Magnetic hydrogels, surface-modified ti implants with anisotropic magnetic hydrogel coatings, and applications thereof

Abstract

The application discloses a magnetic hydrogel, a surface modified Ti implant with an anisotropic magnetic hydrogel coating and application thereof, and relates to the following steps: uniformly depositing Fe3O4 nanoparticles on carboxylated cellulose nanofibers, and modifying the same with polydopamine to prepare magnetic cellulose nanoparticles with high biocompatibility and superparamagnetism, wherein the nanoparticles can sensitively respond to a weak magnetic field; mixing the nanoparticles and calcium phosphate oligomers into a GelMA hydrogel solution, coating the solution on polydopamine modified Ti material, and performing photo-crosslinking after forming an anisotropic structure in a magnetic field, so as to finally obtain the surface modified Ti implant with the anisotropic magnetic hydrogel coating. The magnetic hydrogel coating has an anisotropic structure and mechanical property, and can simulate the anisotropy of bone to a certain extent.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to a magnetic hydrogel, a surface-modified Ti implant with an anisotropic magnetic hydrogel coating, and their applications. Background Technology

[0002] Pure titanium and its alloys are widely used in dental implants, orthopedics, and total joint restorations due to their excellent biocompatibility, mechanical strength, chemical stability, durability, and corrosion resistance. However, their surface properties still have many defects, which affect their long-term application. Previous studies have shown that bone marrow mesenchymal stem cells (BMSCs) and their differentiated progeny (such as osteoblasts) and osteocytes are mechanosensitive in bone, and the extracellular mechanical microenvironment has a significant impact on the adhesion, migration, and differentiation of BMSCs. Different mechanical stimuli can activate different signaling molecules and pathways, guiding BMSC differentiation. Appropriate intensity of mechanical stimulation can change the differentiation direction of BMSCs from adipogenic differentiation to osteogenic differentiation, but the technology for simulating the mechanical environment of BMSCs still needs further development.

[0003] Researchers both domestically and internationally have invented various surface modification techniques and methods. From the perspective of the cellular microenvironment, traditional modification strategies can be mainly divided into two categories: physical microenvironment and biochemical microenvironment. Strategies for regulating the physical microenvironment mainly include increasing the surface roughness of titanium materials, introducing porous structures and anisotropic biomimetic structures, etc., while strategies for regulating the biochemical microenvironment include immobilizing bioactive molecules and constructing inorganic or organic coatings. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a magnetic hydrogel, a surface-modified Ti implant with an anisotropic magnetic hydrogel coating, and its applications. This invention involves depositing Fe3O4 nanoparticles on carboxylated cellulose nanofibers (CNF) via co-precipitation, and preparing magnetic cellulose nanoparticles (PMNPs) by surface modification with polydopamine. These PMNPs, along with calcium phosphate oligomers (CPO), are incorporated into a GelMA hydrogel solution (GelMA / CPO / PMNP). This 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 (Ti-GC-aPMNP). This invention, by preparing an anisotropic magnetic hydrogel coating on Ti implants and combining it with a dynamic magnetic field, can achieve an organic combination of anisotropic morphology and mechanical stimulation on Ti implants. Based on this, an in vitro cell culture platform can be constructed to study the effects of anisotropic morphology and dynamic mechanical stimulation on Ti implants on BMSCs adhesion, osteogenic differentiation and final osseointegration.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides a magnetic hydrogel, which is prepared through the following steps:

[0006] S1. Preparation of polydopamine-coated magnetic cellulose nanoparticles (PMNPs):

[0007] S1-1. Dissolve carboxylated cellulose nanofibers in deionized water and sonicate to obtain a cellulose nanofiber solution.

[0008] S1-2, Add ferrous chloride tetrahydrate and ferric chloride hexahydrate, stir, add ammonia water dropwise while stirring, heat and stir the reaction, separate with a magnet after the reaction is completed, wash the magnetic product to obtain magnetic nanocellulose MNP;

[0009] S1-3. Disperse MNP in Tris solution, then add dopamine hydrochloride, sonicate to mix, and then shake on a shaker in the dark. After the reaction is complete, wash and freeze-dry the product to obtain PMNP.

[0010] S2. Preparation of methacrylamide gelatin (GelMA);

[0011] S3. Preparation of calcium phosphate oligomer CPO:

[0012] Dissolve CaCl2·2H2O in ethanol, add triethylamine while stirring, then add an alcoholic solution of H3PO4 dropwise. After the addition is complete, centrifuge and wash the solid product to obtain CPO.

[0013] S4. Precipitant solution for preparing magnetic hydrogel:

[0014] Under light-protected conditions, GelMA, CPO and photoinitiator were dissolved in PBS solution, and then PMNP was added and mixed evenly to obtain the precursor solution of magnetic hydrogel.

[0015] S5. Prepare magnetic hydrogels using any of the following methods:

[0016] a. The precursor solution of the magnetic hydrogel is coated and cured with blue-violet light to obtain a magnetic hydrogel with randomly distributed magnetic particles.

[0017] b. The precursor liquid of the magnetic hydrogel is coated and then subjected to magnetic orientation treatment in a magnetic field, followed by blue-violet light curing to obtain anisotropic magnetic hydrogel.

[0018] Preferably, step S1 specifically includes:

[0019] S1-1. Dissolve 200 mg of carboxylated cellulose nanofibers in 100 mL of deionized water and sonicate for 30 minutes to obtain a cellulose nanofiber solution.

