A magnetic response composite hydrogel for integrated repair of bone defects and a preparation method thereof

Abstract

The application provides a magnetic response composite hydrogel for integrated repair of bone defects and a preparation method thereof, and belongs to the technical field of medical materials. The composite hydrogel can efficiently inhibit tumor cell activity under the time sequence regulation of an external alternating magnetic field, and then provide a suitable microenvironment and key components for bone tissue regeneration, so that signal conversion and function connection from tumor killing to bone repair are realized. The material has good injectability and self-healing ability, can minimally invasively fill irregular defects, and has significant advantages in simplifying the clinical treatment procedure, shortening the treatment course, reducing the risk of tumor recurrence and improving the repair quality, and provides a new and integrated treatment strategy for tumor-related bone defects.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a magnetically responsive composite hydrogel for integrated repair of bone defects and its preparation method. Background Technology

[0002] The clinical treatment of bone defects has long been a major challenge in the fields of orthopedics, oral and maxillofacial surgery, and reconstructive surgery. The causes of these defects are diverse, including severe trauma, osteomyelitis, congenital malformations, and tumor resection. Among these, bone defects caused by invasion or surgical resection of malignant tumors (such as osteosarcoma and oral squamous cell carcinoma) are the most complex and challenging to treat. Such treatments typically carry a dual and often contradictory set of goals: first, to completely remove any remaining tumor cells to minimize the risk of recurrence; and second, to reconstruct large segments of bone tissue to restore the bone's mechanical support, anatomical structure, and related physiological functions.

[0003] Traditional mainstream treatment strategies generally adopt a "staged approach," which involves first performing extensive tumor resection, followed by postoperative radiotherapy or chemotherapy if necessary, and then conducting secondary bone repair surgery after confirming that the tumor has not recurred. Repair methods typically rely on autologous bone grafting, allogeneic bone grafting, or artificial bone substitutes. However, this approach has significant drawbacks: the treatment cycle is greatly prolonged, requiring patients to endure multiple surgical traumas; while radiotherapy kills residual tumor cells, it can damage surrounding normal bone and soft tissues, potentially causing radiation-induced osteonecrosis and hindering healing; systemic chemotherapy brings significant systemic toxic side effects. More importantly, the local microenvironment created by anti-tumor therapy (such as ischemia, inflammation, and inhibition of cell activity) is often unfavorable for the survival, vascularization, and osteogenic integration of subsequent transplanted bone, leading to an increased risk of nonunion or transplant failure. Summary of the Invention

[0004] The purpose of this invention is to provide a magnetically responsive composite hydrogel for integrated repair of bone defects and its preparation method, which solves the technical problem in existing tumor-related bone defect treatments where anti-tumor and bone repair processes are mutually restrictive and it is difficult to achieve temporal synergistic regulation in a single material system.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a magnetothermal-sensitized calcium-iron synergistic composite hydrogel, comprising a hydrogel matrix, calcium peroxide nanoparticles dispersed in the hydrogel matrix, and magnetite nanoparticles.

[0007] Preferably, the hydrogel matrix is ​​a three-dimensional network structure formed by a crosslinking reaction of carboxymethyl chitosan and aldehyde-modified polyethylene glycol.

[0008] This invention also provides a method for preparing the above-mentioned magnetothermal sensitization-calcium-iron synergistic composite hydrogel, comprising the following steps:

[0009] Aldehyde-modified polyethylene glycol solution, iron oxide magnetic nanoparticle suspension and calcium peroxide nanoparticle suspension were sequentially added to carboxymethyl chitosan solution and thoroughly mixed to obtain the composite hydrogel.

[0010] Preferably, the concentration of the carboxymethyl chitosan solution is 40 mg / mL to 80 mg / mL;

[0011] The mass ratio of the carboxymethyl chitosan to the aldehyde-modified polyethylene glycol is 3:1 to 4.5;

[0012] The concentration of the aldehyde-modified polyethylene glycol solution is 200 mg / mL to 400 mg / mL;

[0013] The concentration of the magnetic nanoparticle suspension of iron oxide is 100 mg / mL to 150 mg / mL;

[0014] The concentration of the calcium peroxide nanoparticle suspension is 4 mg / mL to 6 mg / mL;

[0015] The volume ratio of the carboxymethyl chitosan solution, the aldehyde-modified polyethylene glycol solution, the iron oxide magnetic nanoparticle suspension, and the calcium peroxide nanoparticle suspension is 60~80:40~60:15~25:4~6.

[0016] The mixing and stirring are carried out at room temperature for 1 to 5 minutes; in the composite hydrogel, the mass of the ferric oxide magnetic nanoparticles accounts for 1.0% to 2.0% of the total mass of the hydrogel.

[0017] Preferably, the calcium peroxide nanoparticles are prepared by the following steps:

[0018] Calcium chloride solution was added dropwise to anhydrous methanol under vigorous stirring, followed by the addition of ammonia and hydrogen peroxide solution to carry out the reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain the calcium peroxide nanoparticles.

[0019] The concentration of the calcium chloride solution is 1.5 mol / L to 2.5 mol / L;

[0020] The reaction was carried out by stirring for 30 to 60 minutes after the addition of hydrogen peroxide solution.

[0021] The centrifugation speed is 10,000 rpm to 15,000 rpm, and the time is 5 minutes to 15 minutes;

[0022] The prepared calcium peroxide nanoparticles have a particle size of 80 nm to 150 nm.

[0023] Preferably, the magnetite nanoparticles are prepared by the following steps:

[0024] Ferric chloride and ferrous chloride were dissolved in distilled water, and ammonia was added while heating and stirring. Then, sodium citrate solution was added all at once to continue the reaction. After the reaction was completed, the mixture was magnetically separated, washed and dried to obtain sodium citrate-modified magnetite nanoparticles.

[0025] The molar ratio of ferric chloride, ferrous chloride, and sodium citrate is 2.5~2.7 : 1.0~1.2 : 4.5~5.5;

[0026] The reaction temperature after adding ammonia is 70℃~90℃, and the reaction temperature after adding sodium citrate is 80℃~90℃.

