Preparation method of 3D printing cerium hydroxyapatite bone repair scaffold material

Abstract

The application discloses a preparation method of a 3D printing cerium hydroxyapatite bone repair scaffold material, and comprises the following steps: adding phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate salt powder into manganese-chelated deferoxamine grafted methylacryl gelatin, stirring, adding cerium hydroxyapatite nanowires which are surface-modified by citric acid, and stirring to obtain 3D printing slurry; and the 3D printing slurry is extruded by a 3D printer while being cross-linked by 365nm ultraviolet irradiation to obtain the 3D printing cerium hydroxyapatite bone repair scaffold material; the material has good biocompatibility, can promote vascularization and immune regulation, and can effectively regulate M2 phenotype differentiation of RAW264.7, so that more BMP-2 and PDGF-bb growth factors are secreted, osteogenesis differentiation and angiogenesis are promoted, and bone defect repair is promoted from the perspective of immune regulation.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material. Background Technology

[0002] Non-healing bone defects severely threaten people's mobility and even life and health. The emergence and development of regenerative medicine and tissue engineering have brought new hope for the repair and treatment of non-healing bone defects. Based on the combination of biodegradable materials and the biomedical field, the core goal of bone tissue engineering is to prepare bone filling materials with good biocompatibility, non-cytotoxicity, osteoconductivity, osteoinductive properties, cell adhesion, high mechanical strength, angiogenesis promotion, and degradation rate matching the new bone formation rate by controlling the unique physical and chemical characteristics of biomaterials.

[0003] Currently, traditional tissue-engineered scaffolds used for bone defect repair suffer from the following problems: ① Internal porosity is difficult to control effectively, hindering cell survival within the bone framework; ② Insufficient vascularization leads to inadequate nutrition and necrosis within the newly formed callus; ③ Poor osteoinductivity results in slow bone synthesis and calcification at the defect site; ④ The in vivo degradation rate of the scaffold does not match the rate of new bone formation; ⑤ Irregular bone defect shapes lead to mismatches between the filling material and the defect site. These problems severely limit the development and clinical application of tissue-engineered materials. Therefore, designing and preparing filling materials with excellent tissue-engineered bone repair properties is crucial. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material, addressing the shortcomings of the prior art. This invention involves preparing a 3D printing slurry by combining manganese-chelated deferoxamine-grafted methacrylamide gelatin and cerium hydroxyapatite nanowires modified with citric acid. During extrusion, the slurry is cross-linked under 365nm ultraviolet light to obtain a cerium hydroxyapatite bone repair scaffold material with bone repair function. This material exhibits good biocompatibility, promotes vascularization, and modulates immunity, effectively promoting bone defect repair.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a 3D printed cerium hydroxyapatite bone repair scaffold material, comprising:

[0006] Phenyl (2,4,6-trimethylbenzoyl) lithium phosphate salt powder was added to manganese-chelated deferoxamine-grafted methacryloyl gelatin and stirred. Cerium hydroxyapatite nanowires with citric acid surface modification were then added and stirred to obtain a 3D printing slurry. The 3D printing slurry was extruded using a 3D printer while being cross-linked by 365nm ultraviolet irradiation to obtain a 3D printed cerium hydroxyapatite bone repair scaffold material. The mass of the cerium hydroxyapatite nanowires with citric acid surface modification was 0.2–0.4 times the volume of the manganese-chelated deferoxamine-grafted methacryloyl gelatin. The mass unit of the cerium hydroxyapatite nanowires with citric acid surface modification is g, and the volume unit of the manganese-chelated deferoxamine-grafted methacryloyl gelatin is mL.

[0007] The above method is characterized in that the preparation method of the manganese-chelated deferoxamine-grafted methacrylamide gelatin specifically includes:

[0008] Step 101: Add ethanesulfonic acid to the PBS buffer of methacrylamide gelatin to obtain system A;

[0009] Step 102: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to system A, stir and react at room temperature for 0.5-2 h, add deferoxamine, and stir and react at room temperature for 20-26 h.

[0010] Step 103: Dialyze the product obtained from the reaction, freeze-dry it, and obtain deferoxamine-grafted methacrylamide gelatin;

[0011] Step 104: Dissolve the deferoxamine-grafted methacrylamide gelatin in an aqueous manganese chloride solution and stir the mixture at room temperature under nitrogen atmosphere for 4-8 hours to obtain manganese-chelated deferoxamine-grafted methacrylamide gelatin.

[0012] The above method is characterized in that, in step 102, the mass ratio of the methacrylated gelatin, ethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and deferoxamine is 1:1:0.19:0.057:(0.05~0.35); in step 104, the volume of the manganese chloride aqueous solution is 10 times the mass of the deferoxamine-grafted methacrylated gelatin, the volume of the manganese chloride aqueous solution is in mL, the mass of the deferoxamine-grafted methacrylated gelatin is in g, and the concentration of the manganese chloride aqueous solution is 80~480μM.

[0013] The method described above is characterized in that, in step 102, the mass ratio of the methacrylated gelatin, ethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and deferoxamine is 1:1:0.19:0.057:(0.05~0.15); and in step 104, the concentration of the manganese chloride aqueous solution is 80~320μM.

[0014] The above method is characterized in that the preparation method of cerium hydroxyapatite nanowires with citric acid surface modification specifically includes:

[0015] Step 201: Provide system B containing oleic acid; system B is system B obtained by dissolving oleic acid in deionized water and methanol;

[0016] Step 202: Adjust the pH of system B to 10.0, add an aqueous solution containing calcium chloride and cerium nitrate, stir, add an aqueous solution of sodium dihydrogen phosphate, and obtain mixed system C;

[0017] Step 203: Continue stirring the mixture C for 5-15 minutes, seal, and react at 180°C for 20-26 hours;

[0018] Step 204: After the system temperature drops to room temperature after the reaction, collect the precipitate, wash it with anhydrous ethanol, centrifuge and dry it to obtain cerium hydroxyapatite nanowires.

[0019] Step 205: Dissolve cerium hydroxyapatite nanowires in sodium citrate ethanol solution, stir at room temperature for 2-4 hours, collect the precipitate, wash with deionized water, centrifuge and dry to obtain cerium hydroxyapatite nanowires with citric acid surface modification.