[0020] S1-2. After blowing the cellulose nanofiber solution with nitrogen for 20 minutes, add 180 mg of ferrous chloride tetrahydrate and 470 mg of ferric chloride hexahydrate, stir for 30 minutes, add ammonia water dropwise under stirring to adjust the pH to 10, stir the reaction at 70℃ for 30 minutes, add 100 mL of deionized water to end the reaction, then separate with a magnet, wash the magnetic product with anhydrous ethanol and deionized water in turn to obtain magnetic nanocellulose MNP;

[0021] S1-3. Disperse 100 mg of MNP in 50 mL of Tris solution, then add 50 mg of dopamine hydrochloride, sonicate for 5 minutes to mix, then shake overnight on a shaker at 50 rpm in the dark. Wash the product with deionized water and anhydrous ethanol, freeze dry, and obtain PMNP.

[0022] Preferably, step S2 specifically includes:

[0023] At 50°C, 5g of type A gelatin was added to 50ml of PBS and stirred until completely dissolved. 6mL of methacrylic anhydride was added under light protection. After reacting for 2 hours, 200mL of PBS preheated to 60°C was added to terminate the reaction. The reaction product was dialyzed at 50°C in a dialysis bag with a molecular cutoff of 11 kDa for 4 days. The dialysate in the dialysis bag was collected, lyophilized, and GelMA was obtained.

[0024] Preferably, step S3 specifically includes:

[0025] 2.94 g of CaCl2·2H2O was dissolved in 0.4 L of ethanol. Under magnetic stirring, 55.45 mL of triethylamine was added. Then, an alcoholic solution of H3PO4 was added dropwise at room temperature under magnetic stirring. The reaction was carried out for 12 h. After centrifugation, the solid product was washed to obtain CPO.

[0026] The alcoholic solution of H3PO4 was obtained by dissolving 1.045 mL of H3PO4 in 20.00 mL of ethanol.

[0027] Preferably, step S4 specifically includes:

[0028] S4-1. Under light-protected conditions at 60℃, GelMA, CPO, and photoinitiator LAP were dissolved in PBS solution to make the mass-volume concentrations of GelMA, CPO, and photoinitiator LAP 15%, 2%, and 0.2%, respectively.

[0029] S4-2. Add PMNP and mix thoroughly to achieve a PMNP mass-volume concentration of 0.38-6%, thus obtaining the precursor solution for the magnetic hydrogel.

[0030] Preferably, step S5 specifically includes:

[0031] Magnetic hydrogels can be prepared using any of the following methods:

[0032] a. After coating the precursor solution of the magnetic hydrogel, it is cured by irradiation with blue-violet light for 30-90s to obtain a magnetic hydrogel with randomly distributed magnetic particles.

[0033] b. After coating the precursor solution of the magnetic hydrogel, it is magnetically oriented in a uniform magnetic field of 60-100mT for 2-10s, and then cured by blue-violet light irradiation for 30-90s to obtain anisotropic magnetic hydrogel.

[0034] In a second aspect, the present invention provides a surface-modified titanium material having an anisotropic magnetic hydrogel coating, comprising a titanium material body and a magnetic hydrogel coating disposed on the surface of the titanium material body. The magnetic hydrogel coating is prepared by: coating a precursor liquid of the magnetic hydrogel as described above onto the surface of the titanium material body, then performing magnetic orientation treatment in a magnetic field, and curing with blue-violet light to form the magnetic hydrogel coating.

[0035] Preferably, the surface-modified titanium material with anisotropic magnetic hydrogel coating is prepared by the following method:

[0036] 1) Polydopamine modification of titanium material:

[0037] After cleaning the titanium material body, it was immersed in an alkaline solution under heating, dried, and then immersed in a Tris solution. Dopamine hydrochloride was added to the Tris solution, and the reaction was carried out on a shaker in the dark. The product was cleaned and dried to obtain the polydopamine-modified titanium material body.

[0038] 2) The precursor liquid of the magnetic hydrogel described above is dropped onto the surface of the polydopamine-modified titanium material body, coated evenly, and then subjected to magnetic orientation treatment in a magnetic field. The magnetic hydrogel coating is cured by blue-violet light to obtain the surface-modified titanium material with an anisotropic magnetic hydrogel coating.

[0039] Preferably, the surface-modified titanium material with anisotropic magnetic hydrogel coating is prepared by the following method:

[0040] 1) Polydopamine modification of titanium material:

[0041] The titanium body was first washed with acetone, anhydrous ethanol and deionized water, then soaked in 5M NaOH solution at 60℃ for 24h, dried at 60℃ and then soaked in 10mM Tris solution at pH 8.5. Dopamine hydrochloride was added to the Tris solution to make the concentration of dopamine hydrochloride 1mg / mL, and incubated overnight on a shaker in the dark. The product was washed with deionized water and dried to obtain polydopamine modified titanium body.

[0042] 2) The precursor liquid of the magnetic hydrogel described above is dropped onto the surface of the polydopamine-modified titanium material, and the coating is uniform. Then, it is magnetically oriented in a uniform magnetic field of 60-100mT for 2-10s, and then cured by blue-violet light irradiation for 30-90s to form the magnetic hydrogel coating, thus obtaining a surface-modified titanium material with an anisotropic magnetic hydrogel coating.

[0043] A third aspect of the present invention provides an application of the surface-modified titanium material with an anisotropic magnetic hydrogel coating as described above, which is used as a Ti implant to simulate the bone physical microenvironment and to construct an in vitro cell culture platform for studying the effects of mechanical stimulation on BMSCs adhesion and osteogenic differentiation.