[0027] The prepared magnetite nanoparticles have a particle size of 30 nm to 80 nm.

[0028] The present invention also provides the use of the above-described composite hydrogel or the composite hydrogel prepared by the above-described preparation method in the preparation of a medicament for the treatment or adjuvant treatment of oral squamous cell carcinoma.

[0029] The present invention also provides the application of the above-mentioned composite hydrogel or the composite hydrogel prepared by the above-mentioned preparation method in the preparation of biomaterials for bone defect repair.

[0030] Preferably, the bone defect is a jawbone defect.

[0031] The present invention also provides a programmed magnetically controlled therapy system, comprising an implantable material portion and a magnetic field application portion;

[0032] The implant material is the above-mentioned composite hydrogel or a composite hydrogel prepared by the above-mentioned preparation method.

[0033] The magnetic field applying part is used to apply an alternating magnetic field to the implantation site, and the intensity of the alternating magnetic field is 0.5 kW to 2.0 kW.

[0034] The beneficial effects of this invention are:

[0035] The composite hydrogel provided by this invention can, under the temporal regulation of an applied alternating magnetic field, first efficiently inhibit tumor cell activity, and then provide a suitable microenvironment and key components for bone tissue regeneration, thereby achieving signal conversion and functional integration from tumor killing to bone repair. This material possesses excellent injectability and self-healing capabilities, enabling minimally invasive filling of irregular defects. It demonstrates significant advantages in simplifying clinical treatment procedures, shortening treatment duration, reducing the risk of tumor recurrence, and improving repair quality, providing a novel and integrated treatment strategy for tumor-related bone defects. Attached Figure Description

[0036] Figure 1 The figures show the morphology and particle size characterization of CaO2 nanoparticles, where: Figure 1 A is a scanning electron microscope image; Figure 1 B is a transmission electron microscope image; Figure 1 C represents the particle size distribution.

[0037] Figure 2 Characterization diagram of Fe3O4 nanoparticles modified with sodium citrate, wherein: Figure 2 A is a transmission electron microscope image; Figure 2 B is the particle size distribution diagram; Figure 2 C is the Fourier transform infrared spectrum; Figure 2 D is the hysteresis loop diagram;

[0038] Figure 3 The figures show the structure and properties of pure hydrogels with different ratios, where: Figure 3 A is a comparison image from a scanning electron microscope; Figure 3 B represents the strain scan curve; Figure 3 C is a comprehensive comparison chart of various performance aspects;

[0039] Figure 4 The figures show the magnetocaloric properties, structure, and performance characterization of the composite hydrogel, where: Figure 4 A shows the heating curves of Fe3O4 hydrogels with different concentrations; Figure 4 B is a comparison of the heating capacity of hydrogels with different compositions; Figure 4 C represents the temperature rise curves of the composite hydrogel under different magnetic field intensities; Figure 4 D shows the transmission electron microscopy morphology of different hydrogels; Figure 4 E is a radar chart showing the properties of Fe3O4 hydrogels at different concentrations; Figure 4 F is the elemental distribution diagram of the composite hydrogel; Figure 4 G is a diagram demonstrating injectability and self-healing properties;

[0040] Figure 5 The image shows the in vitro antitumor effect of the composite hydrogel, where: Figure 5 A is a fluorescent image of cell viability and death staining; Figure 5 B is a statistical chart of cell viability;

[0041] Figure 6 The images show the in vitro effects of the composite hydrogel on promoting bone growth and angiogenesis, where: Figure 6 A shows the staining results for osteogenic differentiation. Figure 6 B is a fluorescence image of vascular endothelial cells forming tubes;

[0042] Figure 7 The image shows the in vivo antitumor effect of the composite hydrogel, where: Figure 7 A is a photograph of the tumor sample after treatment; Figure 7 B is a bar chart showing the final tumor volume statistics; Figure 7 C is a comparison chart of tumor growth curves;

[0043] Figure 8 Micro-CT analysis of the osteogenic effect of the composite hydrogel in vivo. Detailed Implementation

[0044] The CaO2 / Fe3O4 nanoparticle composite hydrogel system provided by this invention, which combines synergistic anti-tumor and orderly osteogenic functions, precisely transforms therapeutic damage signals (high temperature, free radicals, ion bursts) into bone regeneration signals (mild thermal stimulation, ion action, scaffold guidance) through a sequential treatment logic of "magnetothermal sensitization-calcium-iron synergy". This achieves an integrated process of first thoroughly controlling the tumor and then efficiently forming bone, providing a new paradigm of minimally invasive, precise and efficient treatment for tumor-related bone defects.

[0045] This invention constructs an injectable, magnetically responsive, in-situ moldable composite hydrogel system, comprising:

[0046] 1. Functional nanoparticles: A cascade reaction system of CaO2 nanoparticles and Fe3O4 magnetic nanoparticles;

[0047] 2. Hydrogel matrix: An injectable hydrogel formed by carboxymethyl chitosan and aldehyde-modified polyethylene glycol. This hydrogel has excellent injectability and self-healing ability, and can perfectly fit the irregular three-dimensional defect cavity after jaw surgery to achieve minimally invasive and precise filling. Its three-dimensional network structure effectively ensures the uniform dispersion of nanoparticles and prevents uneven thermotherapy caused by magnetic agglomeration.

[0048] 3. Time-space control mechanism: Activated by an external alternating magnetic field (AMF), triggering a triple synergistic effect of "thermal-chemical-ionization".

[0049] Treatment mechanism and action process (magnetothermal sensitization-calcium-iron synergistic sequential regulation):

[0050] Phase 1: Magnetocaloric activation and sensitization

[0051] Local heating: Under the action of AMF1 (1.5kW), Fe3O4 nanoparticles generate a magnetocaloric effect of approximately 47°C, directly inducing tumor cell apoptosis; 2. Enhanced cell membrane permeability: Sub-high temperature reversibly disrupts the integrity of tumor cell membranes, promoting the endocytosis efficiency of subsequent CaO2 nanoparticles, and significantly increasing intracellular calcium ions; In this invention, the term 'AMF1' specifically refers to the high-intensity alternating magnetic field applied in the first stage (i.e., the anti-tumor stage). It is an abbreviation for 'Alternating Magnetic Field 1', representing the first set of preset magnetic field parameter combinations.