[0020] The method described above is characterized in that, in step 202, the volume ratio of oleic acid, the aqueous solution containing calcium chloride and cerium nitrate, and the aqueous solution containing sodium dihydrogen phosphate is 42:48:180, and the concentration of calcium chloride in the aqueous solution containing calcium chloride and cerium nitrate is 0.3M, and the concentration of cerium nitrate is 6-18mM.

[0021] The method described above is characterized in that, in step 205, the sodium citrate ethanol solution is a sodium citrate ethanol solution obtained by dissolving sodium citrate in anhydrous ethanol, the concentration of sodium citrate in the sodium citrate ethanol solution is 0.35-1.05M, the volume of the sodium citrate ethanol solution is 45-55 times the mass of the cerium hydroxyapatite nanowires, the volume unit of the sodium citrate ethanol solution is mL, and the mass unit of the cerium hydroxyapatite nanowires is g.

[0022] The above method is characterized in that the methacrylated gelatin is obtained by reacting a PBS solution containing gelatin with methacrylic anhydride under stirring conditions at 45-55°C, followed by dialyzing and freeze-drying.

[0023] The method described above is characterized in that the volume of the methacrylic anhydride is 0.5 to 1.5 times the mass of the gelatin, wherein the volume of the methacrylic anhydride is measured in mL and the mass of the gelatin is measured in g.

[0024] The above method is characterized in that the mass of the phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 2 to 3 times the volume of the manganese-chelated deferoxamine-grafted methacrylamide gelatin, the mass unit of the phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is mg, and the volume unit of the manganese-chelated deferoxamine-grafted methacrylamide gelatin is mL.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1. In the preparation method of the 3D printed cerium hydroxyapatite bone repair scaffold material of the present invention, a 3D printing slurry is prepared by manganese chelated deferoxamine-grafted methacrylamide gelatin and cerium hydroxyapatite nanowires with citric acid surface modification. The slurry is cross-linked by 365nm ultraviolet irradiation during extrusion to obtain a cerium hydroxyapatite bone repair scaffold material with bone repair function. This material has good biocompatibility, promotes angiogenesis and has immunomodulatory functions. It can effectively regulate the M2 phenotype differentiation of RAW264.7, causing it to secrete more BMP-2 and PDGF-bb growth factors, promote osteogenic differentiation and angiogenesis, and promote bone defect repair from the perspective of immunomodulation.

[0027] 2. The preparation method of the 3D printed cerium hydroxyapatite bone repair scaffold material of the present invention creatively combines photocrosslinking and 3D printing, which can design the pore size and morphology of the bone repair scaffold, increase the adaptability of the scaffold to the defect site and facilitate the ingrowth of blood vessels. The scaffold has good adaptability to bone defects and osteoconductive ability, and can effectively avoid the toxic side effects caused by chemical crosslinking agent residues.

[0028] 3. The preparation method of the 3D printed cerium hydroxyapatite bone repair scaffold material of the present invention introduces cerium hydroxyapatite nanowires with citric acid modified on the surface into the scaffold material. Its hydrophilicity can effectively improve the anti-collapse of the scaffold material, while giving full play to the properties of cerium hydroxyapatite to promote the proliferation and osteogenic differentiation of mesenchymal stem cells (hBMSCs).

[0029] 4. Preferably, the method of the present invention includes sealing and reacting a system containing oleic acid with an aqueous solution containing calcium chloride and cerium nitrate, followed by reacting with an ethanol solution of sodium citrate to obtain cerium hydroxyapatite nanowires with citric acid surface modification. These nanowires are inorganic phase materials with high hydrophilicity, which can effectively improve the anti-collapse properties of the scaffold material after being introduced into the scaffold.

[0030] 5. Preferably, the method of the present invention includes adding ethanesulfonic acid to PBS buffer of methacrylamide gelatin, followed by adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, reacting, and then adding deferoxamine to obtain deferoxamine-grafted methacrylamide gelatin. The deferoxamine-grafted methacrylamide gelatin is placed in an aqueous manganese chloride solution and reacted in a nitrogen atmosphere to obtain manganese-chelated deferoxamine-grafted methacrylamide gelatin. This effectively achieves amide bond bonding between deferoxamine and gelatin molecules and chelates manganese ions in the network, resulting in a scaffold structure with sustained-release properties, excellent cell adhesion, pro-angiogenesis, bone induction, and immunomodulation.

[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0032] Instruction manual illustrations

[0033] Figure 1 The 1H NMR spectra of gelatin (Gel), methacrylamide gelatin (GelMA) from Example 1-1, and deferoxamine-grafted methacrylamide gelatin (GelMA-DFO) from Example 2-1 are shown.

[0034] Figure 2 Fourier transform infrared spectra of Gel and GelMA in Example 1-1, and GelMA-DFO and GelMA-DFO@Mn(Ⅱ) in Example 2-1.

[0035] Figure 3 This is a SEM image of the cerium hydroxyapatite nanowire CeHANWs-2 from Example 2-2.

[0036] Figure 4 This is a TEM image of the cerium hydroxyapatite nanowire CeHANWs-2 from Example 2-2.

[0037] Figure 5 These are macroscopic and microscopic images of the cerium hydroxyapatite bone repair scaffold material described in Example 2-2.

[0038] Figure 6 The results show the cytotoxicity of the cerium hydroxyapatite bone repair scaffold materials described in Examples 2-1 to 2-4 on bone marrow mesenchymal stem cells (hBMSCs).

[0039] Figure 7The results of the cytotoxicity of cerium hydroxyapatite nanowires described in Examples 2-1 to 2-3 on hBMSCs are shown.

[0040] Figure 8 The results are from the experimental formation of the cerium hydroxyapatite bone repair scaffold material tube described in Examples 2-3.

[0041] Figure 9 The results of polarization qPCR of RAW264.7 macrophages using the cerium hydroxyapatite bone repair scaffold material described in Examples 2-1 to 2-2 are shown. Detailed Implementation

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the experimental reagents and consumables used are commercially available unless otherwise specified.

[0043] Example 1-1

[0044] This embodiment provides a method for preparing methacrylamide gelatin (GelMA), comprising:

[0045] Step 1: Under magnetic stirring at 50°C, 1g of solid gelatin was completely dissolved in 10mL of PBS buffer to obtain a gelatin solution; the gelatin had a molecular weight of 64.8kDa and was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the PBS buffer was phosphate buffer with a pH of 7.4.