[0044] The beneficial effects of this invention are:

[0045] This invention involves uniformly depositing Fe3O4 nanoparticles on carboxylated cellulose nanofibers and modifying them with polydopamine to prepare magnetic cellulose nanoparticles with high biocompatibility and superparamagnetism. These nanoparticles can sensitively respond to weak magnetic fields (not exceeding 20 mT). The nanoparticles are then incorporated into a GelMA hydrogel solution along with calcium phosphate oligomers. This solution is then coated onto a polydopamine-modified Ti material. After forming an anisotropic structure in a magnetic field, photocrosslinking is performed, ultimately yielding a surface-modified Ti implant with an anisotropic magnetic hydrogel coating. This magnetic hydrogel coating possesses anisotropic structure and mechanical properties, and can simulate the anisotropy of bone to a certain extent.

[0046] This invention, by preparing an anisotropic magnetic hydrogel coating on Ti implants and combining it with a dynamic magnetic field, can achieve an organic combination of anisotropic morphology and mechanical stimulation on Ti implants. Based on this magnetic hydrogel coating, this invention can construct an extracellular cell culture platform for studying the effects of anisotropic morphology and dynamic mechanical stimulation on BMSCs adhesion and osteogenic differentiation. Attached Figure Description

[0047] Figure 1 The system preparation process of this invention is as follows: (A) the preparation process of polydopamine-modified titanium material body; (B) the preparation process of dopamine-modified magnetic cellulose; (C) the preparation process of titanium-based anisotropic magnetic hydrogel (i.e., surface-modified titanium material with anisotropic magnetic hydrogel coating).

[0048] Figure 2 The following are the performance test results of the hydrogels in Example 1 of this invention: (A) Microstructure of hydrogels; (B) Magnetic response properties; (C) Hydrogel compression test; (D) XRD patterns of MNP and PDA-coated MNP (PMNP) nanoparticles; (E) VSM pattern of magnetic nanoparticles; (F) FT-IR spectral analysis; (G) Thermogravimetric analysis.

[0049] Figure 3 The following are test results on the effect of the magnetic hydrogel and related materials of the present invention on cell compatibility: (A) Relative cell viability of hydrogels incorporating different concentrations of CPO (1%, 2%, 3%) and PMNP particles (0.75%, 1.5%, 3%, 6%); (B) Effect on cell proliferation; (C) Fluorescence image of FDA / PI staining (green - live cells, red - dead cells), scale bar 500 μm.

[0050] Figure 4 The test results of the magnetic hydrogel and related materials of the present invention on cell morphology are as follows: (A) Fluorescent images of the cytoskeleton and cell nucleus stained after 4 days of culture (green - cytoskeleton, blue - cell nucleus); (B) Fluorescent images of the alignment direction of the magnetic particles of the anisotropic hydrogel coating and the cell alignment direction stained; (C) Box plot of cell spreading area; (D) Cell aspect ratio plot; (E) Statistical graph of the angle θ between the long axis of the cell and the horizontal line (n=10).

[0051] Figure 5 The following are the test results of the effect of dynamic magnetic stimulation and anisotropic magnetic hydrogel on osteogenic differentiation of cells based on the system of this invention: (A, C) Alkaline phosphatase staining and activity analysis of cells at 4 and 7 days after incubation at Ti-PDA, Ti-G, Ti-GC, Ti-G-rPMNP, Ti-GC-rPMNP, Ti-GC-aPMNP, and Ti-GC-aPMNP; (B, D) Collagen staining and quantitative analysis at 14 days; (E) Mineralization staining and quantitative analysis at 14 days. All data are shown as mean ± SD, n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0053] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0055] Example 1

[0056] A magnetic hydrogel and a surface-modified titanium material with an anisotropic magnetic hydrogel coating based thereon, the magnetic hydrogel being prepared by the following steps:

[0057] S1. Preparation of polydopamine-coated magnetic cellulose nanoparticles (PMNPs):

[0058] S1-1. Dissolve 200 mg of carboxylated cellulose nanofibers in 100 mL of deionized water, sonicate for 30 minutes, and obtain a 0.2 wt% cellulose nanofiber solution after complete dissolution.

[0059] S1-2. After blowing the cellulose nanofiber solution with nitrogen for 20 minutes, add 180 mg of ferrous chloride tetrahydrate and 470 mg of ferric chloride hexahydrate, stir vigorously for 30 minutes, add ammonia dropwise under vigorous stirring to adjust the pH to 10, stir the reaction at 70°C for 30 minutes, add 100 mL of deionized water to end the reaction, then separate with a magnet, wash the magnetic product three times with anhydrous ethanol and deionized water in turn to obtain magnetic nanocellulose MNP;

[0060] S1-3. Disperse 100 mg of MNP in 50 mL of Tris solution, then add 50 mg of dopamine hydrochloride, sonicate for 5 minutes to mix, then shake overnight on a shaker at 50 rpm in the dark. Wash the product three times with deionized water and anhydrous ethanol, and freeze dry to obtain PMNP.

[0061] S2. Preparation of methacrylamide gelatin (GelMA):

[0062] At 50°C, 5g of type A gelatin (purchased from Aladdin, brand name 9000-70-8) was added to 50ml of PBS and stirred until completely dissolved. 6mL of methacrylic anhydride (MA) was added under light protection. After reacting for 2 hours, 200mL of PBS preheated to 60°C was added to terminate the reaction. The reaction product was dialyzed at 50°C in a dialysis bag with a molecular cutoff of 11 kDa for 4 days. The dialysate in the dialysis bag was collected, lyophilized, and GelMA was obtained.