[0052] Phase Two: Intracellular Cascade Reactions and Synergistic Tumor Killing

[0053] CaO2 degradation burst: Endocytinated CaO2 rapidly hydrolyzes in the acidic tumor microenvironment, releasing large amounts of Ca2+. 2+ With H2O2, it achieves a dual attack of intracellular calcium ion overload and oxidative stress:

[0054] Calcium overload (IIT): a sudden increase in intracellular calcium. 2 The concentration of ⁺ disrupts calcium homeostasis, triggering an irreversible "calcium overload" effect. Calcium overload further leads to mitochondrial dysfunction and triggers the apoptosis pathway.

[0055] Fenton reaction (CDT): H2O2 reacts with Fe 2 ⁺ A highly efficient Fenton reaction occurs: Fe 2+ + H2O2 → Fe 3+ + ·OH + OH⁻ generate highly toxic hydroxyl radicals, which oxidize lipids, DNA, and proteins, forming an oxidative stress storm.

[0056] Synergistic lethal effect: MHT, IIT, and CDT are not simply additive, but form an interlocking and mutually reinforcing amplification loop: thermotherapy promotes CaO2 endocytosis, enhancing the substrate supply for IIT and CDT; oxidative stress generated by CDT further disrupts the integrity of organelles (such as the endoplasmic reticulum and mitochondria), exacerbating the imbalance of calcium homeostasis and amplifying the effect of IIT; while calcium overload itself weakens the cell's antioxidant capacity, making it more sensitive to ROS attacks. This synergistic lethal effect of "oxidative stress-calcium overload" greatly improves the killing efficiency of tumor cells, and may show unique advantages, especially for tumor cells that are prone to resistance to traditional therapies.

[0057] Phase 3: Repairing Signal Transition and Orderly Bone Regeneration

[0058] Ca 2+ Promotes bone formation: Ca that was not completely consumed during the anti-tumor phase 2+It directly participates in and accelerates the formation and deposition of hydroxyapatite crystal nuclei, and continuously stimulates osteogenic differentiation and matrix mineralization of bone marrow mesenchymal stem cells. It promotes the expression of Runx2, ALP, and OPG osteogenic genes, and increases the amount of mineralized nodules formed by 2-4 times.

[0059] Fe 3+ Promoting angiogenesis: Fe 3+ This drives the formation of vascular networks on the basis of osteogenic formation, providing nutritional support for bone repair;

[0060] Mild magnetothermal stimulation (AMF2 1kW): The mild thermal effect (40-43℃) can induce a series of osteogenic effects, including upregulating heat shock protein expression and enhancing alkaline phosphatase activity, thereby effectively promoting mesenchymal stem cell mineralization to drive bone repair and achieving a programmed conversion of "damage-repair" signals. In this invention, the term 'AMF2' specifically refers to the high-intensity alternating magnetic field applied in the second stage (i.e., the bone repair stage). It is an abbreviation for 'Alternating Magnetic Field 2', representing a second set of preset magnetic field parameter combinations.

[0061] Scaffold-guided bone ingrowth: The porous hydrogel structure provides a template for cell ingrowth, which is gradually replaced by new bone tissue.

[0062] Specifically, this invention provides a magnetothermal-sensitized calcium-iron synergistic composite hydrogel. This composite hydrogel is an injectable, magnetically responsive biomaterial system comprising a hydrogel matrix and calcium peroxide nanoparticles and magnetite nanoparticles uniformly dispersed within the hydrogel matrix. The hydrogel matrix forms a three-dimensional network scaffold that accommodates and immobilizes the functional nanoparticles, while the calcium peroxide nanoparticles and magnetite nanoparticles serve as core functional units, respectively providing a calcium ion source and magnetothermal conversion capability.

[0063] In this invention, the hydrogel matrix is ​​preferably a three-dimensional network structure formed by a crosslinking reaction of carboxymethyl chitosan and aldehyde-modified polyethylene glycol. Carboxymethyl chitosan is a water-soluble derivative of chitosan, possessing good biocompatibility, biodegradability, and gelling properties. Its abundant amino and carboxyl groups on its molecular chain can participate in various reactions. Aldehyde-modified polyethylene glycol is a polyethylene glycol derivative with aldehyde groups modified at its ends, exhibiting good water solubility and biocompatibility. The crosslinking reaction mainly refers to the Schiff base reaction between the amino groups on the carboxymethyl chitosan chain and the aldehyde groups at the ends of the aldehyde-modified polyethylene glycol chain, forming dynamic imine bonds, thereby constructing an injectable hydrogel with self-healing properties. The mass ratio of carboxymethyl chitosan to aldehyde-modified polyethylene glycol is preferably 3:1 to 3:4.5, for example, 3:1, 3:1.5, 3:2, 3:3, or 3:4.5; more preferably 3:1.5 to 3:3; and most preferably 3:2.

[0064] This invention also provides a method for preparing the above-mentioned magnetothermal-sensitized-calcium-iron synergistic composite hydrogel. This method is simple and easy to operate. The preparation method includes the following steps: sequentially adding a specified concentration of aldehyde-modified polyethylene glycol solution, a suspension of magnetite nanoparticles, and a suspension of calcium peroxide nanoparticles to a specified concentration of carboxymethyl chitosan solution, and then thoroughly mixing and stirring under suitable conditions to obtain the composite hydrogel.