[0046] Step 2: Under magnetic stirring at 50°C, slowly add 1 mL of methacrylic anhydride to the gelatin solution, react for 180 min, and then add 40 mL of PBS buffer to terminate the reaction; the PBS buffer in Step 2 is the same as in Step 1.

[0047] Step 3: Transfer the reaction product to an 8000-14000 Da dialysis bag, dialyze with deionized water for 7 days, and freeze-dry to obtain methacrylamide gelatin (GelMA).

[0048] Examples 1-2

[0049] This embodiment provides a method for preparing methacrylamide gelatin (GelMA), comprising:

[0050] Step 1: Under magnetic stirring at 55℃, 1g of solid gelatin was completely dissolved in 10mL of PBS buffer to obtain a gelatin solution; the gelatin had a molecular weight of 64.8kDa and was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the PBS buffer was phosphate buffer with a pH of 7.4.

[0051] Step 2: Under magnetic stirring at 55°C, slowly add 0.5 mL of methacrylic anhydride to the gelatin solution, react for 180 min, and then add 40 mL of PBS buffer to terminate the reaction; the PBS buffer in Step 2 is the same as in Step 1.

[0052] Step 3: Transfer the reaction product to an 8000-14000 Da dialysis bag, dialyze with deionized water for 7 days, and freeze-dry to obtain methacrylamide gelatin (GelMA).

[0053] The methacrylamide gelatin in this embodiment has essentially the same properties as that in Example 1-1.

[0054] Examples 1-3

[0055] This embodiment provides a method for preparing methacrylamide gelatin (GelMA), comprising:

[0056] Step 1: Under magnetic stirring at 45℃, 1g of solid gelatin was completely dissolved in 10mL of PBS buffer to obtain a gelatin solution; the gelatin had a molecular weight of 64.8kDa and was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the PBS buffer was phosphate buffer with a pH of 7.4.

[0057] Step 2: Under magnetic stirring at 45°C, slowly add 1.5 mL of methacrylic anhydride to the gelatin solution, react for 180 min, and then add 40 mL of PBS buffer to terminate the reaction; the PBS buffer in Step 2 is the same as in Step 1.

[0058] Step 3: Transfer the reaction product to an 8000-14000 Da dialysis bag, dialyze with deionized water for 7 days, and freeze-dry to obtain methacrylamide gelatin (GelMA).

[0059] The methacrylamide gelatin in this embodiment has essentially the same properties as that in Example 1-1.

[0060] Example 2-1

[0061] This embodiment provides a method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material. This 3D-printed cerium hydroxyapatite bone repair scaffold material can enhance bone regeneration function through vascularization and immune modulation. The method includes the following steps:

[0062] Step 1: Providing manganese-chelated deferoxamine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)), specifically including:

[0063] Step 101: Dissolve 1g of GelMA from Step 1-1 in 10mL of PBS buffer, add 1g of ethanesulfonic acid (MES), and adjust the pH to 5.0 with NaOH to obtain system A; the PBS buffer is a phosphate buffer with a pH of 7.4.

[0064] Step 102: Add 191.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 57.5 mg of N-hydroxysuccinimide (NHS) to system A, stir and react at room temperature for 1 h, then add 50 mg of deferoxamine (DFO), and stir and react at room temperature for 24 h.

[0065] Step 103: Transfer the product obtained from the reaction to an 8000-14000 Da dialysis bag, dialyze with deionized water for 5 days, freeze dry, and obtain deferoxamine-grafted methacrylamide gelatin GelMA-DFO-1.

[0066] Step 104: Dissolve 1g of the deferoxamine-grafted methacrylamide gelatin GelMA-DFO-1 in 10mL of 80μM manganese chloride aqueous solution, and stir the mixture at room temperature for 6h under nitrogen atmosphere to obtain manganese chelated deferoxamine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)-1).

[0067] Step 2: Provide cerium hydroxyapatite nanowires (CeHA@CA) with citric acid surface modification, specifically including:

[0068] Step 201: Under stirring conditions, 42 mL of oleic acid, 54 mL of deionized water and 24 mL of methanol are mixed evenly to obtain system B;

[0069] Step 202: Add 60 mL of 17 M NaOH aqueous solution to system B to adjust the pH of the system to 10.0. Then, slowly add 48 mL of an aqueous solution containing calcium chloride and cerium nitrate with a concentration of 0.3 M calcium chloride and a concentration of 6 mM cerium nitrate to the system. After stirring for 30 min, add 180 mL of 0.5 M sodium dihydrogen phosphate aqueous solution to obtain mixed system C.

[0070] Step 203: Continue stirring the mixture C for 10 minutes, then seal and react at 180°C for 24 hours;

[0071] Step 204: After the system temperature drops to room temperature after the reaction, collect the precipitate, wash it with anhydrous ethanol, centrifuge and dry it to obtain cerium hydroxyapatite nanowires CeHANWs-1.

[0072] Step 205: Dissolve 0.2g of cerium hydroxyapatite nanowires CeHANWs-1 in 10mL of 0.35M sodium citrate ethanol solution, stir at room temperature for 3h, collect the precipitate, wash with deionized water, centrifuge and dry to obtain cerium hydroxyapatite nanowires CeHA@CA-1 with citric acid surface modification.

[0073] Step 3: Provide cerium hydroxyapatite bone repair scaffold material, specifically including:

[0074] Step 301: Add 25 mg of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) powder to 10 mL of GelMA-DFO@Mn(Ⅱ)-1 from Step 1, stir for 30 minutes at room temperature, then add 2 g of CeHA@CA-1 from Step 2, stir for 30 minutes at room temperature to obtain 3D printing slurry;

[0075] Step 302: Place the 3D printing paste in the 3D printer. The nozzle diameter is 0.7mm, the extrusion amount is 50%, the layer height and wall thickness are both 0.7mm, the fill density is 60%, the printing temperature is 25℃, and the printing speed is 10mm / s. Irradiate the product generation platform of the 3D printer with 365nm ultraviolet light. The extrudate is irradiated at the same time as it is extruded. After extrusion is completed, continue to irradiate for 60 minutes to allow cross-linking, and obtain the 3D printed cerium hydroxyapatite bone repair scaffold material, named Sample 1.