[0063] S3. Preparation of calcium phosphate oligomer CPO:

[0064] 2.94 g of CaCl2·2H2O was dissolved in 0.4 L of ethanol. Under magnetic stirring, 55.45 mL of triethylamine was added, followed by dropwise addition of an alcoholic solution of H3PO4 at room temperature and under magnetic stirring. The reaction was carried out for 12 h, and the product was centrifuged at 6000 rpm. The solid product was washed three times with ethanol to remove residual triethylamine, yielding CPO. The product was then resuspended in deionized water to form a homogeneous emulsion with a concentration of approximately 3 mg / mL.

[0065] The alcoholic solution of H3PO4 was obtained by dissolving 1.045 mL of H3PO4 in 20.00 mL of ethanol.

[0066] S4. Precipitant solution for preparing magnetic hydrogel:

[0067] S4-1. Under light-protected conditions at 60℃, a homogeneous emulsion of GelMA and CPO and the photoinitiator LAP (lithium phenyl (2,4,6-trimethylbenzoyl)phosphate) were dissolved in PBS solution to make the mass-volume concentrations of GelMA, CPO and photoinitiator LAP 15% (w / v), 2% (w / v) and 0.2% (w / v) respectively.

[0068] S4-2. Add a certain amount of PMNP and mix evenly to obtain precursor solutions of magnetic hydrogels with different PMNP contents, such that the mass-volume concentration of PMNP is 0, 0.38, 0.75, 1.5, 3, and 6% (w / v).

[0069] S5. Prepare magnetic hydrogels using any of the following methods:

[0070] a. After coating the precursor solution of the magnetic hydrogel, it is cured by irradiation with blue-violet light for 60 seconds to obtain a magnetic hydrogel with randomly distributed magnetic particles.

[0071] b. After coating the precursor liquid of the magnetic hydrogel, it is magnetically oriented in a uniform magnetic field of 80mT for 5s and then cured by blue-violet light irradiation for 60s to obtain anisotropic magnetic hydrogel.

[0072] The preparation of magnetic hydrogels will be further explained below in conjunction with specific titanium materials.

[0073] This embodiment also provides a surface-modified titanium material with an anisotropic magnetic hydrogel coating, which is prepared by the following method:

[0074] 1) Polydopamine modification of titanium material:

[0075] The titanium substrate (10mm x 10mm titanium foil) was washed three times each with acetone, anhydrous ethanol, and deionized water. Then, it was soaked in a 5M NaOH solution at 60℃ for 24 hours, dried at 60℃, and then soaked in a 10mM Tris solution (pH adjusted to 8.5 with HCl). Dopamine hydrochloride was added to the Tris solution to make the concentration of dopamine hydrochloride 1mg / mL. The mixture was incubated overnight on a shaker in the dark. The product was washed with deionized water and dried to obtain the polydopamine-modified titanium substrate, denoted as Ti-PDA.

[0076] 2) Add more than 25 μL of the precursor liquid of the magnetic hydrogel prepared in step S4 to the surface of the polydopamine modified titanium material, coat it evenly, and then magnetically oriented it for 5 s in a uniform magnetic field of 80 mT, with the magnetic field direction parallel to the titanium foil plane. Then, irradiate it with blue-violet light for 60 s to crosslink and cure it to form a magnetic hydrogel coating, thus obtaining a surface-modified titanium material with an anisotropic magnetic hydrogel coating, that is, oriented magnetic particles, denoted as Ti-GC-aPMNP.

[0077] In contrast, the following method was used to prepare surface-modified titanium materials with a magnetic hydrogel coating featuring randomly distributed magnetic particles:

[0078] More than 25 μL of the precursor liquid of the magnetic hydrogel prepared in step S4 was added to the surface of the polydopamine-modified titanium material. The coating was spread evenly and cross-linked and cured by blue-violet light for 60 s to form a magnetic hydrogel coating. The resulting surface-modified titanium material with a magnetic hydrogel coating with randomly distributed magnetic particles was obtained, which is called non-directional magnetic particles, denoted as Ti-GC-rPMNP.

[0079] Furthermore, using the same method as Ti-GC-rPMNP, surface-modified titanium materials with pure GelMA hydrogel coatings were prepared: Ti-G (without adding CPO and PMNP in step S4) and surface-modified titanium materials with hydrogel coatings containing only CPO: Ti-GC (without adding PMNP in step S4), which were used for subsequent detection and testing, respectively.

[0080] Performance characterization and testing

[0081] 1. The successful synthesis of the magnetic hydrogel was confirmed using SEM, XRD, and VSM. FT-IR spectroscopy analysis showed that nano-iron oxide (Fe3O4) was successfully deposited on carboxylated cellulose nanofibers via co-precipitation. Thermogravimetric analysis was used to determine the thermal stability of the particles. Thermogravimetric experimental parameters were: temperature: room temperature to 700℃, heating rate: 10 K / min, experimental atmosphere: nitrogen, gas flow rate: 100 mL / min.

[0082] 2. The mechanical properties of the hydrogel were determined using a compression test. Simply put, the hydrogel disk was placed on an Instalon 5567 platform, and a compression test was performed. The gel was compressed to 60% strain. The deformation rate was 1 mm / min. The shear stress of the gel is the stress change corresponding to 60% strain.