[0065] To obtain the composite hydrogel with optimal performance, various parameters in the preparation process were optimized. The concentration of the carboxymethyl chitosan solution is preferably 40 mg / mL to 80 mg / mL, for example, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, or 80 mg / mL; more preferably 55 mg / mL to 65 mg / mL; and most preferably 60 mg / mL. The concentration of the aldehyde-modified polyethylene glycol solution is preferably 200 mg / mL to 400 mg / mL, for example, 200 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, or 400 mg / mL; more preferably 280 mg / mL to 320 mg / mL; and most preferably 300 mg / mL. The concentration of the magnetite nanoparticle suspension is preferably 100 mg / mL to 150 mg / mL, for example, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, or 150 mg / mL; more preferably 115 mg / mL to 125 mg / mL; and most preferably 120 mg / mL. The concentration of the calcium peroxide nanoparticle suspension is preferably 4 mg / mL to 6 mg / mL, for example, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, or 6 mg / mL; more preferably 4.8 mg / mL to 5.2 mg / mL; and most preferably 5 mg / mL.

[0066] The volume ratio of each solution is one of the key factors affecting the final properties of the hydrogel. The preferred volume ratio of the carboxymethyl chitosan solution, the aldehyde-modified polyethylene glycol solution, the iron(III) oxide magnetic nanoparticle suspension, and the calcium peroxide nanoparticle suspension is (60-80):(40-60):(15-25):(4-6). For example, a specific and preferred volume ratio could be 75:50:20:5.

[0067] The mixing and stirring process is typically carried out at room temperature (e.g., 15°C to 30°C), and the stirring time is preferably 1 to 5 minutes, such as 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes; more preferably 2 to 4 minutes. The magnetocaloric properties of the composite hydrogel can be adjusted by controlling the amount of the added magnetite magnetic nanoparticles. In the composite hydrogel, the mass percentage of the magnetite magnetic nanoparticles to the total mass of the hydrogel is preferably 1.0% to 2.0%, such as 1.0%, 1.3%, 1.6%, or 2.0%; more preferably 1.4% to 1.8%; and most preferably 1.6%.

[0068] Furthermore, the present invention provides a preferred method for preparing the calcium peroxide nanoparticles. The method includes: adding a calcium chloride aqueous solution of a certain concentration dropwise to anhydrous methanol under vigorous stirring; subsequently adding ammonia and hydrogen peroxide solution sequentially to react; collecting the product by centrifugation after the reaction is complete; and washing and drying to finally obtain the calcium peroxide nanoparticles. The concentration of the calcium chloride solution is preferably 1.5 mol / L to 2.5 mol / L, for example, 1.5 mol / L, 1.8 mol / L, 2.0 mol / L, 2.2 mol / L, or 2.5 mol / L; more preferably 1.9 mol / L to 2.1 mol / L; and most preferably 2.0 mol / L. The stirring time after adding the hydrogen peroxide solution is preferably 30 minutes to 60 minutes, for example, 30 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes; more preferably 40 minutes to 50 minutes. The centrifugation speed is preferably 10,000 rpm to 15,000 rpm, for example, 10,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, or 15,000 rpm; the time is 5 minutes to 15 minutes, for example, 5 minutes, 8 minutes, 10 minutes, 12 minutes, or 15 minutes. The calcium peroxide nanoparticles prepared by this method have a particle size distribution between 80 nm and 150 nm, for example, 100 nm, 120 nm, or 140 nm; more preferably 100 nm to 130 nm; and most preferably about 120 nm.

[0069] Furthermore, this invention provides a preferred preparation method for the aforementioned magnetite magnetic nanoparticles. This method is an improved co-precipitation method, specifically comprising: dissolving ferric chloride and ferrous chloride in distilled water in a specific ratio; adding ammonia water under heating and stirring conditions; subsequently adding sodium citrate solution in one step to continue the reaction; collecting the black precipitate by magnetic separation after the reaction is complete; and obtaining magnetite magnetic nanoparticles with a surface modified with sodium citrate after washing and drying. The preferred molar ratio of ferric chloride, ferrous chloride, and sodium citrate is (2.5-2.7):(1.0-1.2):(4.5-5.5). For example, a specific and preferred molar ratio can be 2.6:1.1:5.0. Controlling the reaction temperature is crucial for the crystallinity and size of the nanoparticles. The preferred reaction temperature after adding ammonia water is 70℃~90℃, for example, 75℃, 80℃, or 85℃; the preferred reaction temperature after adding sodium citrate is 80℃~90℃, for example, 82℃, 85℃, or 88℃. The magnetic nanoparticles of iron oxide prepared by this method have a particle size distribution between 30 nm and 80 nm, for example, 40 nm, 50 nm or 60 nm; more preferably 45 nm to 55 nm; most preferably about 50 nm, and have good superparamagnetism.

[0070] Based on the unique properties of this composite hydrogel, the present invention also provides its application in the field of biomedicine.

[0071] In a first aspect, the present invention provides the application of the above-described composite hydrogel, or the composite hydrogel prepared by the above-described method, in the preparation of a medicament for the treatment or adjuvant treatment of oral squamous cell carcinoma. Oral squamous cell carcinoma is a common malignant tumor of the head and neck, and its treatment often involves surgical resection, which is prone to recurrence after surgery. By injecting or filling the cavity after tumor resection with the composite hydrogel of the present invention, and activating it under an external alternating magnetic field, the multiple synergistic effects of its magnetocaloric effect, calcium overload, and chemokinetic therapy can be utilized to effectively kill or inhibit any residual tumor cells, thereby playing a role in treatment or adjuvant therapy and reducing the risk of recurrence.

[0072] Secondly, this invention provides the application of the above-mentioned composite hydrogel, or the composite hydrogel prepared by the above-mentioned method, in the preparation of biomaterials for bone defect repair. Bone defect repair is a common need in orthopedics, oral and maxillofacial surgery, and plastic surgery. The composite hydrogel can not only serve as a three-dimensional scaffold for cell growth, but the calcium ions released during its degradation can also be directly used as raw materials for osteoogenesis. Furthermore, under suitable and mild magnetic field stimulation, it can further promote the activity of osteoblasts and the mineralization of the bone matrix.