[0076] Example 2-2

[0077] This embodiment provides a method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material. This 3D-printed cerium hydroxyapatite bone repair scaffold material can enhance bone regeneration function through vascularization and immune modulation. The method includes the following steps:

[0078] Step 1: Providing manganese-chelated deferoxamine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)), specifically including:

[0079] Step 101: Dissolve 1g of GelMA described in Example 1-1 in 10mL of PBS buffer, add 1g of MES, and adjust the pH to 5.0 with NaOH to obtain system A; the PBS buffer is phosphate buffer with a pH of 7.4;

[0080] Step 102: Add 191.7 mg of EDC and 57.5 mg of NHS to system A, stir and react at room temperature for 1 h, then add 100 mg of DFO and stir and react at room temperature for 24 h.

[0081] Step 103: Transfer the product obtained from the reaction to an 8000-14000 Da dialysis bag, dialyze with deionized water for 5 days, freeze dry, and obtain deferoxamine-grafted methacrylamide gelatin GelMA-DFO-2.

[0082] Step 104: Dissolve 1g of the deferoxamine-grafted methacrylamide gelatin GelMA-DFO-2 in 10mL of 160μM manganese chloride aqueous solution, and stir the mixture at room temperature for 6h under nitrogen atmosphere to obtain manganese-chelated deferoxamine-grafted methacrylamide gelatin GelMA-DFO@Mn(Ⅱ)-2.

[0083] Step 2: Provide cerium hydroxyapatite nanowires (CeHA@CA) with citric acid surface modification, specifically including:

[0084] Step 201: Under stirring conditions, 42 mL of oleic acid, 54 mL of deionized water and 24 mL of methanol are mixed evenly to obtain system B;

[0085] Step 202: Add 60 mL of 17 M NaOH aqueous solution to system B to adjust the pH of the system to 10.0. Then, slowly add 48 mL of an aqueous solution containing calcium chloride and cerium nitrate with a concentration of 0.3 M calcium chloride and a concentration of 12 mM cerium nitrate to the system. After stirring for 30 min, add 180 mL of 0.5 M sodium dihydrogen phosphate aqueous solution to obtain mixed system C.

[0086] Step 203: Continue stirring the mixture C for 10 minutes, then seal and react at 180°C for 24 hours;

[0087] Step 204: After the system temperature drops to room temperature after the reaction, collect the precipitate, wash it with anhydrous ethanol, centrifuge and dry it to obtain cerium hydroxyapatite nanowires CeHANWs-2.

[0088] Step 205: Dissolve 0.2g of cerium hydroxyapatite nanowires CeHANWs-2 in 10mL of 0.7M sodium citrate ethanol solution, stir at room temperature for 3h, collect the precipitate, wash with deionized water, centrifuge and dry to obtain cerium hydroxyapatite nanowires CeHA@CA-2 with citric acid surface modification.

[0089] Step 3: Provide cerium hydroxyapatite bone repair scaffold material, specifically including:

[0090] Step 301: Add 25 mg of LAP powder to 10 mL of GelMA-DFO@Mn(Ⅱ)-2 from Step 1, stir for 30 minutes at room temperature, then add 3 g of CeHA@CA-2 from Step 2, stir for 30 minutes at room temperature to obtain 3D printing slurry;

[0091] Step 302: Place the 3D printing paste in the 3D printer. The nozzle diameter is 0.7mm, the extrusion amount is 50%, the layer height and wall thickness are both 0.7mm, the fill density is 60%, the printing temperature is 25℃, and the printing speed is 10mm / s. Irradiate the product generation platform of the 3D printer with 365nm ultraviolet light. The extrudate is irradiated at the same time as it is extruded. After extrusion is completed, continue to irradiate for 60 minutes to allow cross-linking. The 3D printed cerium hydroxyapatite bone repair scaffold material is obtained and named Sample 2.

[0092] Example 2-3

[0093] This embodiment provides a method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material. This 3D-printed cerium hydroxyapatite bone repair scaffold material can enhance bone regeneration function through vascularization and immune modulation. The method includes the following steps:

[0094] Step 1: Providing manganese-chelated deferoxamine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)), specifically including:

[0095] Step 101: Dissolve 1g of GelMA described in Example 1-1 in 10mL of PBS buffer, add 1g of MES, and adjust the pH to 5.0 with NaOH to obtain system A; the PBS buffer is phosphate buffer with a pH of 7.4;

[0096] Step 102: Add 191.7 mg of EDC and 57.5 mg of NHS to system A, stir and react at room temperature for 1 h, then add 150 mg of DFO, and stir and react at room temperature for 24 h.

[0097] Step 103: Transfer the product obtained from the reaction to an 8000-14000 Da dialysis bag, dialyze with deionized water for 5 days, freeze dry, and obtain deferoxamine-grafted methacrylamide gelatin GelMA-DFO-3.

[0098] Step 104: Dissolve 1g of the deferoxamine-grafted methacrylamide gelatin GelMA-DFO-3 in 10mL of 320μM manganese chloride aqueous solution, and stir the mixture at room temperature for 6h under nitrogen atmosphere to obtain manganese-chelated deferoxamine-grafted methacrylamide gelatin GelMA-DFO@Mn(Ⅱ)-3.

[0099] Step 2: Provide cerium hydroxyapatite nanowires (CeHA@CA) with citric acid surface modification, specifically including:

[0100] Step 201: Under stirring conditions, 42 mL of oleic acid, 54 mL of deionized water and 24 mL of methanol are mixed evenly to obtain system B;

[0101] Step 202: Add 60 mL of 17 M NaOH aqueous solution to system B to adjust the pH of the system to 10.0. Then, slowly add 48 mL of an aqueous solution containing calcium chloride and cerium nitrate with a concentration of 0.3 M calcium chloride and a concentration of 18 mM cerium nitrate to the system. After stirring for 30 min, add 180 mL of 0.5 M sodium dihydrogen phosphate aqueous solution to obtain mixed system C.

[0102] Step 203: Continue stirring the mixture C for 10 minutes, then seal and react at 180°C for 24 hours;

[0103] Step 204: After the system temperature drops to room temperature after the reaction, collect the precipitate, wash it with anhydrous ethanol, centrifuge and dry it to obtain cerium hydroxyapatite nanowires CeHANWs-3.

