A magnetothermal asymmetric hydrogel, its preparation method, and its application in bone repair.

Abstract

This invention provides a magnetothermal asymmetric hydrogel, its preparation method, and its application in bone repair, belonging to the field of medical materials technology. By constructing a magnetothermal responsive hydrogel with a spatially asymmetric structure, this invention achieves precise confinement of a mild thermal effect to the target repair area, effectively reducing the risk of thermal diffusion to non-target tissues. This material not only provides a suitable three-dimensional microenvironment for cell growth but also controllably promotes bone regeneration under an external alternating magnetic field. Therefore, it offers a solution for clinical bone defects, especially those in complex locations adjacent to sensitive organs, that combines good biocompatibility, excellent osteogenic activity, and precise therapeutic function.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a magnetothermal asymmetric hydrogel, its preparation method, and its application in bone repair. Background Technology

[0002] Bone defects are a common clinical challenge, and their repair outcomes directly impact patients' functional recovery and quality of life. Ideal bone repair materials need to possess good biocompatibility, osteoconductivity or osteoinductive capacity, and the ability to provide temporary mechanical support. Hydrogels, due to their high water content, tunable physicochemical properties, and three-dimensional network structure similar to the extracellular matrix, have attracted widespread attention in tissue engineering and can be used as cell carriers or growth factor sustained-release platforms for bone regeneration.

[0003] In recent years, strategies combining physical stimuli (such as heat, electricity, and magnetism) with biomaterials to actively regulate cell behavior and promote tissue regeneration have shown great potential. Among these, magnetothermal therapy has gained popularity due to its non-contact nature and deep penetration. This method typically incorporates magnetic nanoparticles into biomaterials. Under the influence of an alternating magnetic field, the magnetic particles generate heat, thereby producing a thermal effect on surrounding cells or tissues. Moderate warmth has been shown to promote osteoblast proliferation, differentiation, and related gene expression, accelerating the bone healing process.

[0004] However, current bone repair materials based on magnetic nanoparticles typically have their magnetic components uniformly or randomly distributed within the hydrogel matrix. When an alternating magnetic field is applied, heat diffuses uniformly or unpredictably throughout the material and surrounding tissue. For repair sites such as craniofacial bones, which may be adjacent to important and heat-sensitive tissues like the brain, this indiscriminate heat diffusion poses a risk of damaging nearby normal tissues, limiting the safe application of magnetothermal therapy in such critical anatomical locations.

[0005] Therefore, developing magnetic biomaterials that can achieve precise spatial control of heat is key to promoting the safe use of magnetothermal therapy for bone repair, especially for repairing bone defects in complex locations. Summary of the Invention

[0006] The purpose of this invention is to provide a magnetothermal asymmetric hydrogel, its preparation method, and its application in bone repair. This invention solves the technical problems of existing magnetothermal materials used for bone repair, which are difficult to achieve precise and safe thermotherapy in deep tissues and cannot effectively promote bone regeneration while avoiding thermal damage to adjacent sensitive tissues.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a magnetothermal asymmetric hydrogel for bone repair, comprising the following steps: S1. Prepare composite magnetic microspheres containing iron oxide nanoparticles and a polymer coating layer; S2. Dissolve methacrylamide gelatin and photoinitiator in phosphate buffer to obtain the precursor solution; S3. Add the composite magnetic microspheres to the precursor liquid and disperse them evenly to obtain a composite precursor solution; S4. Inject the composite precursor solution into the mold, place one side of the mold close to the permanent magnet, and apply a magnetic field to guide the directional migration of the composite magnetic microspheres. S5. Under the guidance of a magnetic field, the composite precursor solution is irradiated with ultraviolet light to crosslink and solidify the methacrylamide gelatin, thereby obtaining the bone repair mild magnetothermal asymmetric hydrogel.

[0008] Preferably, in step S1, the polymer coating layer is made of mPEG-PLGA.

[0009] Preferably, step S1 specifically includes: S1.1. Ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved together in deionized water, concentrated ammonia is added to adjust the pH to 9-11 to generate iron oxide nanoparticles, oleic acid is then added for modification, and hydrophobically modified iron oxide nanoparticles are obtained after magnetic separation, washing and drying. S1.2. Using a dichloromethane solution containing mPEG-PLGA and the hydrophobically modified iron oxide nanoparticles as the oil phase and an aqueous solution containing 1%~5% polyvinyl alcohol as the external aqueous phase, an emulsion is formed under the conditions of homogenization speed of 2000rpm~6000rpm and homogenization time of 0.5min~3min. S1.3. The emulsion is stirred at a constant temperature of 20~30℃ for 4~8 hours to evaporate the organic solvent, and then centrifuged, washed and freeze-dried to obtain the composite magnetic microspheres.

[0010] Preferably, in step S1.1, the step of adding oleic acid for modification includes: stirring the oleic acid and the iron oxide nanoparticle suspension at 50~70°C for 20~40 minutes, and then adding dilute hydrochloric acid dropwise to adjust the pH to 5~7 until the foam on the liquid surface completely disappears; In step S1.1, the molar ratio of ferric chloride hexahydrate to ferrous chloride tetrahydrate is 1.5~2.5:1; In step S1.2, the concentration of mPEG-PLGA in the oil phase is 40 mg / mL to 60 mg / mL; the concentration of the hydrophobically modified iron oxide nanoparticles in the oil phase is 20 mg / mL to 30 mg / mL. In step S1.2, after the emulsion is formed, the solvent is evaporated by constant temperature stirring at a speed of 200 rpm to 600 rpm.

[0011] Preferably, the saturation magnetization of the composite magnetic microspheres obtained in step S1 is 5 emu / g to 15 emu / g; In step S2, the concentration of the methacrylamide gelatin in the precursor solution is 10%~20% w / v; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, and its concentration in the precursor solution is 2%~6% w / v.