[0073] Preferably, the bone defect is a jawbone defect. Jawbone defects are often caused by trauma, tumor resection, or inflammation, and require high repair standards. The injectability of this composite hydrogel can well adapt to the irregular shape of jawbone defects, achieving minimally invasive filling and repair.

[0074] Finally, the present invention also provides a programmed magnetically controlled therapy system. This system is a key operational platform for realizing the above-mentioned sequential integrated treatment of "anti-tumor first, then promoting repair". The system includes an implantable material part and a magnetic field application part. The implantable material part is the above-mentioned composite hydrogel or a composite hydrogel prepared by the above-mentioned preparation method. The magnetic field application part refers to a device capable of generating an alternating magnetic field with the required parameters, such as a high-frequency alternating magnetic field generator, an induction heating device, etc., which is used to apply an alternating magnetic field to the body part in which the composite hydrogel is implanted. The intensity of the alternating magnetic field can be adjusted in the range of 0.5 kW to 2.0 kW, for example, 0.5 kW, 1.0 kW, 1.5 kW or 2.0 kW; more preferably 0.8 kW to 1.8 kW. By programmatically changing the magnetic field intensity and application time (for example, applying a higher intensity magnetic field first to activate the anti-tumor function, and then applying a lower intensity magnetic field to promote bone repair), the functional switching of the implantable material in the body can be precisely controlled to achieve synergy between treatment and repair.

[0075] 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.

[0076] Example

[0077] Synthesis and characterization of calcium peroxide nanoparticles

[0078] To prepare a 2 mol / L calcium chloride aqueous solution, take 1 mL of the calcium chloride solution and add it dropwise to 60 mL of vigorously stirred anhydrous methanol. After magnetic stirring for 10 minutes, add 300 μL of ammonia (25%) to the reaction system and stir for 5 minutes. Then, slowly add 200 μL of hydrogen peroxide (30%) and stir for 45 minutes. The system color changes from colorless to pale blue. Transfer the solution to a high-speed centrifuge (12000 rpm, 10 min) for centrifugation and discard the supernatant. Wash three times with a methanol / ethanol mixture and finally freeze-dry for later use. Results are shown below: Figure 1 A scanning electron microscope image of CaO2 nanoparticles; Figure 1 Transmission electron microscopy image of CaO2 nanoparticles; Figure 1 Particle size distribution diagram of CCaO2 nanoparticles. Figure 1 A and Figure 1 As can be seen from B, the prepared CaO2 nanoparticles are regular spherical and uniformly dispersed; Figure 1C indicates that the obtained CaO2 nanoparticles are quasi-monodisperse with a particle size of 120.05 nm.

[0079] Synthesis and characterization of sodium citrate-modified Fe3O4 nanoparticles

[0080] FeCl3·6H2O (26.0 g) and FeCl2·4H2O (9.56 g) were dissolved in 30 mL of distilled water. The solution was filtered and poured into 70 mL of distilled water at 70 °C with vigorous stirring. Then, 50 mL of ammonia solution (25%) was added dropwise to the mixture, followed by a single, continuous addition of 48 mL of sodium citrate (2.0 M). The reaction was stirred for 1 hour, then the temperature was raised to 85 °C and stirred for 30 minutes. The black precipitate was separated with the aid of a magnet and further washed three times, successively with distilled water and ethanol, and then dried for later use. The results are shown below: Figure 2 A transmission electron microscope image of Fe3O4 nanoparticles modified with sodium citrate; Figure 2 B. Particle size distribution of Fe3O4 nanoparticles modified with sodium citrate; Figure 2 Fourier transform infrared spectrum of Fe3O4 nanoparticles modified with sodium citrate; Figure 2 D. Hysteresis loop diagram of Fe3O4 nanoparticles modified with sodium citrate. (From...) Figure 2 A and Figure 2 B indicates that the prepared sodium citrate-modified Fe3O4 nanoparticles are regular spherical with a particle size of 50.34 nm; Figure 2 C indicates that the characteristic absorption peak at 566 cm⁻¹ is attributed to the Fe-O bond vibration of the Fe₃O₄ core; the asymmetric and symmetric vibrational absorptions of the carboxylate group at 1620 cm⁻¹ and 1355 cm⁻¹ confirm that citric acid has been successfully modified onto the nanoparticle surface. Magnetic property testing ( Figure 2 D) shows that the saturation magnetization of sodium citrate-modified Fe3O4 is 54.02 emu / g, and the coercivity and remanence are close to zero, exhibiting superparamagnetism, indicating that sodium citrate-modified Fe3O4 has a strong magnetic response capability.

[0081] Hydrogel Synthesis: Injectable hydrogel formed from carboxymethyl chitosan (CMCS) and aldehyde-modified polyethylene glycol (PEG-CHO)

[0082] Optimize the ratio of CMCS and PEG-CHO

[0083] Nanoparticle-free pure hydrogels were prepared with CMCS and PEG-CHO ratios of 3:1, 3:1.5, 3:2, 3:3, and 3:4.5. Hydrogels with different ratios were prepared by adding 20, 30, 40, 60, and 90 μL of PEG-CHO (300 mg / mL) to 60 μL of CMCS (60 mg / mL) and stirring at room temperature. The morphology of the hydrogels was observed using a scanning electron microscope (JSM5900LV, Japan). For quantitative pore size analysis, SEM images from multiple representative regions were processed using ImageJ software. Approximately 50 pores were randomly selected from each hydrogel group to calculate the pore size distribution (mean ± standard deviation). The rheological properties were evaluated using a Modular Compact Rheometer 302 (Anton Paar, Austria). Figure 3 A. Scanning electron micrographs of hydrogels with different proportions; Figure 3 B. Strain scanning curves of hydrogels with different proportions; Figure 3 C. Comparison of the overall properties of hydrogels with different proportions;

