and the surface moisture was absorbed using filter paper. The wet weight (W) of each time period was then measured. t ) until the sample mass no longer increases with the extension of immersion time. The swelling ratio (WR) of the hydrogel is calculated as follows:

[0074]

[0075] The initial weight of each group of hydrogels in the saturated swelling state is recorded as W t The same PBS soaking method was used, with three replicates per group. Each sample was soaked for 14 days and taken out at each predetermined time point. The surface water of the hydrogel was absorbed and then weighed again, which was recorded as W. i The calculation formula of the degradation rate (DS) of the hydrogel is as follows:

[0076]

[0077] in, Figure 4 (a) Swelling curve of the hydrogel; (b) Degradation curve of the hydrogel. As shown in the figure, each group of hydrogels reached swelling equilibrium with a similar trend within 48 hours. The swelling rate of the pure GelMA group reached approximately 220%, while the swelling rate of the group doped with Fe3O4 decreased slightly. This is because the addition of magnetic particles changed the pore structure of the hydrogel. The degradation curves show that all groups of hydrogels were effectively degraded within two weeks, and the degradation was particularly obvious in the group loaded with the highest concentration of magnetic particles, which may be caused by the continuous leakage of magnetic particles. Among the experimental groups, the hydrogel loaded with 0.2M magnetic nanoparticles degraded the best, and the degradation rate could be controlled by adjusting the content of magnetic nanoparticles.

[0078] In order to verify the roughness of the titanium substrate before and after modification, the titanium substrate was cleaned and dried naturally at room temperature, and then AFM scanning test was performed. The results are as follows: Figure 5 The test results of the bonding strength between the titanium substrate and the hydrogel before and after modification are shown in Figure 6 shown.

[0079] according to Figure 5 As can be seen in the AFM images, the arithmetic mean is used to reflect the roughness (Ra). The average Ra values for each group were 18.6, 116.8, 41.3, 2.95, and 7.96 nm, respectively. Compared with smooth pure Ti, the surface roughness of the Ti surface after acid corrosion was greatly increased, while the surface roughness of the surface with the PDA layer was reduced. However, the subsequent hydrogel coating resulted in a significant reduction in the surface roughness of the sample, indicating that the hydrogel coating can completely shield the underlying topological structure.

[0080] according to Figure 6 It can be seen that the pure titanium substrate and the polydopamine-modified titanium substrate were modified with magnetic hydrogel coatings, respectively. Among them, the pure titanium substrate modified with magnetic hydrogel coatings was denoted as Ti-Gel, and the titanium substrate modified with magnetic hydrogel coatings and polydopamine was denoted as pTi-PDA-Gel; (a) Force-displacement relationship curve of magnetic hydrogel coating and titanium substrate; (b) Adhesion strength diagram of magnetic hydrogel coating and titanium substrate. On a universal mechanical testing machine, a 30N sensor was used to test the adhesion strength between the Ti sheet surface and the hydrogel coating at a tensile rate of 0.2mm / s. The adhesive was cyanoacrylate adhesive, and the sample contact area S was 706.5mm 2 In the experiment, the maximum pulling force required to separate the Ti sheet from the coating was recorded as Fm, and the adhesive strength Sa (kPa) was calculated using the following formula: Sa = Fm / S.

[0081] The results are as follows Figure 6As shown, the adhesion strength between the untreated pure titanium surface and the hydrogel coating was 6.28 ± 0.58 kPa, while the adhesion strength between the titanium surface etched with double acid and coated with PDA increased to 17.71 ± 1.38 kPa. The results indicate that the roughness of the titanium surface increased after treatment, significantly improving the adhesion between the titanium substrate and the magnetic hydrogel coating.

[0082] In order to verify the ability of the surface modified titanium scaffold with A1-magnetic hydrogel coating (denoted as A1-pTi-PDA-GelMA@Fe3O4) to promote the osteogenic differentiation of bone marrow mesenchymal stem cells, corresponding performance tests were carried out. The test results are shown in the figure. Figure 7-11 shown.