[0012] Preferably, in step S4, the mold is a cylindrical mold with an inner cavity diameter of 4 mm to 6 mm and a height of 2 mm to 3 mm. In step S4, the permanent magnet is a neodymium iron boron permanent magnet; In step S4, the magnetic field guidance is applied for 30 to 120 seconds. In step S5, the wavelength of the ultraviolet light is 405 nm, and the composite precursor solution is irradiated from both the front and back of the mold for 40 to 80 seconds each.

[0013] The present invention also provides a bone repair magnetothermal asymmetric hydrogel prepared by the above preparation method.

[0014] The present invention also provides the application of the above-mentioned bone repair magnetothermal asymmetric hydrogel in the preparation of medical devices or biomaterials for promoting bone tissue repair or regeneration.

[0015] Preferably, the bone tissue repair or regeneration targets craniofacial bone defects.

[0016] Preferably, the application includes joint application with an alternating magnetic field.

[0017] The beneficial effects of this invention are: This invention constructs a bone repair magnetothermal responsive hydrogel with a spatially asymmetric structure, which precisely confines the mild thermal effect to the target repair area, effectively reducing the risk of thermal diffusion to non-target tissues. This material not only provides a suitable three-dimensional microenvironment for cell growth, but also controllably promotes bone regeneration under an external alternating magnetic field. Thus, it provides a solution for clinical bone defects, especially those in complex locations adjacent to sensitive organs, that combines good biocompatibility, excellent osteogenic activity, and precise therapeutic function. Attached Figure Description

[0018] Figure 1 Light microscopy images of Fe3O4@mPEG-PLGA microspheres with different particle sizes (scale bar = 100 μm). Figure 2Scanning electron microscope (SEM) images of Fe3O4@mPEG-PLGA microspheres with different particle sizes (scale bar = 50 μm); Figure 3 Thermogravimetric (TGA) curves of Fe3O4@mPEG-PLGA microspheres with different particle sizes; Figure 4 VSM magnetization curves of MS-3 microspheres; Figure 5 Fourier transform infrared (FTIR) spectra of Fe3O4, Fe3O4@mPEG-PLGA microspheres and Fe3O4@mPEG-PLGA / GelMA composite hydrogel. Figure 6 Energy dispersive spectroscopy (EDS) mapping of Fe3O4@mPEG-PLGA / GelMA composite hydrogel (scale bar = 250 μm); Figure 7 Scanning electron microscope (SEM) cross-sectional images (scale bar = 500 μm) of composite hydrogels with different microsphere contents, and magnified views of their top and bottom surfaces (scale bar = 100 μm). Figure 8 Statistical histogram of average pore size for composite hydrogels with different microsphere contents (based on SEM image analysis). Figure 9 Compressive stress-strain curves of composite hydrogels with different microsphere contents; Figure 10 A statistical graph of Young's modulus for composite hydrogels with different microsphere contents; Figure 11 The swelling rate of composite hydrogels with different microsphere contents in phosphate buffered saline (PBS) changes (3 hours). Figure 12 Thermal imaging (A) and heating curve (B) of composite hydrogels with different microsphere contents under an alternating magnetic field (magnetic field power P = 3.5 kW); Figure 13 Cyclic magnetothermal stability testing of composite hydrogels; Figure 14 Asymmetric heating thermal imaging of the composite hydrogel (A) and heating curve (B). Figure 15 Thermal imaging of the surface temperature distribution of the composite hydrogel under magnetothermal and photothermal stimulation (magnetic field power P = 3.5 kW, near-infrared laser wavelength 808 nm, power P = 1 W / cm²). 2 ); Figure 16 The relative cell viability of BMSCs after co-culturing with different materials (detected by CCK-8 assay). Figure 17Alkaline phosphatase (ALP) staining characterization (A) and quantitative analysis of activity of BMSCs cultured with different materials for 7 days (B); Figure 18 Alizarin Red (ARS) staining characterization of mineralized nodules of BMSCs after 14 days of culture with different materials (A) and quantitative analysis (B); Figure 19 To verify the safety of in vivo magnetothermal therapy with composite hydrogel, (A) in vivo infrared thermography showing the temperature distribution on the top and bottom surfaces of the hydrogel; (B) representative H&E stained sections of brain tissue after surgery. Figure 20 Micro-CT three-dimensional reconstruction images (A) and bone volume fraction statistics (B) of skull defects in rats at 4, 8 and 12 weeks post-surgery. Detailed Implementation

[0019] This invention provides a method for preparing a magnetothermal asymmetric hydrogel for bone repair. The method combines a magnetic component with a photocurable biopolymer solution and uses a magnetic field for spatial guidance before cross-linking, ultimately obtaining a composite hydrogel material with an asymmetric distribution of internal magnetic microspheres.

[0020] The preparation method specifically includes the following steps: S1. Prepare composite magnetic microspheres containing iron oxide nanoparticles and a polymer coating layer.

[0021] In one embodiment of the present invention, the iron(III) oxide nanoparticles, with the chemical formula Fe3O4, are the core magnetic component for preparing the composite magnetic microspheres. The polymer coating layer, composed of a polymer material, serves to coat the iron(III) oxide nanoparticles, forming a core-shell structured composite microsphere.

[0022] S2. Dissolve the methacrylamide gelatin and photoinitiator in a phosphate buffer solution to obtain a precursor solution. Specifically, the methacrylamide gelatin is the matrix material of the hydrogel of the present invention. The photoinitiator is used to initiate the crosslinking reaction of the methacrylamide gelatin under light irradiation, and may be selected from at least one of phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959), or 2-hydroxy-2-methylphenylacetone (Irgacure 1173). The phosphate buffer solution is a conventional physiological buffer system with a pH value typically in the range of 7.0 to 7.6, for example, 7.2, 7.3, 7.4, or 7.5, preferably 7.2 to 7.4.