[0083] 3. Hydrophilicity test: The hydrophilicity of each group of hydrogels and ppyNWs was determined using the water contact angle. The gel slides and the ppyNWs solution coated on the glass slides were dried, placed on the stage, the camera was turned on, and the same volume of water droplets were dropped and photographed. The baseline and lateral tangent were drawn using software to determine the contact angle.

[0084] Test results:

[0085] The microstructure of the hydrogel was characterized by SEM. Figure 2 A). The results show that the pore size of pure 15% GelMA hydrogel is relatively uniform (~5 μm), and the pore size decreases after the addition of particles. After magnetic orientation, an oriented structure appears inside the hydrogel, indicating that the method of constructing hydrogel coatings with anisotropic structures by magnetic orientation is effective.

[0086] PMNP particles in different concentration groups can respond quickly to the magnetic field and align themselves in the direction of the magnetic field. Figure 2 (B) However, in the 0.38% and 0.75% groups, the alignment of PMNPs in the hydrogel was limited due to the low concentration, specifically manifested as few and short chain structures formed by PMNPs. As for the 3.00% group, although there were clearly aligned chain structures, the width of these chain structures was not uniform enough; some were too thick, while others were too thin, indicating a tendency for these chain structures to aggregate, which may affect the mechanical properties of the hydrogel coating. In contrast, the magnetic cellulose in the 1.50% concentration group showed the best alignment effect, with uniform aligned chain structures.

[0087] Compression tests were performed on hydrogels containing magnetic particles of different concentrations (0, 0.38, 0.75, 1.5, 3.0% (w / v)) and orientations (oriented: Alined, A; unoriented: Random, R). Figure 2 C).

[0088] Figure 2 In the C series, GelMA represents a pure hydrogel coating; R-0.38 represents a non-oriented hydrogel coating with a PMNP concentration of 0.38% (w / v); R-0.75, R-1.5, and R-3 have similar meanings, differing only in PMNP concentration. A-0.38 represents an oriented hydrogel coating with a PMNP concentration of 0.38% (w / v); A-0.75, A-1.5, and A-3 have similar meanings, differing only in PMNP concentration. GelMA-2CPO represents a hydrogel coating with a CPO doping concentration of 2% (w / v); R-1.5-2CPO represents a non-oriented hydrogel coating with a PMNP concentration of 1.5% (w / v) and a CPO concentration of 2% (w / v); and A-1.5-2CPO represents an oriented hydrogel coating with a PMNP concentration of 1.5% (w / v) and a CPO concentration of 2% (w / v). Figure 3 In the right figure of C, GelMA-2CPO-1.5PMNP represents a non-oriented hydrogel coating.

[0089] The compression level is set to 60%. Figure 2 The compressive stress-strain curves in Figure C show that 15% (w / v) GelMA hydrogel exhibits high brittleness. When the concentration of PMNP particles is low (0.38%), the hydrogel begins to fracture when the compressive strain reaches 45-55%, manifested as a sharp drop in compressive stress. While the fracture strain increases with a higher PMNP particle concentration (3%), the brittleness of the GelMA hydrogel remains unresolved. The compressive modulus was calculated by determining the slope of the linear fitting curve at 10%-20% of the stress-strain curve. The compressive modulus of pure 15% (w / v) GelMA hydrogel is 8.7 kPa. As the PMNP particle concentration increases from 0.38% to 3%, the compressive modulus increases somewhat, but only by a limited amount, up by 34%. Magnetic-induced particle alignment also increases the compressive modulus in this alignment direction. However, simply adding magnetic particles does not seem to solve the problems of low stiffness and brittleness in GelMA hydrogels.

[0090] Therefore, this invention incorporates calcium phosphate oligomers (CPO) into the hydrogel to enhance its fundamental mechanical properties. CPO is an ultra-small (particle size no greater than 1 nm) calcium phosphate compound that can self-assemble into rod-shaped hydroxyapatite within the hydrogel, achieving strong protein (organic)-mineral (inorganic) interactions, thereby improving the hydrogel's mechanical properties and overcoming its high brittleness. To this end, 2% CPO was added to both pure GelMA hydrogel and a magnetic hydrogel doped with 1.5% PMNP. The stress-strain curves are shown below. Figure 2 As shown in the curves of GelMA-2CPO, R-1.5-2CPO, and A-1.5-2CPO in C, the addition of 2% CPO nanoparticles can significantly improve the mechanical properties of GelMA hydrogel. When the compressive strain reaches 60%, the hydrogel containing CPO still shows no signs of cracking, and after stress relief, it can almost return to its original shape, exhibiting good toughness. Figure 2 As can be seen from the compressive modulus analysis of the hydrogels in C, the addition of 2% (w / v) CPO to GelMA significantly improved the compressive modulus, increasing it from 10-15 kPa to 30-35 kPa. Therefore, adding CPO to GelMA is expected to improve its inherent poor mechanical properties and brittleness.

[0091] This embodiment examined the XRD patterns (5° < 2θ < 85°) of nanoparticles including cellulose nanofibers (CNF), Fe3O4, magnetic cellulose nanoparticles (MNP), and PDA-coated MNP (PMNP). The results showed ( Figure 2 D), Fe3O4, MNP, and PMNP all showed characteristic peaks at 30°, 35.4°, 43°, 56.9°, and 62.5°, corresponding to crystal planes (220), (311), (400), (511), and (440), respectively. These peaks are consistent with the main characteristic peaks of the Fe3O4 standard card, indicating that the main crystal structure of the above three particles is iron(III) oxide.