[0083] 1. Isolation and Culture of Rat Bone Marrow Mesenchymal Stem Cells

[0084] The surgical instruments used in the experiment were ultrasonically cleaned with clean water, and then the sealed instruments were placed in a 120-degree high-temperature and high-pressure sterilizer for 30 minutes for sterilization, and then dried for later use. SD rats weighing approximately 120 g were sacrificed by spinal dislocation, and their body surfaces were disinfected with 75% alcohol. After 5 minutes, the skin and muscle of the rat's legs were cut open using sterile surgical instruments under a sterile environment, and the femur and tibia of the rat were removed and separated, with the ends of the bones cut to expose the bone marrow cavity. Culture medium (containing 10% fetal bovine serum) was aspirated using a 5 mL sterile syringe, and the bone marrow cavities of the femur and tibia were repeatedly perfused until the medullary cavity turned white. The bone marrow aspirate was repeatedly perfused to completely disperse the cells and obtain a uniform cell suspension. The cell suspension was inoculated into a cell culture flask and cultured in a 37°C cell culture incubator (containing 5% CO2) (the medium was changed daily before passaging and every two days thereafter). After 7-10 days of culture, when the cell aggregation in the cell flask was approximately 80%, the cells in the culture flask were digested with a trypsin solution, washed with sterile PBS, and then passaged. In subsequent in vitro cell experiments, cells with good development status of the 3rd to 5th generations were used for experiments.

[0085] 2. Cell survival and proliferation of bone marrow mesenchymal stem cells on titanium scaffolds

[0086] The materials used were sterilized with high temperature and high pressure, and placed in a clean bench for ultraviolet sterilization; two groups of experiments were set up, one group without applying a magnetic field, and the other group with a magnetic field. Bone marrow mesenchymal stem cells passed to the third generation were inoculated on the material surface at a density of 2×104 cells / cm2, with 3 parallels in each group (n=3); they were cultured in a constant temperature incubator at 37°C, 98% constant humidity, and 5% CO2 for 3 days; the culture medium was discarded, and the cells were washed with PBS three times; an FDA / PI mixture was prepared, with a volume ratio of FDA, PI, and serum-free culture medium of 1:1:100; 100 μL of the prepared FDA / PI mixture was added to each well, and the cells were incubated at room temperature in the dark for 10 minutes; after washing with PBS, the cells were observed under a fluorescence microscope and photographed for record.

[0087] The results are as follows Figure 7 As shown in Figure 1, where SMF refers to a static magnetic field that acts synergistically with the scaffold material on cells. Bone marrow mesenchymal stem cells seeded on the surface of this scaffold material exhibited normal morphology, with only a few cells dying, demonstrating normal growth. This demonstrates that the surface-modified titanium scaffold material with a magnetic hydrogel coating produced in this application has good cell compatibility and can support normal cell growth and proliferation.

[0088] 3. Cell morphology and spreading of bone marrow mesenchymal stem cells on titanium scaffolds

[0089] The third generation of bone marrow mesenchymal stem cells were cultured at a rate of 5×10 3 cells / cm 2 The density of cells was inoculated onto the surface of the scaffold material, and two groups of experiments were set up, one of which was not subjected to a magnetic field, and the other was subjected to a magnetic field, with three parallels in each group (n=3); cultured in a constant temperature incubator at 37°C, 98% constant humidity, and 5% CO2 for 24 hours; the culture medium was discarded and the cells were washed three times with PBS; 200 μL of pre-cooled 4% paraformaldehyde solution was added to each well, and the cells were fixed for 30 minutes at 4°C; the paraformaldehyde was aspirated, the cells were washed with PBS, and then 200 μL of 0.2% Triton X-100 solution was added to each well and incubated for 2 minutes to permeate the cells; the Triton solution was aspirated, the cells were washed three times with PBS, and 200 μL of rhodamine-phalloidin solution (5 units / mL) was added to each well, and the cells were incubated in the dark at 4°C overnight to stain the skeleton of the cells; the phalloidin was recovered and carefully washed five times with PBS for 3-5 minutes each time; 200 μL of DAPI solution was incubated at room temperature in the dark for 30 minutes to stain the cell nucleus; the stain was removed by aspiration, and the cells were washed three times with PBS, each time for 3-5 minutes; glycerol was added to seal the slides, and the slides were observed and photographed using a laser scanning confocal microscope.