[0084] The results showed that hydrogels of different proportions all exhibited a three-dimensional porous network structure, but the degree of cross-linking varied. Figure 3 A indicates that when the CMCS:PEG-CHO ratio is 3:1, the pore size is 78.64 μm and the cross-linking degree is more dense; when the CMCS:PEG-CHO ratio is 3:4.5, the pore size increases to 156.67 μm, the structure becomes loose, and uneven pores appear. The pore size for the remaining ratios is between 110-130 μm. Strain scanning was then used to systematically evaluate the mechanical properties of hydrogels formed with different ratios, aiming to screen for the optimal material formulation for subsequent intratumoral injection therapy. The strain scanning results show that ( Figure 3 B) All hydrogels exhibited typical strain-dependent softening behavior in terms of both storage modulus G' and loss modulus G''. In the injection-related low-strain region (<1%), the 3:2 ratio showed a stable G' at 105.42 Pa, with a loss factor tanδ=0.580, the lowest among the five groups, indicating the most stable elastic network and minimal viscous dissipation. This ensures effective maintenance of structural integrity under low-shear conditions within the tumor. When the strain increased to over 100% of the injection-related strain, G' decreased to approximately 90.19 Pa, still higher than G'', showing significant shear-thinning characteristics, which is beneficial for passage through a 25G insulin injection needle. In contrast, the 3:1 ratio, although initially showing a slightly higher G' (114.26 Pa), had a larger tanδ=0.627, indicating a higher proportion of viscous response leading to increased injection resistance and weakened post-injection recovery. The 3:4.5 ratio showed a G' of only 60.85 Pa, with a weak network easily dispersed by tissue hydraulic pressure, resulting in a short retention time within the tumor. The study considered multiple parameters including gel time-pore structure, cell compatibility, and rheology. Figure 3C indicates that the 3:2 ratio achieves the optimal balance between injectability, in-situ retention capacity, and biosafety, and therefore it was selected for subsequent experiments.

[0085] Synthesis of CaO2 / Fe3O4 nanoparticle composite hydrogel

[0086] The hydrogels were placed in a high-frequency sympathetic magnetic field induction heating device (SPG-10A-I, Shenzhen, China) for in-depth study. The temperature at each time interval (every 1 minute) was recorded using an infrared thermal imaging camera (Fluke, USA). Specifically, 50 μL of magnetic Fe3O4 hydrogels with different concentrations (0, 75, 100, 125, 150 mg / mL) were prepared and subjected to magnetic field stimulation of different intensities (1.0, 1.5, 2.0 kW, 8 min). Based on this, calcium peroxide was added to prepare FC Gel, which was then placed in the high-frequency sympathetic magnetic field induction heating device, and the temperature was recorded every 1 minute using an infrared thermal imaging camera.

[0087] 50 μL of PEG-CHO solution (300 mg / mL), 20 μL of Fe3O4 (120 mg / mL), and 5 μL of CaO2 (5 mg / mL) were sequentially added to 75 μL of CMCS (60 mg / mL). After thorough stirring at room temperature, Fe3O4 / CaO2 / CMCS / PEG-CHO hydrogels (FC Gel) were obtained. Additionally, CMCS / PEG-CHO hydrogels (Gel) without CaO2 and Fe3O4 were prepared as a control group. The morphology, rheology, and self-healing ability of FC Gel and Gel were systematically evaluated. The morphology of the hydrogels was observed using scanning electron microscopy (SEM, JSM5900LV, Japan). For quantitative pore size analysis, SEM images from multiple representative regions were processed using ImageJ software, and approximately 50 pores were randomly selected from each hydrogel group to calculate the pore size distribution (mean ± standard deviation). Figure 4 A comparison of the temperature rise curves for 1.0% F Gel, 1.3% F Gel, 1.6% F Gel, and 2.0% F Gel; Figure 4 Comparison of heating curves for B Gel, C Gel, and 1.6% FC Gel; Figure 4 Comparison of heating curves of 1.6%FC Gel under different magnetic field intensities; Figure 4 Transmission electron micrographs of DGel, CGel, 1.0% FGel, 1.3% FGel, 1.6% FGel and 2.0% FGel; Figure 4 Overall performance comparison of E 1.0% F Gel, 1.3% F Gel, 1.6% F Gel and 2.0% F Gel; Figure 4FFC Gel element distribution diagram; Figure 4 Demonstration of the injectability and self-healing properties of GFC Gel; comprehensive analysis of the data shows that the studied gel material has excellent overall performance.

[0088] from Figure 4 A shows that the temperature of the hydrogel increases significantly with increasing Fe3O4 content (from 1.0% to 2.0%). The 1.6% F gel reaches approximately 47.5℃ after being placed in a 1.5 kW magnetic field for 8 minutes, indicating that sodium citrate-modified Fe3O4 has significant magnetocaloric conversion capabilities. Based on this, CaO2 was further incorporated, and the temperature rise of the blank hydrogel (Gel), the hydrogel with only CaO2 (C Gel), and the hydrogel with both CaO2 and Fe3O4 (FC Gel) were compared. Figure 4 B shows that Gel and C show almost no temperature increase, while FC Gel can still reach 47℃, proving that the addition of CaO2 does not affect the temperature rise capability of the hydrogel. Figure 4 C indicates that as the magnetic field strength increases, the highest temperature of the hydrogel reaches 41.9, 47.1 and 51℃ after 8 minutes, respectively. Figure 4 D indicates that the pure gel exhibits a relatively random porous network. The introduction of citric acid-modified Fe3O4 makes the three-dimensional network structure more regular and dense, and the pore size distribution more uniform. Figure 4 E comprehensively compared the performance of each gel in five dimensions: cell viability, gelation time, pore size, heating capacity, and strain. The 1.6% concentration F Gel was superior to other concentrations in multiple performance indicators. Figure 4 F confirms that elements such as calcium, iron, carbon, nitrogen, and oxygen are uniformly distributed in the material, supporting the material's compositional stability. Figure 4 G further demonstrated the gel's injectability and self-healing capabilities, adaptability to different shapes, and hinted at its potential in biomedical applications.