[0104] Step 205: Dissolve 0.2g of cerium hydroxyapatite nanowires CeHANWs-3 in 10mL of 1.05M sodium citrate ethanol solution, stir at room temperature for 3h, collect the precipitate, wash with deionized water, centrifuge and dry to obtain cerium hydroxyapatite nanowires CeHA@CA-3 with citric acid surface modification.

[0105] Step 3: Provide cerium hydroxyapatite bone repair scaffold material, specifically including:

[0106] Step 301: Add 25 mg of phenyl LAP powder to 10 mL of GelMA-DFO@Mn(Ⅱ)-3 from Step 1, stir for 30 minutes at room temperature, then add 4 g of CeHA@CA-3 from Step 2, stir for 30 minutes at room temperature to obtain 3D printing slurry;

[0107] Step 302: Place the 3D printing paste in the 3D printer. The nozzle diameter is 0.7mm, the extrusion amount is 50%, the layer height and wall thickness are both 0.7mm, the fill density is 60%, the printing temperature is 25℃, and the printing speed is 10mm / s. Irradiate the product generation platform of the 3D printer with 365nm ultraviolet light. The extrudate is irradiated at the same time as it is extruded. After extrusion is completed, continue to irradiate for 60 minutes to allow cross-linking, and obtain the 3D printed cerium hydroxyapatite bone repair scaffold material, named Sample 3.

[0108] Examples 2-4

[0109] This embodiment provides a method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material. This 3D-printed cerium hydroxyapatite bone repair scaffold material can enhance bone regeneration function through vascularization and immune modulation. The method includes the following steps:

[0110] Step 1: Providing manganese-chelated deferoxamine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)), specifically including:

[0111] Step 101: Dissolve 1g of GelMA described in Example 1-1 in 10mL of PBS buffer, add 1g of MES, and adjust the pH to 5.0 with NaOH to obtain system A; the PBS buffer is phosphate buffer with a pH of 7.4;

[0112] Step 102: Add 191.7 mg of EDC and 57.5 mg of NHS to system A, stir and react at room temperature for 1 h, then add 350 mg of DFO, and stir and react at room temperature for 24 h.

[0113] Step 103: Transfer the product obtained from the reaction to an 8000-14000 Da dialysis bag, dialyze with deionized water for 5 days, freeze dry, and obtain deferroamine-grafted methacrylamide gelatin GelMA-DFO-4.

[0114] Step 104: Dissolve 1g of the iron-free amine-grafted methacrylamide gelatin GelMA-DFO-4 in 10mL of 480μM manganese chloride aqueous solution, and stir the mixture at room temperature for 6h under nitrogen atmosphere to obtain manganese-chelated iron-free amine-grafted methacrylamide gelatin GelMA-DFO@Mn(Ⅱ)-4.

[0115] Step 2: Provide cerium hydroxyapatite nanowires (CeHA@CA) with citric acid surface modification, specifically including:

[0116] Step 201: Under stirring conditions, 42 mL of oleic acid, 54 mL of deionized water and 24 mL of methanol are mixed evenly to obtain system B;

[0117] Step 202: Add 60 mL of 17 M NaOH aqueous solution to system B to adjust the pH of the system to 10.0. Then, slowly add 48 mL of an aqueous solution containing calcium chloride and cerium nitrate with a concentration of 0.3 M calcium chloride and a concentration of 12 mM cerium nitrate to the system. After stirring for 30 min, add 180 mL of 0.5 M sodium dihydrogen phosphate aqueous solution to obtain mixed system C.

[0118] Step 203: Continue stirring the mixture C for 10 minutes, then seal and react at 180°C for 24 hours;

[0119] Step 204: After the system temperature drops to room temperature after the reaction, collect the precipitate, wash it with anhydrous ethanol, centrifuge and dry it to obtain cerium hydroxyapatite nanowires CeHANWs-4.

[0120] Step 205: Dissolve 0.2g of cerium hydroxyapatite nanowires CeHANWs-4 in 10mL of 0.7M sodium citrate ethanol solution, stir at room temperature for 3h, collect the precipitate, wash with deionized water, centrifuge and dry to obtain cerium hydroxyapatite nanowires CeHA@CA-4 with citric acid surface modification.

[0121] Step 3: Provide cerium hydroxyapatite bone repair scaffold material, specifically including:

[0122] Step 301: Add 25 mg of LAP powder to 10 mL of GelMA-DFO@Mn(Ⅱ)-4 from Step 1, stir for 30 minutes at room temperature, then add 3 g of CeHA@CA-4 from Step 2, stir for 30 minutes at room temperature to obtain 3D printing slurry;

[0123] Step 302: Place the 3D printing paste in the 3D printer. The nozzle diameter is 0.7mm, the extrusion amount is 50%, the layer height and wall thickness are both 0.7mm, the fill density is 60%, the printing temperature is 25℃, and the printing speed is 10mm / s. Irradiate the product generation platform of the 3D printer with 365nm ultraviolet light. The extrudate is irradiated at the same time as it is extruded. After extrusion is completed, continue to irradiate for 60 minutes to allow cross-linking. The 3D printed cerium hydroxyapatite bone repair scaffold material is obtained and named Sample 4.

[0124] Examples 2-5

[0125] This embodiment provides a method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material. This 3D-printed cerium hydroxyapatite bone repair scaffold material can enhance bone regeneration function through vascularization and immune modulation. The method includes the following steps:

[0126] Step 1: Providing manganese-chelated deferoxamine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)), specifically including:

[0127] Step 101: Dissolve 1g of GelMA from Steps 1-2 in 10mL of PBS buffer, add 1g of ethanesulfonic acid (MES), and adjust the pH to 5.0 with NaOH to obtain system A; the PBS buffer is a phosphate buffer with a pH of 7.4.

[0128] Step 102: Add 191.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 57.5 mg of N-hydroxysuccinimide (NHS) to system A, stir and react at room temperature for 0.5 h, then add 50 mg of deferoxamine (DFO), and stir and react at room temperature for 20 h.