[0092] The hysteresis loop of the particles was measured using a vibrating sample magnetometer (VSM). Figure 2 E) The hysteresis loops of MNP and PMNP at 300K show that the coercivity of both particles is negligible, and there is no obvious hysteresis loop, indicating that the particles are superparamagnetic. Both MNP and PMNP have relatively high saturation magnetization (Ms), which are 54.4 emu / g and 42.7 emu / g, respectively.

[0093] FT-IR spectral analysis results show that ( Figure 2 F), 3335cm -1 3000cm -1 1420cm -1 1032cm -1 and 1653cm -1 The peaks at 1600 cm⁻¹ correspond to OH stretching, NH stretching, CN stretching, C-OH stretching, and CC stretching, respectively. -1 1407cm -1 These are two characteristic peaks of the carboxyl group in carboxylate, 543 cm⁻¹. -1 Corresponding to the Fe-O vibration peak of Fe3O4. 1600 cm⁻¹ on CNF. -1 1407cm -1 Two strong absorption peaks indicate that CNF contains a large number of carboxyl groups, which explains why CNF can achieve a uniform size distribution of Fe3O4: a large number of uniformly distributed negatively charged carboxyl groups can attract positively charged iron ions, which greatly increases the nucleation sites of Fe3O4. The weakened carboxyl peak on MNP indicates that the carboxyl groups on CNF are largely consumed. After MNP is coated with polydopamine, the peak at 1032 cm⁻¹... -1 The weakened C-OH stretching at the point of re-enhancement can be attributed to the large number of phenolic hydroxyl groups on polydopamine. Compared to Fe3O4, the Fe-O vibrational peak on MNP, from 580 cm⁻¹, is significantly enhanced. -1 Reduced to 543cm -1 This indicates that a strong interaction force exists between Fe3O4 and cellulose.

[0094] Through thermogravimetric analysis ( Figure 2 G), the results showed that the decomposition temperature of CNF was around 300℃, the Fe3O4 content in MNP was about 77.46%, and the Fe3O4 content in PMNP was about 44.33%.

[0095] Example 2: Determination of the cell compatibility of the surface-modified titanium material in Example 1

[0096] 1. CCK-8 determination:

[0097] 1) Place the surface-modified titanium material prepared in Example 1 at the bottom of a 24-hole plate;

[0098] 2) Then, the third-generation BMSCs were seeded at a density of 4×10⁴ / well on Ti substrate in different 24-well plates and cultured, with the culture medium changed once a day;

[0099] 3) After 1 day and 3 days of culture, add 400 μL of serum-free medium containing 10% CCK-8 solution to each well by changing the medium, and incubate at 37°C for 1 hour.

[0100] 4) Take an appropriate amount of culture and measure the absorbance at 450nm.

[0101] 2. FDA / PI live / dead staining:

[0102] 1) Third-generation BMSCs were seeded at a density of 4×10⁴ / well on different Ti-based materials in 24-well plates and cultured.

[0103] 2) After 4 days, discard the culture medium and wash twice with PBS for 5 minutes each time;

[0104] 3) Prepare the staining working solution by diluting the FDA / PI stock solution 500 times with serum-free culture medium;

[0105] 4) Add 1 mL of staining working solution to each well and incubate for 15 minutes;

[0106] 5) Discard the staining working solution, wash twice with PBS for 5 minutes each time, and observe and photograph under a fluorescence microscope.

[0107] The measurement results are as follows Figure 3 As shown:

[0108] Figure 3In A, GelMA represents a pure hydrogel coating, CPO1, CPO2, and CPO3 represent hydrogel coatings in step S4 of Example 1 with only 1%, 2%, and 3% (w / v) concentration of CPO added, respectively, and PMNP0.75, PMNP1.5, PMNP3, and PMNP6 represent hydrogel coatings in step S4 of Example 1 with only 0.75%, 1.5%, 3%, and 6% (w / v) concentration of PMNP added, respectively.

[0109] Figure 3 In B, Ti-PDA represents a polydopamine-modified titanium substrate, Ti-G represents a surface-modified titanium substrate with a pure GelMA hydrogel coating, and Ti-GC represents a surface-modified titanium substrate with a hydrogel coating containing only CPO. (Where Ti-GC-PMNP is Ti-GC-rPMNP)

[0110] Figure 3 The results showed that 2% CPO was beneficial to the proliferation of MSCs, exhibiting optimal cell proliferation capacity and biocompatibility. Therefore, a CPO concentration of 2% was selected for subsequent experiments. For PMNP particles, at lower concentrations (0.75%, 1.5%), their cell proliferation capacity was comparable to that of the GelMA control group, indicating that lower concentrations of PMNP had good biocompatibility. Overall, the prepared magnetic hydrogel coating Ti-GC-PMNP showed no significant cell proliferation toxicity. Figure 3 B). FDA / PI ( Figure 3 C) The live-dead staining experiment showed that, within each group, the majority of cells were live and there were almost no dead cells. The cells had a high survival rate, indicating that the material had good biocompatibility and no obvious cytotoxicity.

[0111] Therefore, a CPO concentration of 2% and a PMNP concentration of 1.5% were chosen for subsequent experiments.

[0112] Example 3: Detection of the effect of surface-modified titanium material from Example 1 on cell morphology changes.