[0023] S3. Add the composite magnetic microspheres to the precursor liquid and disperse them evenly to obtain a composite precursor solution. In this step, the even dispersion can be achieved by conventional mixing methods in the art, such as mechanical stirring, vortex oscillation, or brief ultrasonic treatment.

[0024] S4. Inject the composite precursor solution into the mold, place one side of the mold close to the permanent magnet, and apply a magnetic field to guide the directional migration of the composite magnetic microspheres. As an example, the mold can be selected or customized according to the desired final shape and size of the hydrogel, and its material is typically translucent, such as polydimethylsiloxane, polypropylene, or glass. The permanent magnet is used to provide the guiding magnetic field, and its types include, but are not limited to, neodymium iron boron permanent magnets, samarium cobalt permanent magnets, or alnico permanent magnets.

[0025] S5. Under the guidance of a magnetic field, the composite precursor solution is irradiated with ultraviolet light to crosslink and solidify the methacrylamide gelatin, thereby obtaining the bone repair mild magnetothermal asymmetric hydrogel.

[0026] Typically, the ultraviolet irradiation is performed using a light source that emits ultraviolet light, such as an LED ultraviolet lamp or a mercury lamp. The wavelength of the ultraviolet light must match the effective absorption band of the selected photoinitiator, for example, it can be 365 nm, 385 nm or 405 nm.

[0027] Preferably, in step S1, the polymer coating layer is made of mPEG-PLGA.

[0028] The mPEG-PLGA is a common amphiphilic biodegradable block copolymer composed of methoxy polyethylene glycol segments and poly(lactic acid-glycolic acid) copolymer segments.

[0029] Preferably, step S1 specifically includes the following sub-steps: S1.1. Ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved together in deionized water, and concentrated ammonia is added to adjust the pH to 9-11 to generate iron oxide nanoparticles. Oleic acid is then added for modification. After magnetic separation, washing and drying, hydrophobically modified iron oxide nanoparticles are obtained.

[0030] This step is a classic co-precipitation method for preparing oleic acid-modified iron(III) oxide nanoparticles. Ferric chloride hexahydrate (FeCl3·6H2O) and ferrous chloride tetrahydrate (FeCl2·4H2O) serve as the iron source. The concentrated ammonia solution typically has a mass concentration of 25%-28%. The oleic acid is used as a surface modifier. The magnetic separation can be performed using a permanent magnet or electromagnet. Washing commonly uses deionized water, ethanol, or acetone as the washing solvent. Drying can be achieved using vacuum drying, freeze drying, or oven drying.

[0031] S1.2. Using a dichloromethane solution containing dissolved mPEG-PLGA and the hydrophobically modified iron oxide nanoparticles as the oil phase, and an aqueous solution containing 1%~5% polyvinyl alcohol as the external aqueous phase, emulsification is performed under homogenization conditions of 2000rpm~6000rpm and 0.5min~3min to form an emulsion. This step uses an emulsification-solvent evaporation method to prepare composite microspheres. The dichloromethane is a commonly used organic solvent for dissolving mPEG-PLGA. The polyvinyl alcohol serves as an emulsion stabilizer for the external aqueous phase. The homogenization speed is a key parameter affecting the droplet size of the emulsion, and is further preferably 4500rpm to 5500rpm. The homogenization time is further preferably 1.8min to 2.2min.

[0032] S1.3. The emulsion is stirred at a constant temperature of 20-30°C for 4-8 hours to evaporate the organic solvent, then centrifuged, washed, and freeze-dried to obtain the composite magnetic microspheres. The constant temperature stirring can be carried out in a water bath or on a magnetic stirrer. The centrifugation is used to collect the solidified microspheres. The freeze-drying is used to remove moisture from the microspheres.

[0033] Preferably, in step S1.1, the modification step of adding oleic acid includes: stirring the oleic acid and the iron oxide nanoparticle suspension at 50-70°C for 20-40 minutes, then adding dilute hydrochloric acid dropwise to adjust the pH to 5-7 until the foam on the liquid surface completely disappears. The concentration of the dilute hydrochloric acid is, for example, 0.5 mol / L to 1.5 mol / L. The pH value is further preferably 5.5 to 6.5.

[0034] In step S1.1, the molar ratio of ferric chloride hexahydrate to ferrous chloride tetrahydrate is 1.5~2.5:1, more preferably 1.8~2.2:1.

[0035] In step S1.2, the concentration of mPEG-PLGA in the oil phase is 40 mg / mL to 60 mg / mL, more preferably 45 mg / mL to 55 mg / mL. The concentration of the hydrophobically modified iron oxide nanoparticles in the oil phase is 20 mg / mL to 30 mg / mL, more preferably 22 mg / mL to 28 mg / mL.

[0036] In step S1.2, after the emulsion is formed, the solvent is evaporated by constant temperature stirring at a speed of 200 rpm to 600 rpm, and more preferably by constant temperature stirring at a speed of 450 rpm to 550 rpm.

[0037] Preferably, the saturation magnetization of the composite magnetic microspheres obtained in step S1 is 5 emu / g to 15 emu / g, more preferably 7 emu / g to 12 emu / g. Saturation magnetization is an important parameter characterizing the strength of a material's magnetism and can be measured using a vibrating sample magnetometer.

[0038] In step S2, the concentration of the methacrylamide gelatin in the precursor solution is 10%~20% w / v, more preferably 12%~18% w / v. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, and its concentration in the precursor solution is 2%~6% w / v, more preferably 3%~5% w / v.