[0129] Step 103: Transfer the product obtained from the reaction to an 8000-14000 Da dialysis bag, dialyze with deionized water for 5 days, and freeze-dry to obtain deferroamine-grafted methacrylamide gelatin GelMA-DFO.

[0130] Step 104: Dissolve 1g of the iron-free amine-grafted methacrylamide gelatin GelMA-DFO in 10mL of 80μM manganese chloride aqueous solution, and stir the mixture at room temperature for 4h under nitrogen atmosphere to obtain manganese chelated iron-free amine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)).

[0131] Step 2: Provide cerium hydroxyapatite nanowires (CeHA@CA) with citric acid surface modification, specifically including:

[0132] Step 201: Under stirring conditions, 42 mL of oleic acid, 54 mL of deionized water and 24 mL of methanol are mixed evenly to obtain system B;

[0133] Step 202: Add 60 mL of 17 M NaOH aqueous solution to system B to adjust the pH of the system to 10.0. Then, slowly add 48 mL of an aqueous solution containing calcium chloride and cerium nitrate with a concentration of 0.3 M calcium chloride and a concentration of 6 mM cerium nitrate to the system. After stirring for 30 min, add 180 mL of 0.5 M sodium dihydrogen phosphate aqueous solution to obtain mixed system C.

[0134] Step 203: Continue stirring the mixture C for 5 minutes, then seal and react at 180°C for 26 hours;

[0135] Step 204: After the system temperature drops to room temperature after the reaction, collect the precipitate, wash it with anhydrous ethanol, centrifuge and dry it to obtain cerium hydroxyapatite nanowires CeHANWs.

[0136] Step 205: Dissolve 0.2g of cerium hydroxyapatite nanowires CeHANWs in 11mL of 0.35M sodium citrate ethanol solution, stir at room temperature for 4h, collect the precipitate, wash with deionized water, centrifuge and dry to obtain cerium hydroxyapatite nanowires CeHA@CA with citric acid surface modification.

[0137] Step 3: Provide cerium hydroxyapatite bone repair scaffold material, specifically including:

[0138] Step 301: Add 20 mg of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) powder to 10 mL of GelMA-DFO@Mn(II) from Step 1, stir for 30 minutes at room temperature, then add 2 g of CeHA@CA from Step 2, stir for 30 minutes at room temperature to obtain 3D printing slurry;

[0139] Step 302: Place the 3D printing paste in the 3D printer with a nozzle diameter of 0.7mm, an extrusion rate of 50%, a layer height and wall thickness of 0.7mm, a fill density of 60%, a printing temperature of 25℃, and a printing speed of 10mm / s. Irradiate the product generation platform of the 3D printer with 365nm ultraviolet light. The extrudate is irradiated simultaneously with the extrusion. After extrusion, continue irradiation for 60 minutes to allow cross-linking, thereby obtaining the 3D printed cerium hydroxyapatite bone repair scaffold material.

[0140] The properties of the 3D-printed cerium hydroxyapatite bone repair scaffold material in this embodiment are basically the same as those in Example 2-1.

[0141] Examples 2-6

[0142] This embodiment provides a method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material. This 3D-printed cerium hydroxyapatite bone repair scaffold material can enhance bone regeneration function through vascularization and immune modulation. The method includes the following steps:

[0143] Step 1: Providing manganese-chelated deferoxamine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)), specifically including:

[0144] Step 101: Dissolve 1g of GelMA from Steps 1-3 in 10mL of PBS buffer, add 1g of ethanesulfonic acid (MES), and adjust the pH to 5.0 with NaOH to obtain system A; the PBS buffer is a phosphate buffer with a pH of 7.4;

[0145] Step 102: Add 191.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 57.5 mg of N-hydroxysuccinimide (NHS) to system A, stir and react at room temperature for 2 h, then add 50 mg of deferoxamine (DFO), and stir and react at room temperature for 26 h.

[0146] Step 103: Transfer the product obtained from the reaction to an 8000-14000 Da dialysis bag, dialyze with deionized water for 5 days, and freeze-dry to obtain deferroamine-grafted methacrylamide gelatin GelMA-DFO.

[0147] Step 104: Dissolve 1g of the iron-free amine-grafted methacrylamide gelatin GelMA-DFO in 10mL of 80μM manganese chloride aqueous solution, and stir the mixture at room temperature for 8h under nitrogen atmosphere to obtain manganese-chelated iron-free amine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)).

[0148] Step 2: Provide cerium hydroxyapatite nanowires (CeHA@CA) with citric acid surface modification, specifically including:

[0149] Step 201: Under stirring conditions, 42 mL of oleic acid, 54 mL of deionized water and 24 mL of methanol are mixed evenly to obtain system B;

[0150] Step 202: Add 60 mL of 17 M NaOH aqueous solution to system B to adjust the pH of the system to 10.0. Then, slowly add 48 mL of an aqueous solution containing calcium chloride and cerium nitrate with a concentration of 0.3 M calcium chloride and a concentration of 6 mM cerium nitrate to the system. After stirring for 30 min, add 180 mL of 0.5 M sodium dihydrogen phosphate aqueous solution to obtain mixed system C.

[0151] Step 203: Continue stirring the mixture C for 15 minutes, then seal and react at 180°C for 20 hours;

[0152] Step 204: After the system temperature drops to room temperature after the reaction, collect the precipitate, wash it with anhydrous ethanol, centrifuge and dry it to obtain cerium hydroxyapatite nanowires CeHANWs.

[0153] Step 205: Dissolve 0.2g of cerium hydroxyapatite nanowires CeHANWs in 9mL of 0.35M sodium citrate ethanol solution, stir at room temperature for 2h, collect the precipitate, wash with deionized water, centrifuge and dry to obtain cerium hydroxyapatite nanowires CeHA@CA with citric acid surface modification.

[0154] Step 3: Provide cerium hydroxyapatite bone repair scaffold material, specifically including:

[0155] Step 301: Add 30 mg of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) powder to 10 mL of GelMA-DFO@Mn(Ⅱ) from Step 1, stir for 30 minutes at room temperature, then add 2 g of CeHA@CA from Step 2, stir for 30 minutes at room temperature to obtain 3D printing slurry;

[0156] Step 302: Place the 3D printing paste in the 3D printer with a nozzle diameter of 0.7mm, an extrusion rate of 50%, a layer height and wall thickness of 0.7mm, a fill density of 60%, a printing temperature of 25℃, and a printing speed of 10mm / s. Irradiate the product generation platform of the 3D printer with 365nm ultraviolet light. The extrudate is irradiated simultaneously with the extrusion. After extrusion, continue irradiation for 60 minutes to allow cross-linking, thereby obtaining the 3D printed cerium hydroxyapatite bone repair scaffold material.