[0090] according to Figure 8It can be seen that the cells on the surface of the scaffold material with and without an external magnetic field showed various morphologies such as spindle and triangle, and more pseudopodia were observed around the stem cells.

[0091] 4. Alkaline phosphatase (ALP) staining and quantitative detection

[0092] ALP staining: The third generation bone marrow mesenchymal stem cells were cultured at a rate of 2×10 4 cells / cm 2 The cells were seeded at different densities on the scaffold materials under different magnetic field conditions (0mT, 0.6mT, 1.2mT, 1.8mT, 2.4mT, 3.0mT), with 3 parallels per group (n=3); they were cultured in a constant temperature incubator at 37°C, 98% constant humidity, 5% CO2 for 4 days and 7 days respectively, and stimulated for 30 minutes every day according to different magnetic field conditions. The culture medium was replaced every two days during this period; the culture medium was discarded, and the attached cells were slowly rinsed with PBS three times for 3-5 minutes each time. in; add pre-chilled 4% paraformaldehyde solution to fix the sample for 20-30 minutes; aspirate and discard the paraformaldehyde, wash with PBS three times, 3-5 minutes each time; prepare the color development solution (buffer: BCIP:NBT = 300:1:2) according to the instructions of the ALP color development kit, and protect from light; add 200 μL of ALP color development solution to each well, incubate at room temperature for 30 minutes, protecting from light throughout the process; aspirate and discard the color development solution, slowly wash with PBS, and photograph and preserve under a stereomicroscope.

[0093] ALP quantitative detection: The third generation bone marrow mesenchymal stem cells were collected at 2×10 4 cells / cm 2 The cells were inoculated at different densities onto scaffold materials under different magnetic field conditions, with 6 parallels (n=6) set up in each group; they were cultured in a constant temperature incubator at 37°C, 98% constant humidity, and 5% CO2 for 4 days and 7 days, respectively, and stimulated for 30 minutes every day according to different magnetic field conditions, and the culture medium was replaced every two days; the culture medium was discarded, and the attached cells were slowly rinsed 3 times with PBS, each time for 3-5 minutes; 200 μL of 1% TritonX-100 solution was added to each well, and the cells were lysed at 4°C for 30 minutes, and the lysate was collected; according to the instructions of the BCA detection kit, the absorbance values were detected and recorded at a wavelength of 562 nm using a microplate reader, and the protein concentrations in the samples of different groups were calculated; according to the ALP activity detection kit, the absorbance values were detected and recorded at a wavelength of 520 nm using a microplate reader, and normalized against the protein concentration previously obtained in each well to calculate the ALP activity of each sample.

[0094] The results are as follows Figure 9As shown, (a) ALP staining; (b) ALP quantitative statistical graph. After 4 days of culture, compared with the control group, the magnetic field stimulation group had significantly higher ALP activity expression, and the activity expression gradually increased with the increase in magnetic field intensity until it reached a peak and then declined slightly. After 7 days of culture, the ALP activity of each group was increased compared to 4 days, and the trend was basically the same as that on the 4th day. Among them, the 2.4mT group had the highest activity expression and the strongest alkaline phosphatase positive expression (purple). The ALP qualitative staining results were also basically consistent with this trend. The above research results show that the magnetic field range of 0-3mT can increase the ALP activity level of bone marrow mesenchymal stem cells, especially the 2.4mT group, which has the most obvious effect, and has a certain promoting effect on their early osteogenic differentiation.