[0039] Preferably, in step S4, the mold is a cylindrical mold with an inner cavity diameter of 4 mm to 6 mm, more preferably 4.5 mm to 5.5 mm; and a height of 2 mm to 3 mm, more preferably 2.2 mm to 2.8 mm.

[0040] In step S4, the permanent magnet is a neodymium iron boron permanent magnet.

[0041] In step S4, the magnetic field is applied for 30 to 120 seconds, more preferably 45 to 90 seconds.

[0042] In step S5, the wavelength of the ultraviolet light is 405 nm. The composite precursor solution is irradiated from both the front and back of the mold for 40 to 80 seconds each, more preferably 50 to 70 seconds.

[0043] The present invention also provides a magnetothermal asymmetric hydrogel prepared by the above preparation method.

[0044] The present invention also provides the application of the above-mentioned magnetothermal asymmetric hydrogel in the preparation of medical devices or biomaterials for promoting bone tissue repair or regeneration.

[0045] In this invention, the medical device refers to instruments, equipment, appliances, etc., used directly or indirectly on the human body, and may be selected from, but not limited to, further preparation of bone repair scaffolds, bone filling materials, implantable patches, medical catheters, or surgical instruments. The biomaterial refers to materials used in contact with living systems for the diagnosis, treatment, repair, or replacement of tissues and organs, and may be used in conjunction with other commonly used materials.

[0046] Preferably, the bone tissue repair or regeneration targets craniofacial bone defects. Craniofacial bone defects refer to interruptions or absences of bone tissue continuity in the skull and facial bone regions.

[0047] Preferably, the application includes combined use with an alternating magnetic field. The alternating magnetic field is generated by an alternating magnetic field generator, such as a high-frequency induction heating device. Its frequency, power, and duration of action can be adjusted according to the treatment needs.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example

[0050] The detailed steps for preparing magnetic composite microspheres (Fe3O4@mPEG-PLGA) are as follows: (1) Synthesis of magnetic iron oxide nanoparticles: Weigh 3.9 g of ferric chloride hexahydrate (FeCl3·6H2O) and 4.9 g of ferrous chloride tetrahydrate (FeCl2·4H2O), and dissolve them together in 300 mL of deionized water. The solution was mechanically stirred at 300 rpm for 10 min at room temperature to obtain a homogeneous, pale yellow, clear solution. Then, 10 mL of 25%-28% concentrated ammonia solution was added dropwise to the solution while continuously stirring to adjust the pH to 10. At this point, the solution color gradually changed from yellow to gray and finally to black. After stirring for another 10 min, the water bath temperature was adjusted to 60°C and stirring continued for 25 min to obtain a ferric oxide nanoparticle suspension. 1.5 g of oleic acid was added to the suspension, and stirring was continued at 300 rpm for 30 min at 60°C, resulting in white-gray foam appearing on the surface. Subsequently, dilute hydrochloric acid was slowly added dropwise while stirring. The foam gradually decreased, and when the pH value dropped to between 6 and 7, the foam completely disappeared, and obvious black flocculent oily aggregates formed in the solution. The upper liquid became transparent, indicating that the modification was complete. Magnetic separation was performed using a neodymium iron boron permanent magnet (60 mm in diameter, 15 mm in height, grade N35). The supernatant was discarded, and the collected black magnetic aggregates were washed multiple times with deionized water and anhydrous ethanol to thoroughly remove unreacted and non-magnetic substances. Finally, the product was dried in a vacuum drying oven at 60°C for 12 hours to obtain hydrophobically modified iron tetroxide nanoparticles (hereinafter referred to as Fe3O4).

[0051] (2) Preparation of microspheres by oil / water (O / W) monoemulsion solvent evaporation method: 100 mg of the biodegradable polymer mPEG-PLGA was dissolved in 2 mL of dichloromethane as the oil phase. 50 mg of the hydrophobically modified Fe3O4 was added to the oil phase and thoroughly dispersed by ultrasonication (40 kHz, 180 W) in an ice bath for 20 min to form a homogeneous primary emulsion. Subsequently, this primary emulsion was dropwise added to 50 mL of an aqueous phase containing 2.5% polyvinyl alcohol (PVA) and homogenized to form an O / W emulsion. Specific parameters such as the speed and time of the homogenizer were used. The resulting microsphere particle size and Fe3O4 encapsulation efficiency are shown in Table 1. Table 1 Microsphere preparation parameters and their corresponding microsphere properties Microsphere number Homogenization and emulsification speed (rpm) Homogenization emulsification time (min) Constant temperature stirring speed (rpm) Microsphere size (μm) <![CDATA[Encapsulation rate of Fe3O4]]> MS-1 6000 2 500 7.53±2.04 11.20% MS-2 4000 2 500 15.62±1.80 12.28% MS-3 3000 1 500 47.68±1.42 13.09% MS-4 3000 1 200 96.76±2.16 8.95% The system was stirred at a constant temperature of 25°C for 6 hours to completely evaporate the organic solvent. Subsequently, the microspheres were collected by centrifugation at 5000 rpm for 10 min, washed three times with deionized water to remove PVA, and finally lyophilized to obtain Fe3O4@mPEG-PLGA composite microsphere powder.

[0052] Observe the color and appearance of the microspheres using an optical microscope. Figure 1 MS-1 and MS-2 microspheres are light black, while MS-3 microspheres are the darkest dark brown, suggesting a higher encapsulation efficiency for Fe3O4. Conversely, although MS-4 microspheres have the largest particle size at 96.76 μm, their color is lighter than that of MS-3 microspheres with a particle size of 47.68 μm.