[0157] The properties of the 3D-printed cerium hydroxyapatite bone repair scaffold material in this embodiment are basically the same as those in Example 2-1.

[0158] Performance evaluation:

[0159] Figure 1The 1H NMR spectra of the gelatin (Gel), methacrylamide gelatin (GelMA) described in Examples 1-1, and deferoxamine-grafted methacrylamide gelatin (GelMA-DFO) of Example 2-1 are shown below. Figure 1 It can be seen that methacrylamide gelatin (GelMA) exhibits characteristic peaks at 5.35 and 5.6 (a and b) belonging to -CH2, and at 1.8 (c) belonging to -CH3, proving that the double bond grafting was successful. Calculations based on the lysine methylene peak indicate a double bond grafting rate of 65%. The peaks d, g, h, and i in the ferricamine-grafted methacrylamide gelatin (GelMA)-DFO diagram further confirm the successful grafting of GelMA with ferricamine.

[0160] Figure 2 Fourier transform infrared (FTIR) spectra of the gelatin (Gel) described in Examples 1-1, methacrylamide gelatin (GelMA), deferoxamine-grafted methacrylamide gelatin (GelMA-DFO) described in Examples 2-1, and manganese-chelated deferoxamine-grafted methacrylamide gelatin (GelMA-DFO@Mn(Ⅱ)). Figure 2 It can be seen that 1633cm -1 The characteristic peak at 1539 cm⁻¹ belongs to the C=O bond vibration, indicating that the gelatin structure has not changed after modification. -1 and 1231cm -1 The characteristic peaks correspond to the stretching vibrations of the δN-H bond and the CN bond, indicating successful double bond grafting. (3392 cm⁻¹) -1 -OH at 1739cm -1 The C=O peak shifted to the left, proving that Mn(Ⅱ) chelation was successful.

[0161] Figure 3 The image shows a SEM image of the cerium hydroxyapatite nanowires (CeHANWs-2) described in Example 2-2. Figure 4 This is a TEM image of CeHANWs-2 as described in Example 2-2. According to... Figure 3 and Figure 4 It is evident that cerium hydroxyapatite nanowires were successfully synthesized, and the wire bundles are uniform.

[0162] Figure 5 a and b are photographs of the appearance of the 3D-printed cerium hydroxyapatite bone repair scaffold material described in Example 2-2. Figure 5 'a' is the top view. Figure 5 b is a side view, according to Figure 5 As can be seen from a and 5b, the scaffold material obtained by the present invention through the combination of 3D printing and photocrosslinking has uniform pore size and no collapse. Figure 5 c is an SEM image of the cross-section of the support material. Figure 5 d is a magnified view of a portion of the image, based on... Figure 5As can be seen in c and 5d, the cerium hydroxyapatite nanowires are tightly bound to the hydrogel, and this binding helps to enhance the mechanical properties of the material.

[0163] Figure 6 The cytotoxicity results of the 3D-printed cerium hydroxyapatite bone repair scaffold material described in Examples 2-1 to 2-4 on bone marrow mesenchymal stem cells (hBMSCs) were obtained by the following test method: The sample was sterilized, and 1g of the sterilized sample was placed in 10mL of DMEM culture medium (the DMEM culture medium contained 10% serum and 1% penicillin antibodies) for 72h to obtain the extract. hBMSCs cells were then incubated at 10... 4 Cells were seeded at a density of 1 cell / well in 96-well plates and incubated in a CO2 incubator for 24 h. The original culture medium was replaced with extract medium, while the control group was incubated with fresh medium. Incubation continued for another 24 h. 10 μL of CCK-8 was added to each well, and incubation was continued for 3 h. Absorbance at 540 nm was measured, and cell viability was calculated using the following formula: Cell viability (%) = [(A...] 样 -A 空 ) / (A 对 -A 空 )]×100%, where A 样 A represents the absorbance of the sample. 对 For the absorbance of the control, A 空 The absorbance of the culture medium is shown in the following figures. Figure 6 As shown. (Through) Figure 6 It is evident that samples 1, 2, and 3 all promoted the proliferation of hBMSCs cells, with sample 2 exhibiting the strongest effect. This may be because the concentration of manganese ions, which promotes cell proliferation, was within an appropriate range, and deferoxamine did not exhibit cytotoxicity within the corresponding range. Sample 4 did not promote cell proliferation, possibly because the concentrations of manganese ions and deferoxamine were too high, and their cytotoxicity led to cell death.

[0164] Figure 7 The results of the cytotoxicity of cerium hydroxyapatite nanowires (CeHANWs) to hBMSCs described in Examples 2-1 to 2-3 are as follows: the test method is the same as... Figure 6 The method is the same, from Figure 7 As can be seen, CeHANWs-1, CeHANWs-2, and CeHANWs-3 all have significant effects on promoting the proliferation of hBMSCs. Among them, CeHANWs-2 has the best effect on promoting the proliferation of hBMSCs cells. This may be because the doping amount of cerium ions in CeHANWs-2 has the peak effect on cell proliferation.

[0165] Figure 8The experimental results of the formation of the 3D-printed cerium hydroxyapatite bone repair scaffold material tubes described in Examples 2-3 were obtained through the following testing methods: The sample was sterilized, and 1g of the sterilized sample was placed in 10mL of ECM-specific culture medium and extracted at 37℃ for 72h to obtain the extract. 300μL of Matrigel was spread evenly in a 48-well plate at 4℃ and incubated for 30min to cure. Well-grown human tracheal endothelial cells (HUVECs) were then placed on the Matrigel (cell density approximately 3×10⁻⁶). 4 Add 100 μL of extraction solution to each well (number of cells / well), and incubate for 3 hours at 37°C in a 5% CO2 incubator. Observe the results. The control group is GelMA. Figure 8 As can be seen, the number of nodes, total length, number of enclosed regions, and area of ​​blood vessels generated in sample 3 (right figure) are all better than those in the blank control (left figure), indicating that the cerium hydroxyapatite scaffold material of the present invention has good angiogenesis-promoting effects.