[0089] In vitro cell experiments

[0090] SCC7 cells were seeded in 24-well plates (2 × 10⁻⁶ cells / well). 4 Cells / well), and incubated with DMEM (10% FBS) for 24 hours. FC Gel+AMF1 (1.5kW, 10min), FC Gel, Gel, Control and other treatment groups were co-cultured with cells in Trans-well chambers for 24 h. Cell viability in each group was detected using CCK-8 and a cell viability test kit (Beyotime, Shanghai, China). Figure 5 A fluorescence images of SCC7 cell survival / death under different treatment conditions; Figure 5 B. Quantitative statistical analysis of SCC7 cell viability under different treatment conditions; Figure 5A shows that SCC7 cells treated with FC Gel exhibited significant red fluorescence, which was significantly enhanced after the application of AMF1, demonstrating that the calcium overload-magnetic-thermal synergy enhanced cytotoxicity. Figure 5 B showed that using FC gel alone reduced cell viability to 60.04%, and when heated to 47.2°C, cell viability decreased to 30.45%, while there was no statistically significant difference between the gel-only group and the control group.

[0091] ALP staining was performed using the alkaline phosphatase detection kit from Beyotime Biotechnology Co., Ltd. The simplified procedure is as follows: 3T3-E1 was stained at 5 × 10⁻⁶... 4 Cells were seeded at a density of 10 cells / well in 12-well plates and incubated at 37°C for 24 hours. Osteogenic induction was then performed for 7 days. The induction solution was discarded, and the cells were washed with PBS, fixed with paraformaldehyde for 15 minutes, stained with staining working solution, and imaged under a microscope. 3T3-E1 cells were seeded at a density of 5 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of 10 cells / well in 12-well plates and incubated at 37°C for 24 hours. Osteogenic induction was then performed for 14 days. The induction solution was discarded, and the cells were washed with PBS, fixed with paraformaldehyde for 15 minutes, stained with alizarin red, and photographed under a microscope for recording.

[0092] The matrix gel was thawed overnight at 4°C the day before the experiment. During the experiment, the pre-cooled 24-well plate was removed, and 20 μL of matrix gel was added to each well, spread evenly, and incubated at 37°C for 30 minutes to solidify. Then, 500 μL of HUVEC cell suspension (approximately 1.5 × 10⁻⁶ cells) was added to each well. 5 (100 cells) were placed in a 37°C, 5% CO2 incubator for incubation; finally, the tube formation was observed under a microscope, and photographs were taken under a microscope at the 8-hour mark for recording. Figure 6 A. ALP staining and ARS staining of 3T3-E1 cells at day 7 and day 14 of osteogenic differentiation in different treatment groups; Figure 6 B. Fluorescence images of HUVEC cells in different treatment groups after 8 hours; Figure 6 A indicates that the FC Gel + AMF2 treatment group exhibited significant advantages at all stages of osteogenic differentiation. In the early differentiation stage, alkaline phosphatase staining analysis after 7 days of culture showed that the ALP staining in the FC Gel + AMF2 group was significantly higher than that in the Control, Gel, and FC Gel groups. Entering the late differentiation stage, Alizarin Red S (ARS) staining analysis after 14 days of culture further indicated that the FC Gel + AMF2 group formed the most abundant mineralized nodules, suggesting its strongest ability to promote mineralization. Figure 6 B indicates that the FC Gel + AMF2 group exhibits excellent tube-promoting ability.

[0093] In vivo anti-tumor

[0094] 4 × 10 6 One SCC7 cell was subcutaneously seeded into the groin of a C57BL / 6 (female, 5 weeks old) tumor until the tumor grew to 70 mm. 3 Around day 14, mice were randomly divided into four groups: Group 1 (control group), receiving an intratumoral injection of 50 μL PBS on day 0, with no other treatment. Group 2, each mouse received an intratumoral injection of 50 μL of empty hydrogel. Group 3, each mouse received an intratumoral injection of 50 μL of FC Gel on day 0. Group 4, each mouse received an intratumoral injection of 50 μL of FC Gel on day 0, and was given a magnetic field (1.5 kW, 8 min) on days 1, 3, 5, and 7. Mice were observed after these treatments until day 14. Starting from day 1, mice were weighed and tumor volume was measured every two days. Mouse weight and tumor size were measured using calipers, and tumor size was calculated using the equation (V = a × b). 2 / 2). V is the tumor volume, a is the tumor length, and B is the tumor width. Mice were sacrificed after 14 days, and their tumors were removed for further analysis. Figure 7 A. Physical images of representative tumor samples after different treatments; Figure 7 B. Bar chart showing tumor volume after different treatments; Figure 7 C. Comparison of tumor volume growth curves in different treatment groups.

[0095] Figure 7 A and Figure 7 B shows that the Control group and the Gel group had the largest residual tumors at the end, while the FC Gel group and the FCGel+AMF1 group both inhibited tumor growth. Figure 7 C shows changes in tumor volume, revealing different trends of rapid increase (Control and Gel groups), slow increase (FC Gel group), and continuous decrease (FC Gel + AMF1 group). This indicates that FC Gel, under AMF1 activation, promotes tumor cell apoptosis and inhibits tumor cell proliferation.

[0096] In vivo bone formation

[0097] A 1.2 mm defect was created in the mandible of female C57BL / 6 mice (n=4). Mice were randomly assigned to four groups: Group 1, control group (no treatment); Group 2, implanted gel followed by suturing; Group 3, implanted FC gel followed by suturing; Group 4, implanted FC gel followed by suturing, with magnetic field applied on days 1, 3, 5, and 7 (1.0 kW, 8 min). Micro-CT analysis was performed at 2 and 4 weeks post-surgery. Figure 8The results showed that the control group had the largest bone defect area, while the FC Gel+AMF2 group had the best repair effect, achieving complete healing by week 4. This indicates that FC Gel significantly promotes bone repair under AMF2 activation.