[0053] Using scanning electron microscopy (SEM), Figure 2 Observing the morphology and appearance of the microspheres, MS-3 microspheres are more rounded and uniform in shape, MS-1 and MS-2 microspheres have uneven particle size and large pores and collapses on the surface, while MS-4 microspheres are rough and uneven in shape with dotted depressions on the surface.

[0054] Thermogravimetric analysis (TGA) Figure 3 Quantitative data confirmed that the Fe3O4 residue (i.e., drug loading rate) of the four microspheres (MS-1, MS-2, MS-3, and MS-4) at 600℃ was 11.20%, 12.28%, 13.09%, and 8.95%, respectively. MS-3 microspheres exhibited the highest drug loading rate, consistent with its morphological observations. In summary, it can be inferred that the mismatch between parameters such as rotation speed during preparation and the target particle size led to structural instability and decreased encapsulation efficiency in the MS-1, MS-2, and MS-4 microspheres.

[0055] Figure 4 The magnetic properties of the selected MS-3 microspheres were characterized using a vibrating sample magnetometer (VSM). Figure 4As shown, its magnetization curve exhibits a typical "S" shape, proving that the prepared microspheres possess superparamagnetism. The measured saturation magnetization of the microspheres is 8.37 emu / g, meeting the basic requirements for efficient magnetocaloric conversion.

[0056] The characterization results above demonstrate that MS-3 microspheres (particle size 47.68 ± 1.42 μm) exhibit the best overall performance in terms of particle size uniformity, structural integrity, Fe3O4 loading rate (13.09%), and superparamagnetism. Therefore, MS-3 microspheres were selected in this invention to construct asymmetric mild magnetocaloric responsive hydrogels.

[0057] The preparation and performance characterization of the asymmetric mild magnetocaloric responsive hydrogel (Fe3O4@mPEG-PLGA / GelMA) are detailed below: (1) Preparation of precursor stock solution Methacrylamide gelatin (GelMA) was dissolved in phosphate buffer to prepare a 15% (w / v) solution. A 4% (w / v) concentration of the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) was added to this solution, and the mixture was stirred at 37°C in the dark until completely dissolved, yielding a homogeneous GelMA / LAP precursor solution.

[0058] (2) Preparation and screening of composite hydrogels To investigate the effect of microsphere content on material properties, a component design was conducted. The precursor solution was taken, and MS-3 microspheres of varying masses were added, along with phosphate buffer, to ensure that each component reached the target concentrations shown in Table 2 in the final composite precursor solution used for curing. Specifically, the final working concentrations of GelMA and LAP were fixed at 6.75% and 0.2% (w / v), respectively, while the microsphere content was varied (from 0% to 10%). The microspheres were uniformly dispersed by shaking to form a series of composite precursor solutions. These solutions were injected into specific molds, with one side of the mold pressed against a permanent magnet. The magnetically guided composite precursor solutions were then irradiated with 405 nm ultraviolet light from both directions for 60 seconds each, allowing the GelMA network to fully cross-link and cure.

[0059] Table 2. Composition and gelling properties of composite hydrogels with different Fe3O4@mPEG-PLGA microsphere contents Group <![CDATA[Fe3O4]]> GelMA LAP gelling properties control group 0.00% 6.75% 0.2% success 1.25% 0.14% 6.75% 0.2% success 2.50% 0.28% 6.75% 0.2% success 5.00% 0.56% 6.75% 0.2% success 7.50% 0.84% 6.75% 0.2% fail 10.00% 1.12% 6.75% 0.2% fail Experiments showed that when the microsphere content was ≥7.50%, the solution could not cross-link into a gel due to excessively high viscosity and light-shielding effect. Based on subsequent performance tests, the group with a microsphere content of 5.00% exhibited the best overall performance and was therefore determined to be the optimal content for constructing the hydrogel.

[0060] (3) Investigation on the magnetic interaction time of asymmetric hydrogels A composite precursor solution with a microsphere content of 5.00% was injected into a cylindrical mold (material: transparent silicone rubber; inner cavity dimensions: diameter 5 mm, height 2.5 mm). Then, one side of the mold was placed tightly against the surface of a neodymium iron boron permanent magnet (grade N35, diameter 25 mm, thickness 5 mm), and the superparamagnetism of the magnetic microspheres was used to guide their directional migration towards the magnetic source. To optimize the formation of the asymmetric structure, different magnetic treatment times (0, 15, 30, and 60 seconds) were applied, and the mold was irradiated with 405 nm ultraviolet light from both sides for 60 seconds each. The results showed that after 30 seconds of magnetic treatment, preliminary enrichment of magnetic microspheres towards the magnetic source side was observed, forming a distribution with a certain degree of asymmetry; after 60 seconds, the magnetic microspheres achieved high enrichment on one side of the gel, forming a typical asymmetric structure with a clear interface. Therefore, 60 seconds was ultimately determined as the optimal magnetic treatment time for constructing this asymmetric hydrogel. Therefore, 60 seconds was ultimately determined to be the optimal magnetic interaction time for constructing this asymmetric hydrogel.

[0061] Figure 5 The FTIR spectra showed that the hydrophobic Fe3O4 exhibited characteristic Fe-O bond peaks at 578 cm⁻¹ and 465 cm⁻¹, and characteristic absorption peaks of oleic acid at 2923, 2855, and 1627 cm⁻¹, confirming its successful synthesis. The Fe3O4@mPEG-PLGA microspheres showed characteristic C=O and CO peaks at 1759 cm⁻¹ and 1182 cm⁻¹, while the -OH peak weakened, confirming successful mPEG-PLGA encapsulation. The composite hydrogel spectrum further revealed amide characteristic peaks of GelMA: amino N–H stretching vibration at 3410 cm⁻¹, amide I C=O stretching vibration at 1648 cm⁻¹, and amide II C–N stretching and N–H bending vibrations at 1537 cm⁻¹, confirming successful construction.