[0095] 5. Collagen Secretion Staining and Quantitative Detection

[0096] Collagen secretion staining: The third generation bone marrow mesenchymal stem cells were cultured at 2×10 4 cells / cm 2 The cells were inoculated at different densities onto scaffold materials under different magnetic field conditions (0 mT, 0.6 mT, 1.2 mT, 1.8 mT, 2.4 mT, and 3.0 mT), with three parallels set up in each group (n=3); they were cultured in a constant temperature incubator at 37°C, 98% constant humidity, and 5% CO2 for 7 days and 14 days, respectively, during which magnetic stimulation was continuously applied using the corresponding magnetic field conditions, and the culture medium was replaced every two days; the culture medium was discarded, and the adhered cells were slowly rinsed with PBS three times, each time for 3-5 minutes; pre-cooled 4% paraformaldehyde solution was added to fix the samples for 20-30 minutes; the samples were washed with PBS three times, each time for 3-5 minutes; 200 μL of Sirius red staining solution was added to each well and incubated in the dark at room temperature for 1 hour; after staining, the samples were washed with PBS, and then photographed and preserved under a stereomicroscope.

[0097] Quantitative detection of collagen secretion: After photographing, add 200 μL of 0.1 M sodium hydroxide solution to each well of each stained sample to decompose the dye and gently shake to dissolve it for 10 minutes. Centrifuge the resulting mixed solution at 11,000 rpm for 10 minutes to obtain the supernatant. Collect the supernatant into a 96-well plate and measure the absorbance at 540 nm using a microplate reader. Record and save.

[0098] The results are as follows Figure 10As shown in the figure, (a) collagen secretion staining; (b) quantitative statistical graph of collagen secretion. On day 7, the 2.4mT group showed a significant trend of promoting collagen secretion compared with other groups (excluding the 3.0mT group) (p < 0.001), with no significant difference from the 3.0mT group. After 14 days, the 2.4mT group and the 3.0mT group also showed a significant difference (p < 0.05), with other trends generally consistent with day 7. The qualitative staining of collagen with Sirius Red was consistent with the quantitative detection results. This shows that the magnetic field stimulation of 0-3mT we used can promote the osteogenic differentiation of bone marrow mesenchymal stem cells and increase collagen secretion levels, with the 2.4mT magnetic field being the most effective in promoting collagen secretion.

[0099] 6. Mineralization staining and quantitative detection

[0100] Mineralization staining: The third generation bone marrow mesenchymal stem cells were cultured at 2×10 4 cells / cm 2 The cells were seeded at different densities onto scaffold materials under different magnetic field conditions (0 mT, 0.6 mT, 1.2 mT, 1.8 mT, 2.4 mT, and 3.0 mT), with three parallels (n=3) set up for each group; they were cultured in a 37°C, 98% constant humidity, 5% CO2 constant temperature incubator for 14 days and 21 days, respectively, with the culture medium replaced every two days; the culture medium was discarded, and the adhered cells were slowly rinsed with PBS three times, each time for 3-5 minutes; pre-cooled 4% paraformaldehyde solution was added to fix the samples for 20-30 minutes; PBS was washed three times, each time for 3-5 minutes; 200 μL of 0.1% alizarin red staining solution with a pH of 4.1 was added to each well, and the samples were incubated for 60 minutes at 37°C in a light-proof shaking incubator; after staining, the samples were washed with double distilled water, and then photographed and preserved under a stereomicroscope.

[0101] Quantitative detection of mineralization: After taking pictures, 100 μL of 10% acetic acid solution was added to each well of the 24-well plate and incubated at room temperature for 30 minutes; the cells were gently scraped off, the mixed solution was transferred to an EP tube, and vortexed for 30 seconds to allow the solution to fully react; the fully reacted solution was placed in a water bath and water bathed at 85°C for 10 minutes; the sample was cooled to room temperature and centrifuged for 10 minutes at a speed of 12,000 r / min to remove insoluble crystals; the supernatant was transferred to a new EP tube, and the same volume of 10% ammonia solution as the supernatant was added to each sample to neutralize the acetic acid in the solution; the sample supernatant was added to a 96-well plate, and the absorbance value at a wavelength of 405 nm was detected using an enzyme reader and recorded and saved.