[0166] Figure 9 The polarization qPCR results of RAW264.7 macrophages using the cerium hydroxyapatite bone repair scaffold material described in Examples 2-1 and 2-2 were obtained. The testing method included: placing well-grown RAW264.7 cells into six-well plates at a cell density of approximately 5 × 10⁻⁶ cells / well. 4 Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. Then, 1 mL of LPS (10 μg / mL) was added to induce RAW264.7 cells to polarize towards M1. For the control group, 1 mL of cell culture medium was added. Cells were cultured for 24 hours, and then the LPS induction solution and cell culture medium were aspirated and added... Figure 6 In the method described above, the extraction solution was replaced with fresh culture medium in the control group. Cells were cultured for 24 hours, and RNA was extracted. The extracted RNA was reverse transcribed, and the results were tested using a qPCR instrument. The results are as follows: Figure 9 As shown. According to Figure 9 As can be seen, compared with LPS induction, the expression level of iNOS, a marker of M1 macrophages, in RAW264.7 cells treated with sample 1 and sample 2 was significantly reduced, while the expression level of CD206, a marker of M2 macrophages, was significantly increased. This shows that the cerium hydroxyapatite scaffold material described in this invention can effectively promote the polarization of RAW264.7 cells from M1 to M2, thereby enhancing the repair capacity of bone defects from an immunological perspective.

[0167] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a 3D-printed cerium hydroxyapatite bone repair scaffold material, characterized in that, include: Phenyl (2,4,6-trimethylbenzoyl) lithium phosphate salt powder was added to manganese-chelated deferoxamine-grafted methacrylamide gelatin and stirred. Cerium hydroxyapatite nanowires with citric acid surface modification were then added and stirred to obtain a 3D printing slurry. The 3D printing slurry was extruded using a 3D printer and simultaneously cross-linked under 365nm ultraviolet light to obtain a 3D printed cerium hydroxyapatite bone repair scaffold material. The mass of the cerium hydroxyapatite nanowires with citric acid surface modification was 0.2–0.4 times the volume of the manganese-chelated deferoxamine-grafted methacrylamide gelatin. The mass unit of the cerium hydroxyapatite nanowires with citric acid surface modification is g, and the volume unit of the manganese-chelated deferoxamine-grafted methacrylamide gelatin is mL. The preparation method of the manganese-chelated deferoxamine-grafted methacrylamide gelatin specifically includes: Step 101: Add MES to the PBS buffer of methacrylamide gelatin to obtain system A; Step 102: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to system A, stir and react at room temperature for 0.5-2 h, add deferoxamine, and stir and react at room temperature for 20-26 h; the mass ratio of methacrylamide gelatin, MES, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and deferoxamine is 1:1:0.19:0.057:(0.05-0.15); Step 103: Dialyze the product obtained from the reaction, freeze-dry it, and obtain deferoxamine-grafted methacrylamide gelatin; Step 104: Dissolve the deferoxamine-grafted methacrylamide gelatin in an aqueous manganese chloride solution, and stir the mixture at room temperature under nitrogen atmosphere for 4–8 hours to obtain manganese-chelated deferoxamine-grafted methacrylamide gelatin; the volume of the manganese chloride aqueous solution is 10 times the mass of the deferoxamine-grafted methacrylamide gelatin, the volume of the manganese chloride aqueous solution is in mL, the mass of the deferoxamine-grafted methacrylamide gelatin is in g, and the concentration of the manganese chloride aqueous solution is 80–320 μM.

2. The method according to claim 1, characterized in that, The specific methods for preparing cerium hydroxyapatite nanowires with citric acid surface modification include: Step 201: Provide system B containing oleic acid; system B is system B obtained by dissolving oleic acid in deionized water and methanol; Step 202: Adjust the pH of system B to 10.0, add an aqueous solution containing calcium chloride and cerium nitrate, stir, add an aqueous solution of sodium dihydrogen phosphate, and obtain mixed system C; Step 203: Continue stirring the mixture C for 5-15 minutes, seal, and react at 180°C for 20-26 hours; Step 204: After the system temperature drops to room temperature after the reaction, collect the precipitate, wash it with anhydrous ethanol, centrifuge and dry it to obtain cerium hydroxyapatite nanowires. Step 205: Dissolve cerium hydroxyapatite nanowires in sodium citrate ethanol solution, stir at room temperature for 2-4 hours, collect the precipitate, wash with deionized water, centrifuge and dry to obtain cerium hydroxyapatite nanowires with citric acid surface modification.

3. The method according to claim 2, characterized in that, In step 202, the volume ratio of oleic acid, the aqueous solution containing calcium chloride and cerium nitrate, and the aqueous solution containing sodium dihydrogen phosphate is 42:48:

180. In the aqueous solution containing calcium chloride and cerium nitrate, the concentration of calcium chloride is 0.3M, and the concentration of cerium nitrate is 6-18mM.

4. The method according to claim 2, characterized in that, In step 205, the sodium citrate ethanol solution is a sodium citrate ethanol solution obtained by dissolving sodium citrate in anhydrous ethanol. The concentration of sodium citrate in the sodium citrate ethanol solution is 0.35-1.05M, and the volume of the sodium citrate ethanol solution is 45-55 times the mass of the cerium hydroxyapatite nanowires. The volume unit of the sodium citrate ethanol solution is mL, and the mass unit of the cerium hydroxyapatite nanowires is g.

5. The method according to claim 1, characterized in that, The methacrylamide gelatin is obtained by reacting a PBS solution containing gelatin with methacrylic anhydride under stirring conditions at 45-55°C, followed by dialyzing and freeze-drying.

6. The method according to claim 5, characterized in that, The volume of the methacrylic anhydride is 0.5 to 1.5 times the mass of the gelatin, and the volume of the methacrylic anhydride is measured in mL, while the mass of the gelatin is measured in g.

7. The method according to claim 1, characterized in that, The mass of the phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 2 to 3 times the volume of the manganese-chelated deferoxamine-grafted methacrylamide gelatin. The mass unit of the phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is mg, and the volume unit of the manganese-chelated deferoxamine-grafted methacrylamide gelatin is mL.

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