[0062] Figure 6 EDS-mapping results of the Fe3O4@mPEG-PLGA / GelMA composite hydrogel showed that the Fe element signal was highly enriched in the top region of the hydrogel, while the signal was weak in the bottom region. This result directly confirms that, after magnetic guidance, the magnetic microspheres formed a significant spatial asymmetric distribution inside the gel.

[0063] Figure 7 SEM cross-sectional images of the hydrogels show that the asymmetry of the internal structure of the hydrogels becomes more pronounced with increasing microsphere content. The 5.00% content group exhibits the most typical asymmetric structure, with a dense structure on the top surface due to microsphere enrichment and a loose, porous network on the bottom surface. This structure is conducive to achieving differentiated biological functions.

[0064] Figure 8The pore size distribution is based on SEM cross-sectional images of the hydrogel. As the microsphere content increases from 0% to 5.00%, the average pore size of the hydrogel decreases, reaching 227.84 μm, 166.94 μm, 112.43 μm, and 95.49 μm, respectively. However, even the smallest pore size of 95.49 μm in the 5.00% group still provides a relatively suitable physical microenvironment for cell adhesion, proliferation, and differentiation.

[0065] Figure 9 The compressive stress-strain curves show that the stress values ​​of each sample at strain increase with the increase of microsphere content, specifically 0.294 MPa (control group), 0.259 MPa (1.25%), 0.377 MPa (2.50%), and 0.507 MPa (5.00%). The data indicate that the 5.00% content group has the highest load-bearing capacity within the tolerable deformation range.

[0066] Figure 10 The Young's modulus statistics show that the stiffness of the composite hydrogel significantly increases with increasing microsphere content. The Young's moduli for each group are 0.021 MPa (control group), 0.0244 MPa (1.25%), 0.0292 MPa (2.50%), and 0.0326 MPa (5.00%), respectively. The Young's modulus of the hydrogel with 5.00% microsphere content is approximately 1.5 times that of the pure GelMA hydrogel. These results indicate that increasing the microsphere content effectively strengthens the three-dimensional network of the GelMA hydrogel, thereby improving its overall mechanical properties.

[0067] Figure 11 The swelling rate curves of composite hydrogels with different microsphere contents in PBS over 3 hours show that the swelling rate of the hydrogels significantly decreases with increasing microsphere content, reaching 10.45% (control group), 8.12% (1.25%), 6.12% (2.50%), and 4.45% (5.00%), respectively. The swelling rate of the 5.00% microsphere content group is reduced to half that of pure GelMA hydrogels, confirming that the addition of microspheres increases the cross-linking density of the gel network, thereby enhancing its structural stability.

[0068] Figure 12 The magnetocaloric heating diagrams and curves of AB show that the magnetocaloric heating rate and the final temperature increase with increasing microsphere content. The 5.00% microsphere content group can stably rise from room temperature to above 41°C within 8 minutes under an alternating magnetic field of 3.5 kW, which is within the mild magnetocaloric temperature range that can stimulate osteoblast activity.

[0069] Based on the characterization of hydrogels with different microsphere contents in terms of morphology, pore size, mechanical properties, swelling, and magnetocaloric properties, the results show that the 5.00% content group exhibits the best overall performance. Therefore, this group was selected for subsequent in vitro and in vivo experiments to verify its asymmetric heating characteristics and osteogenic effects.

[0070] Figure 13 The results showed that the temperature rise curves of the 5.00% microsphere composite hydrogel highly overlapped during four consecutive heating-cooling cycles under an alternating magnetic field of 3.5 kW, reaching a treatment temperature above 41℃ each time. This demonstrates that the Fe3O4@mPEG-PLGA / GelMA composite hydrogel possesses excellent magnetothermal stability and reliability, meeting the needs of multiple treatments.

[0071] Figure 14 Asymmetric heating thermal imaging and heating curves of the AB composite hydrogel under an alternating magnetic field of 3.5 kW show that the temperature of the top surface enriched with hydrogel magnetic microspheres rapidly rises to 42.1 °C, while the bottom surface, affected by heat conduction, only rises to 36.1 °C. This temperature difference of more than 5 °C significantly confirms that this asymmetric structure can effectively confine the thermal effect to the target side, greatly reducing the risk of thermal damage to sensitive tissues on the bottom surface (such as brain tissue).

[0072] Figure 15 The thermal imaging comparison of magnetothermal and photothermal heating uniformity clearly shows a significant difference in temperature distribution between the two stimulation modes. Using hydrogels of the same size, the surface temperature distribution of the hydrogel under magnetothermal stimulation is uniform; in contrast, the hydrogel in the photothermal stimulation group shows a radial attenuation of heat from the center outwards, forming a clear temperature gradient. The results indicate that the magnetothermal therapy employed in this invention has a significant advantage in heating uniformity, achieving a precise and uniform heating effect within the target area.

[0073] The detailed steps for in vitro and in vivo osteogenic validation are as follows: (1) In vitro biocompatibility evaluation To assess the cell compatibility of the materials, cell proliferation activity was detected using the CCK-8 assay. Four groups were established: a control group containing only cells and culture medium; a microsphere group where a 5 mg / mL suspension of Fe3O4@mPEG-PLGA microspheres was added to the upper chamber of a 24-well plate trans-well insert for indirect co-culture with cells cultured in the lower chamber; a hydrogel group where sterilized composite hydrogel discs (5 mm in diameter, 2.5 mm thick) containing 5% microspheres were placed in a 24-well plate trans-well insert for indirect co-culture with cells cultured in the lower chamber; and a hydrogel combined with magnetothermal group where identical hydrogel discs were placed in inserts for indirect co-culture, and an alternating magnetic field was applied once daily using a high-frequency induction heating device (model: SPG-10-I) at a power setting of 3.5 kW for 8 minutes each time. All groups were tested using a 2.0 × 10⁻⁶ m-well plate. 4 Third-generation rat bone marrow mesenchymal stem cells (BMSCs) were seeded at a density of 1 cell / well. On days 1, 2, and 3 of co-culture, CCK-8 reagent was added to the corresponding parallel wells of each group. After incubation at 37°C for 30 minutes, the absorbance was measured at 450 nm using an ELISA reader, and the relative cell proliferation rate at each time point was calculated.