[0113] 1) After inoculating BMSCs onto the surface-modified titanium material prepared in Example 1 and culturing for 4 days, discard the culture medium and wash twice with PBS for 5 minutes each time;

[0114] 2) Fix with 4% paraformaldehyde for 30 minutes, then wash twice with PBS;

[0115] 3) Perforate the cell membrane with 0.2% Triton X-100 for 2 minutes, then wash twice with PBS;

[0116] 4) Add rhodamine-phalloidin, incubate overnight at 4°C in the dark, and perform cytoskeleton staining;

[0117] 5) After recovering the staining solution on the second day, wash three times with PBS for 5 minutes each time;

[0118] 6) Add DAPI to stain cell nuclei for 30 minutes, recover the staining solution, and wash with PBS 3 times, 5 minutes each time;

[0119] 7) Mount the slide with 200 μL of glycerol, store at 4°C, and observe and photograph it under a fluorescence confocal microscope;

[0120] 8) Use ImageJ software to perform statistical analysis on cell area, ratio of major to minor axis, and angle of major axis.

[0121] Results: MSCs in all groups were in a spread-out state. Figure 4 A) The cells extend more filamentous pseudopodia outwards, and compared to the control group Ti-PDA (polydopamine-modified titanium body, without hydrogel layer), the cells of the GelMA hydrogel-coated modified group are generally taller. Figure 4 C) Higher spreading area. The anisotropy of the hydrogel coating also has a significant impact on cell spreading. The anisotropic groups (Ti-GC-aPMNP, Ti-GC-aPMNP+DMF) have higher cell spreading areas. (Ti-GC-aPMNP+DMF represents anisotropic hydrogels with an applied magnetic field)

[0122] Effects of anisotropy and dynamic mechanical stimulation on MSC polarization (including cell aspect ratio and cell orientation):

[0123] Cell aspect ratio: Cell aspect ratio shows ( Figure 4 (D) Cells in the Ti-PDA and Ti-G groups had the lowest relative aspect ratios, mainly concentrated around 1.6; in contrast, the Ti-GC group had a higher cell aspect ratio, mainly distributed between 2.1 and 3.1, while the Ti-GC-rPMNP group also had a similar cell aspect ratio distribution, indicating that randomly distributed magnetic nanoparticles had no significant effect on cell morphology. Consistent with expectations, the introduction of anisotropic structures onto the hydrogel coating further significantly increased the cell aspect ratio. However, the similar cell aspect ratio distributions in the Ti-GC-aPMNP and Ti-GC-aPMNP+DMF groups indicate that the dynamic mechanical stimulation used in this study had no significant effect on cell morphology.

[0124] Cell orientation: The angle θ between the long axis of 10 cells and the horizontal line was randomly calculated. For example... Figure 4E shows that the angle θ of cells in the isotropic group is distributed over a wide range, appearing as a wide box plot; while in the anisotropic group, the cell angle θ is concentrated near the alignment direction of the magnetic particles in their respective hydrogel coatings (Ti-GC-aPMNP: 58.6°, Ti-GC-aPMNP+DMF: 135.8°), indicating that the cells have obvious directional alignment.

[0125] Example 4: Detection of the effect of the surface-modified titanium material prepared in Example 1 on osteogenic differentiation of cells.

[0126] 1) After inoculating BMSCs onto surface-modified titanium material and culturing for 4 or 7 days, wash once with PBS;

[0127] 2) Then, fix with 4% paraformaldehyde for 15 minutes;

[0128] 3) Wash three times with PBS, five minutes each time;

[0129] 4) Early osteogenic differentiation was determined by the ALP kit, intermediate osteogenic differentiation by the Sirius red kit, and late osteogenic differentiation by the Alizarin Red kit.

[0130] Measurement results:

[0131] ALP staining and quantitative detection results showed that ( Figure 5 In the Ti-GC-rPMNP, Ti-GC-aPMNP, and Ti-GC-aPMNP+DMF groups, regardless of whether it was day 4 or 7, the Ti-GC-aPMNP+DMF group had the largest ALP staining positive area and the highest ALP activity, significantly higher than the Ti-GC-rPMNP group. Secondly, Sirius red staining and quantitative detection (… Figure 5 B, 5D). The results showed that compared with the Ti-PDA group, the collagen secretion in the Ti-GC group was significantly increased (p<0.05). Furthermore, the anisotropic topography and dynamic mechanical stimulation of the hydrogel further increased the collagen secretion of MSCs (p<0.05). Third, the quantification results of alizarin red staining at 14 days ( Figure 5 E) indicates that extracellular collagen mineralization levels and ALP activity and collagen secretion show a consistent trend.