[0098] As can be seen from the above embodiments, the present invention provides a functionally tunable composite hydrogel material. In vitro experiments have confirmed that this material can effectively inhibit tumor cell viability under specific magnetic field conditions, while simultaneously exhibiting the potential to promote osteoblast differentiation and angiogenesis. In vivo animal experiments further demonstrate that this material can significantly inhibit tumor growth in activation mode and effectively guide new bone formation in a jawbone defect model, preliminarily verifying the feasibility of its integrated anti-tumor and bone repair function.

[0099] 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 magnetothermal-sensitized-calcium-iron synergistic composite hydrogel, characterized in that, It includes a hydrogel matrix, calcium peroxide nanoparticles dispersed in the hydrogel matrix, and magnetite nanoparticles; The hydrogel matrix is ​​a three-dimensional network structure formed by cross-linking carboxymethyl chitosan and aldehyde-modified polyethylene glycol. The preparation method of the magnetothermal-sensitized-calcium-iron synergistic composite hydrogel includes the following steps: Aldehyde-modified polyethylene glycol solution, iron oxide magnetic nanoparticle suspension and calcium peroxide nanoparticle suspension were sequentially added to carboxymethyl chitosan solution and thoroughly mixed and stirred to obtain the composite hydrogel. The calcium peroxide nanoparticles are prepared by the following steps: Calcium chloride solution was added dropwise to anhydrous methanol under vigorous stirring, followed by the addition of ammonia and hydrogen peroxide solution to carry out the reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain the calcium peroxide nanoparticles. The magnetic nanoparticles of iron oxide were prepared by the following steps: Ferric chloride and ferrous chloride were dissolved in distilled water, and ammonia was added while heating and stirring. Then, sodium citrate solution was added all at once to continue the reaction. After the reaction was completed, the mixture was magnetically separated, washed and dried to obtain sodium citrate-modified magnetite nanoparticles.

2. The preparation method of the magnetothermal-sensitized-calcium-iron synergistic composite hydrogel according to claim 1, characterized in that, Includes the following steps: Aldehyde-modified polyethylene glycol solution, iron oxide magnetic nanoparticle suspension and calcium peroxide nanoparticle suspension were sequentially added to carboxymethyl chitosan solution and thoroughly mixed and stirred to obtain the composite hydrogel. The calcium peroxide nanoparticles are prepared by the following steps: Calcium chloride solution was added dropwise to anhydrous methanol under vigorous stirring, followed by the addition of ammonia and hydrogen peroxide solution to carry out the reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain the calcium peroxide nanoparticles. The magnetic nanoparticles of iron oxide were prepared by the following steps: Ferric chloride and ferrous chloride were dissolved in distilled water, and ammonia was added while heating and stirring. Then, sodium citrate solution was added all at once to continue the reaction. After the reaction was completed, the mixture was magnetically separated, washed and dried to obtain sodium citrate-modified magnetite nanoparticles.

3. The preparation method according to claim 2, characterized in that, The concentration of the carboxymethyl chitosan solution is 40 mg / mL to 80 mg / mL; The mass ratio of the carboxymethyl chitosan to the aldehyde-modified polyethylene glycol is 3:1 to 4.5; The concentration of the aldehyde-modified polyethylene glycol solution is 200 mg / mL to 400 mg / mL; The concentration of the magnetic nanoparticle suspension of iron oxide is 100 mg / mL to 150 mg / mL; The concentration of the calcium peroxide nanoparticle suspension is 4 mg / mL to 6 mg / mL; The volume ratio of the carboxymethyl chitosan solution, the aldehyde-modified polyethylene glycol solution, the iron oxide magnetic nanoparticle suspension, and the calcium peroxide nanoparticle suspension is 60~80:40~60:15~25:4~6. The mixing and stirring are carried out at room temperature for 1 to 5 minutes; in the composite hydrogel, the mass of the ferric oxide magnetic nanoparticles accounts for 1.0% to 2.0% of the total mass of the hydrogel.

4. The preparation method according to claim 2, characterized in that, The calcium peroxide nanoparticles are prepared by the following steps: Calcium chloride solution was added dropwise to anhydrous methanol under vigorous stirring, followed by the addition of ammonia and hydrogen peroxide solution to carry out the reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain the calcium peroxide nanoparticles. The concentration of the calcium chloride solution is 1.5 mol / L to 2.5 mol / L; The reaction was carried out by stirring for 30 to 60 minutes after the addition of hydrogen peroxide solution. The centrifugation speed is 10,000 rpm to 15,000 rpm, and the time is 5 minutes to 15 minutes; The prepared calcium peroxide nanoparticles have a particle size of 80 nm to 150 nm.

5. The preparation method according to claim 2, characterized in that, The magnetic nanoparticles of iron oxide were prepared by the following steps: Ferric chloride and ferrous chloride were dissolved in distilled water, and ammonia was added while heating and stirring. Then, sodium citrate solution was added all at once to continue the reaction. After the reaction was completed, the mixture was magnetically separated, washed and dried to obtain sodium citrate-modified magnetite nanoparticles. The molar ratio of ferric chloride, ferrous chloride, and sodium citrate is 2.5~2.7 : 1.0~1.2 : 4.5~5.5; The reaction temperature after adding ammonia is 70℃~90℃, and the reaction temperature after adding sodium citrate is 80℃~90℃. The prepared magnetite nanoparticles have a particle size of 30 nm to 80 nm.

6. The use of the composite hydrogel of claim 1 or the composite hydrogel prepared by any one of claims 2 to 5 in the preparation of a medicament for the treatment or adjuvant treatment of oral squamous cell carcinoma.

7. The application of the composite hydrogel according to claim 1 or the composite hydrogel prepared by any one of claims 2 to 5 in the preparation of biomaterials for bone defect repair.

8. The application according to claim 7, characterized in that, The bone defect repair mentioned above is for the repair of jawbone defects.

9. A programmed magnetically controlled therapy system, characterized in that, Includes the implant material and the magnetic field application part; The implant material is the composite hydrogel as described in claim 1 or the composite hydrogel prepared by any one of claims 2 to 5. The magnetic field application section is used to apply an alternating magnetic field to the implantation site.

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