[0074] (2) Evaluation of in vitro osteogenicity: To further evaluate the osteogenic induction capacity of the material, alkaline phosphatase (ALP) and alizarin red (ARS) staining and quantification experiments were performed. The experimental groupings followed the CCK-8 setup, but to focus on the scaffold itself and the combined magnetocaloric effect, the microsphere group alone was removed, resulting in the following three groups for comparison: Control group: containing only cells and osteogenic induction solution; Hydrogel group: sterile composite hydrogel discs (5 mm in diameter, 2.5 mm thick) containing 5% microspheres were placed in permeable inserts of 12-well plates for indirect co-culture with cells; Hydrogel combined with magnetocaloric group: identical hydrogel discs were placed in inserts for indirect co-culture, with alternating magnetic field stimulation applied every 3 days during culture (equipment: SPG-10-I high-frequency induction heater, parameters: power 3.5 kW, stimulation for 8 minutes). All groups were seeded with third-generation rat bone marrow mesenchymal stem cells (BMSCs) at a density of 5.0 × 10⁴ cells / well. On day 7, cells were stained with alkaline phosphatase (ALP) using BCIP / NBT substrate chromogenic solution, and the absorbance of cell lysates was measured at 405 nm using a microplate reader to quantitatively analyze early osteogenic differentiation activity. On day 14, after cell fixation, cells were stained with 1% Alizarin Red (ARS) for 30 minutes to visualize calcium nodules. The bound dye was extracted with 10% cetylpyridinium chloride solution, and the absorbance was measured at 562 nm to quantitatively assess late mineralization capacity.

[0075] (3) Evaluation of in vivo bone repair To evaluate the efficacy and safety of in vivo bone repair, 45 male SD rats were selected. A critical-sized bone defect of 5 mm in diameter was created in the skull and the rats were randomly divided into three groups (n=15): (i) Control group: no filling was performed on the defect; (ii) Hydrogel group: sterile asymmetric hydrogel discs with 5% microsphere content, 5 mm in diameter and 2.5 mm thick, with the magnetic enrichment surface facing upwards, were implanted; (iii) Hydrogel combined with magnetothermal group: the same hydrogel was implanted, and on postoperative days 7, 14, 21, and 28, rats in the hydrogel combined with magnetothermal group received magnetothermal stimulation for 8 minutes under anesthesia using a high-frequency induction heating device (model: SPG-10-I, power set at 3.5 kW). Infrared thermal imaging was used to monitor the temperature distribution of the defect site with the magnetothermal surface facing upwards and the non-magnetothermal surface (i.e., bottom surface) facing upwards, to verify its in vivo asymmetric heating characteristics. At weeks 4, 8, and 12 post-surgery, rats were euthanized after intraperitoneal injection of an overdose of sodium pentobarbital. Skull samples were collected for Micro-CT scanning at 10 μm resolution and 3D reconstruction, and the bone volume fraction of the defect area was calculated. Brain tissue below the defect was harvested, fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to systematically evaluate the bone formation effect and biosafety.

[0076] Figure 16 CCK-8 assay results showed that different materials had varying effects on the proliferation of BMSCs. After 3 days of culture, the cell viability of the microsphere group was 94.2% of that of the control group, remaining at a high level; while the cell viability of the hydrogel group and the combined magnetothermal group increased to 108.0% and 116.5% of the control group, respectively. These results indicate that encapsulating microspheres in GelMA hydrogel can effectively improve its biocompatibility, and the combined magnetothermal stimulation can further synergistically promote cell proliferation.

[0077] The results are as follows Figure 17 As shown in Figures AB, ALP activity in the hydrogel group was significantly higher than that in the control group. The hydrogel combined with magnetothermal group showed the deepest ALP staining, with a quantitative activity value twice that of the control group, indicating that magnetothermal stimulation can synergistically and significantly enhance the early osteogenic differentiation of BMSCs.

[0078] like Figure 18 As shown in Figures AB, the hydrogel combined with magnetothermal therapy resulted in the formation of numerous, dense, deep red calcium nodule deposits. Its relative absorbance (ARS) was significantly higher than other groups, being 1.61 times that of the control group, demonstrating that combined magnetothermal therapy can greatly promote extracellular matrix mineralization and drive the osteogenic process into the maturation stage.

[0079] like Figure 19As shown in Figure A, the temperature at the center of the defect on the upward-facing magnetothermal surface precisely rises to 42.5°C; while the temperature in the corresponding area on the bottom surface facing the brain tissue only rises to 38.3°C, forming a safe temperature difference of more than 4°C, thus achieving asymmetrical heating within the body. Figure 19 B showed that, compared with the control group, no edema, degeneration, or abnormal inflammatory cell infiltration was observed in the brain tissue sections of the hydrogel combined with magnetothermal group, confirming the good in vivo biocompatibility of this mild magnetothermal therapy.