[0132] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A surface-modified titanium material with an anisotropic magnetic hydrogel coating, characterized in that, It includes a titanium body and a magnetic hydrogel coating disposed on the surface of the titanium body. This surface-modified titanium material is prepared by the following method: 1) Polydopamine modification of titanium material: After cleaning the titanium material body, it was immersed in an alkaline solution under heating, dried, and then immersed in a Tris solution. Dopamine hydrochloride was added to the Tris solution, and the reaction was carried out on a shaker in the dark. The product was cleaned and dried to obtain the polydopamine-modified titanium material body. 2) The precursor liquid of magnetic hydrogel is dropped onto the surface of polydopamine modified titanium material, coated evenly, and then magnetically oriented in a uniform magnetic field of 60-100mT for 2-10s, and then cured by blue-violet light irradiation for 30-90s to form the magnetic hydrogel coating, thus obtaining a surface-modified titanium material with an anisotropic magnetic hydrogel coating. The precursor solution of the magnetic hydrogel is prepared by the following steps: S1. Preparation of polydopamine-coated magnetic cellulose nanoparticles (PMNPs): S1-1. Dissolve carboxylated cellulose nanofibers in deionized water and sonicate to obtain a cellulose nanofiber solution. S1-2, Add ferrous chloride tetrahydrate and ferric chloride hexahydrate, stir, add ammonia water dropwise while stirring, heat and stir the reaction, separate with a magnet after the reaction is completed, wash the magnetic product to obtain magnetic nanocellulose MNP; S1-3. Disperse MNP in Tris solution, then add dopamine hydrochloride, sonicate to mix, and then shake on a shaker in the dark. After the reaction is complete, wash and freeze-dry the product to obtain PMNP. S2. Preparation of methacrylamide gelatin (GelMA); S3. Preparation of calcium phosphate oligomer CPO: Dissolve CaCl2·2H2O in ethanol, add triethylamine while stirring, then add an alcoholic solution of H3PO4 dropwise. After the addition is complete, centrifuge and wash the solid product to obtain CPO. S4. Precipitant solution for preparing magnetic hydrogel: Under light-protected conditions, GelMA, CPO, and photoinitiator LAP were dissolved in PBS solution, and then PMNP was added and mixed thoroughly to obtain the precursor solution for the magnetic hydrogel.

2. The surface-modified titanium material with an anisotropic magnetic hydrogel coating according to claim 1, characterized in that, Step S1 is as follows: S1-1. Dissolve 200 mg of carboxylated cellulose nanofibers in 100 mL of deionized water and sonicate for 30 minutes to obtain a cellulose nanofiber solution. S1-2. After blowing the cellulose nanofiber solution with nitrogen for 20 minutes, add 180 mg of ferrous chloride tetrahydrate and 470 mg of ferric chloride hexahydrate, stir for 30 minutes, add ammonia water dropwise under stirring to adjust the pH to 10, stir the reaction at 70℃ for 30 minutes, add 100 mL of deionized water to end the reaction, then separate with a magnet, wash the magnetic product with anhydrous ethanol and deionized water in turn to obtain magnetic nanocellulose MNP; S1-3. Disperse 100 mg of MNP in 50 mL of Tris solution, then add 50 mg of dopamine hydrochloride, sonicate for 5 minutes to mix, and then shake overnight on a shaker at 50 rpm in the dark. Wash the product with deionized water and anhydrous ethanol, and freeze-dry to obtain PMNP.

3. The surface-modified titanium material with an anisotropic magnetic hydrogel coating according to claim 1, characterized in that, Step S2 is as follows: At 50°C, 5g of type A gelatin was added to 50ml of PBS and stirred until completely dissolved. 6mL of methacrylic anhydride was added under light protection. After reacting for 2 hours, 200mL of PBS preheated to 60°C was added to terminate the reaction. The reaction product was dialyzed at 50°C in a dialysis bag with a molecular cutoff of 11 kDa for 4 days. The dialysate in the dialysis bag was collected, lyophilized, and GelMA was obtained.

4. The surface-modified titanium material with an anisotropic magnetic hydrogel coating according to claim 1, characterized in that, Step S3 is as follows: 2.94 g of CaCl2·2H2O was dissolved in 0.4 L of ethanol. Under magnetic stirring, 55.45 mL of triethylamine was added. Then, an alcoholic solution of H3PO4 was added dropwise at room temperature under magnetic stirring. The reaction was carried out for 12 hours. After centrifugation, the solid product was washed to obtain CPO. The alcoholic solution of H3PO4 was obtained by dissolving 1.045 mL of H3PO4 in 20.00 mL of ethanol.

5. The surface-modified titanium material with an anisotropic magnetic hydrogel coating according to claim 1, characterized in that, Step S4 is as follows: S4-1. Under light-protected conditions at 60℃, GelMA, CPO, and photoinitiator LAP were dissolved in PBS solution to achieve mass-volume concentrations of 15%, 2%, and 0.2%, respectively. S4-2. Add PMNP and mix thoroughly to achieve a PMNP mass-volume concentration of 0.38-6%, thus obtaining the precursor solution for the magnetic hydrogel.

6. The surface-modified titanium material with an anisotropic magnetic hydrogel coating according to claim 1, characterized in that, It is prepared by the following method: 1) Polydopamine modification of titanium material: The titanium body was first washed with acetone, anhydrous ethanol and deionized water, then soaked in 5M NaOH solution at 60℃ for 24h, dried at 60℃ and then soaked in Tris solution at 10 mM pH 8.

5. Dopamine hydrochloride was added to the Tris solution to make the concentration of dopamine hydrochloride 1 mg / mL, and incubated overnight on a shaker in the dark. The product was washed with deionized water and dried to obtain polydopamine modified titanium body. 2) The precursor liquid of the magnetic hydrogel is dropped onto the surface of the polydopamine-modified titanium material, coated evenly, and then magnetically oriented in a uniform magnetic field of 60-100mT for 2-10s, and then cured by blue-violet light irradiation for 30-90s to form the magnetic hydrogel coating, thus obtaining a surface-modified titanium material with an anisotropic magnetic hydrogel coating.

7. An application of a surface-modified titanium material with an anisotropic magnetic hydrogel coating as described in any one of claims 1-6, used as a Ti implant to simulate the bone physical microenvironment, and to construct an in vitro cell culture platform for studying the effects of mechanical stimulation on BMSCs adhesion and osteogenic differentiation.

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