[0102] The results are as follows Figure 11As shown, (a) mineralization staining image; (b) mineralization quantitative statistical graph. Compared with the control group, each magnetic field group had different mineralization-promoting effects. On day 14, the 2.4mT group showed significant differences from the 0mT, 0.6mT, and 1.2mT groups (p < 0.001), but no significant differences from the 1.8mT and 3.0mT groups. After 21 days, the 2.4mT group reached its strongest calcium deposition-promoting effect and showed significant differences from each of the remaining groups (p < 0.001 or p < 0.01). The results of the study indicate that applying a magnetic field can promote mineralization in the middle and late stages of bone marrow mesenchymal stem cell osteogenic differentiation, and the effect is more pronounced at a magnetic field of 2.4mT.

[0103] Therefore, the surface-modified titanium scaffold with a magnetic hydrogel coating provided in this application comprises: a titanium substrate and a polydopamine coating and a hydrogel precursor solution coating composited on the titanium substrate; the polydopamine coating is located between the titanium substrate and the hydrogel precursor solution coating; and the hydrogel precursor solution is made of freeze-dried GelMA, Fe3O4 nanoparticles, and ammonium persulfate. The surface-modified titanium scaffold with a magnetic hydrogel coating has excellent mechanical properties and stability. During the osteogenic differentiation of stem cells, an external magnetic field of a certain intensity can increase the ALP activity level of the cells, increase the level of collagen secretion, and promote mineralization, thereby accelerating the bone regeneration process. It has broad application prospects in the preparation of bone transplant materials and bone repair materials.

[0104] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0105] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A surface-modified titanium stent with a magnetic hydrogel coating, characterized in that: include: A titanium substrate and a polydopamine coating and a hydrogel precursor solution coating composited on the titanium substrate; The polydopamine coating is located between the titanium substrate and the hydrogel precursor solution coating; The hydrogel precursor solution is prepared from freeze-dried GelMA, Fe3O4 nanoparticles and ammonium persulfate.

2. The surface-modified titanium stent with a magnetic hydrogel coating according to claim 1, characterized in that: The GelMA lyophilized solution accounted for 10-20% (w / v) of the hydrogel precursor solution.

3. The surface-modified titanium stent with a magnetic hydrogel coating according to claim 1, characterized in that: The particle size of the Fe3O4 nanoparticles is 100-200nm.

4. The surface-modified titanium stent with a magnetic hydrogel coating according to claim 1, characterized in that: The concentration of the Fe3O4 nanoparticles is 0.1-0.2 mol / L.

5. A method for preparing a surface-modified titanium stent with a magnetic hydrogel coating according to any one of claims 1 to 4, characterized in that: The preparation method comprises: Providing a polydopamine-modified titanium substrate; Freeze-dried GelMA and Fe3O4 nanoparticles were added to phosphate buffer, mixed evenly, and then ammonium persulfate was added for reaction to obtain a hydrogel precursor solution; The hydrogel precursor solution is dropped onto the surface of the polydopamine-modified titanium substrate to obtain the surface-modified titanium stent with the magnetic hydrogel coating.

6. The method for preparing a surface-modified titanium stent with a magnetic hydrogel coating according to claim 5, characterized in that: When freeze-dried GelMA and Fe3O4 nanoparticles are added to a PBS solution for reaction, the reaction temperature is 40-50°C and the reaction time is 30-60 minutes.

7. The method for preparing a surface-modified titanium stent with a magnetic hydrogel coating according to claim 5, wherein: When ammonium persulfate is added for reaction, the reaction temperature is 40-50° C. and the reaction time is 30-60 minutes.

8. The method for preparing a surface-modified titanium stent with a magnetic hydrogel coating according to claim 5, wherein: When the hydrogel precursor solution is added dropwise to the surface of the polydopamine-modified titanium substrate, the reaction temperature is 50-60° C. and the reaction time is 3-4 hours.

9. Use of a surface-modified titanium stent with a magnetic hydrogel coating according to any one of claims 1 to 4 or a surface-modified titanium stent with a magnetic hydrogel coating prepared by the preparation method according to any one of claims 5 to 8 in the preparation of bone transplant materials or bone repair materials.

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