[0080] like Figure 20 As shown in Figure A, the defect in the control group was clearly defined with almost no new bone bridging. In the hydrogel group, new bone formation was only observed at the defect edges, with a clearly translucent defect area in the center. In the hydrogel combined with magnetothermal group, the defect area was completely filled and bridged by high-density new bone tissue, with an outline closely resembling the original skull. Figure 20 As shown in Figure B, the bone volume fraction increased with time, and the hydrogel combined with magnetothermal group maintained a significant advantage at all time points (p<0.01). By week 12, its bone volume fraction (0.311) was 2.57 times that of the control group (0.121), indicating that the synergistic promoting effect of magnetothermal stimulation continued to enhance with the repair process.

[0081] In summary, this invention constructs a composite material with a spatially asymmetric structure by loading Fe3O4@mPEG-PLGA magnetic microspheres into GelMA hydrogel. This material not only solves the safety challenges of deep hyperthermia through its unique spatial structure design, but also significantly enhances osteogenic bioactivity through synergistic magnetothermal stimulation. Ultimately, it achieves safe and efficient bone defect repair in animal models, providing a highly promising solution for clinical translation.

[0082] As shown in the above embodiments, this invention provides a magnetothermal responsive hydrogel with a spatially asymmetric structure and its construction method. Under the guidance of an external permanent magnet, the hydrogel's internal magnetic components can be directionally enriched on one side, thus forming an asymmetric structure. Under the action of an alternating magnetic field, this structure successfully achieves differentiated heating on both sides of the hydrogel, precisely limiting the effective treatment temperature to a preset side and significantly reducing thermal exposure to simulated sensitive tissues on the dorsal side. In vitro experiments have confirmed that this material system has good biocompatibility and can synergistically promote the activity, differentiation, and mineralization function of osteoblast-related cells through magnetothermal stimulation. In a rat skull defect model, this material combined with intermittent magnetothermal therapy can safely and effectively promote the formation of new bone and defect healing without causing observable thermal damage to adjacent brain tissue. Therefore, this invention provides a potential new strategy that balances safety and efficacy for bone defect repair in adjacent heat-sensitive areas.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a bone repair magneto-thermal asymmetric hydrogel, characterized in that, Includes the following steps: S1. Prepare composite magnetic microspheres containing iron oxide nanoparticles and a polymer coating layer; S2. Dissolve methacrylamide gelatin and photoinitiator in phosphate buffer to obtain the precursor solution; S3. Add the composite magnetic microspheres to the precursor liquid and disperse them evenly to obtain a composite precursor solution; S4. Inject the composite precursor solution into the mold, place one side of the mold close to the permanent magnet, and apply a magnetic field to guide the directional migration of the composite magnetic microspheres. S5. Under the guidance of a magnetic field, the composite precursor solution is irradiated with ultraviolet light to crosslink and solidify the methacrylamide gelatin, thereby obtaining the bone repair mild magnetothermal asymmetric hydrogel.

2. The production method according to claim 1, characterized by, In step S1, the polymer coating layer is made of mPEG-PLGA.

3. The preparation method according to claim 2, characterized in that, Step S1 specifically includes: S1.

1. Ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved together in deionized water, concentrated ammonia is added to adjust the pH to 9-11 to generate iron oxide nanoparticles, oleic acid is then added for modification, and hydrophobically modified iron oxide nanoparticles are obtained after magnetic separation, washing and drying. S1.

2. Using a dichloromethane solution containing mPEG-PLGA and the hydrophobically modified iron oxide nanoparticles as the oil phase, and an aqueous solution containing 1%~5% polyvinyl alcohol as the external aqueous phase, an emulsion is formed under the conditions of homogenization speed of 2000 rpm~6000 rpm and homogenization time of 0.5 min~3 min. S1.

3. The emulsion is stirred at a constant temperature of 20~30℃ for 4~8 hours to evaporate the organic solvent, and then centrifuged, washed and freeze-dried to obtain the composite magnetic microspheres.

4. The production method according to claim 3, characterized by, In step S1.1, the step of adding oleic acid for modification includes: stirring the oleic acid and the iron oxide nanoparticle suspension at 50~70℃ for 20~40 minutes, and then adding dilute hydrochloric acid dropwise to adjust the pH to 5~7 until the foam on the liquid surface completely disappears; In step S1.1, the molar ratio of ferric chloride hexahydrate to ferrous chloride tetrahydrate is 1.5~2.5:1; In step S1.2, the concentration of mPEG-PLGA in the oil phase is 40 mg / mL to 60 mg / mL; the concentration of the hydrophobically modified iron oxide nanoparticles in the oil phase is 20 mg / mL to 30 mg / mL. In step S1.2, after the emulsion is formed, the solvent is evaporated by constant temperature stirring at a speed of 200 rpm to 600 rpm.

5. The preparation method according to claim 2, characterized in that, The saturation magnetization of the composite magnetic microspheres obtained in step S1 is 5 emu / g to 15 emu / g; In step S2, the concentration of the methacrylamide gelatin in the precursor solution is 10%~20% w / v; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, and its concentration in the precursor solution is 2%~6% w / v.

6. The method of claim 1, wherein, In step S4, the mold is a cylindrical mold with an inner cavity diameter of 4 mm to 6 mm and a height of 2 mm to 3 mm. In step S4, the permanent magnet is a neodymium iron boron permanent magnet; In step S4, the magnetic field guidance is applied for 30 to 120 seconds. In step S5, the wavelength of the ultraviolet light is 405 nm, and the composite precursor solution is irradiated from both the front and back of the mold for 40 to 80 seconds each.

7. The bone repair magnetothermal asymmetric hydrogel prepared by any one of claims 1 to 6.

8. The use of the bone repair magnetothermal asymmetric hydrogel of claim 7 in the preparation of medical devices or biomaterials for promoting bone tissue repair or regeneration.

9. Use according to claim 8, characterized in that, The bone tissue repair or regeneration targets craniofacial bone defects.

10. Use according to claim 8, characterized in that, The applications include combined use with alternating magnetic fields.

Images