Modified zinc oxide-based bone implant scaffold material, molding method and preparation method thereof

Abstract

The present invention belongs to the field of new material technology, and specifically discloses a modified zinc oxide-based bone implant scaffold material, a molding method and a preparation method thereof, wherein the perfusion material comprises defect engineering modified zinc oxide nanoparticles loaded by hydrogel. The molding method is to perfuse the perfusion material of defect engineering modified zinc oxide nanoparticles loaded by hydrogel into a 3D printed bone implant scaffold, solidify under ultraviolet light, and then dry to obtain a perfused and solidified 3D printed bone implant scaffold. The scaffold using the perfusion material of the present invention has high-efficiency antibacterial ability and the effect of promoting bone repair. At the same time, the present invention also improves some inherent defects of zinc oxide materials from the perspective of defect engineering and increases the types of bone repair materials.

Description

Technical Field

[0001] The invention belongs to the technical field of new materials, and in particular relates to a modified zinc oxide-based bone implant scaffold material, a molding method and a preparation method thereof. Background Art

[0002] Primary hip and knee replacement is a common procedure (more than 1 million / year in the United States) and the number of surgeries is rising due to demographic changes. At the same time, the number of reoperations and related complications is also increasing year by year. Among them, implant-related infection is the most common and serious complication after orthopedic surgery, with an incidence of 0.7-4.2%. For temporomandibular total joint replacement and craniomaxillofacial surgery, the incidence of postoperative infection is even higher. These all have serious consequences for patients and impose high costs on the health system.

[0003] Implant materials have a higher risk of infection because they often provide a bastion for bacterial adhesion, and in the early postoperative period, the body's immune defense at the implant-tissue interface is reduced. Postoperative infection not only aggravates the inflammatory response and hinders bone integration, but also the colonized bacteria can spread to the joint / medullary cavity, leading to arthritis / osteomyelitis.

[0004] Surgical debridement combined with antibiotic administration is the first choice for solving implant-related infection. Although the related medical expenses are not low, the treatment effect is far from satisfactory and the recurrence rate is quite high. In severe cases, the failed implant must be removed and amputation may even be required. Therefore, the ideal orthopedic implant material must have efficient and rapid antibacterial properties while overcoming the inherent bacterial resistance of antibiotics to minimize the risk of implant-related infection. In addition, it should also induce bone regeneration in vivo during the recovery process. Therefore, it is necessary to develop bone repair scaffold materials that can effectively and quickly resist bacteria to promote bone repair without producing bacterial resistance.

[0005] ZnO is a direct wide bandgap semiconductor. Due to its good electrical, optical and catalytic properties, it has good application prospects in many fields. ZnO has inherent piezoelectric properties and is a piezoelectric material with great application potential. However, in practical applications, due to the wide bandgap of ZnO, it is not easy for electrons to jump from the valence band to the conduction band, and the conductivity is low; at the same time, the electron holes of ZnO are easy to recombine quickly, resulting in a decrease in its catalytic efficiency. Therefore, it is urgent to modify ZnO.

[0006] In the prior art, a variety of bone repair materials have been disclosed, but these materials mainly consider improving the osteogenic ability and mechanical properties of the scaffold. At present, there is no disclosure of a bone repair material that combines defect engineering modified zinc oxide with a 3D printed bone repair matrix to form a bone repair material with high antibacterial ability and the effect of promoting bone repair. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a modified zinc oxide-based bone implant scaffold material, a preparation method and a molding method thereof. The scaffold using the perfusion material has high-efficiency antibacterial ability and the effect of promoting bone repair. At the same time, the present invention also improves some inherent defects of zinc oxide materials from the perspective of defect engineering and increases the types of bone repair materials.

[0008] The technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a modified zinc oxide-based bone implant scaffold material, comprising defect-engineered modified zinc oxide nanoparticles supported by a hydrogel, wherein the hydrogel is one of methacrylated gelatin, methacrylated chitosan, and methacrylated hyaluronic acid.

[0010] It should be noted that ion doping is one of the common methods to improve the photocatalytic performance of materials. Ion doping will cause the material to form impurity energy levels, which will reduce the band gap and the energy required for electron excitation, thereby improving the performance of the catalytic material. At the same time, ion doping often introduces defects. Defect engineering can not only optimize the adsorption of materials and regulate the electronic structure of materials, but also improve the conductivity of semiconductor electrocatalysts by promoting charge separation. It is an effective way to improve the catalytic activity of materials.

[0011] In a second aspect, the present invention provides a method for forming a bone implant scaffold material, wherein a perfusion material of defect-engineered modified zinc oxide nanoparticles loaded by a hydrogel is perfused into a 3D-printed bone implant scaffold, cured under ultraviolet light, and then dried to obtain a perfused and cured 3D-printed bone implant scaffold.

[0012] In combination with the second aspect, the present invention provides a first implementation manner of the second aspect, wherein the curing time of the ultraviolet light is 10-60s.

[0013] In combination with the second aspect, the present invention provides a second implementation of the second aspect, wherein the 3D printed bone implant scaffold is a polyetheretherketone scaffold or a hydroxyapatite scaffold.

[0014] In a third aspect, the present invention also provides a method for preparing a modified zinc oxide-based bone implant scaffold material, which comprises firstly adding alkaline solution to a mixed solution of zinc salt and heteroatom metal salt to form a reaction solution, then treating the reaction solution by a hydrothermal method to remove impurity ions and then drying to obtain defect-engineered modified zinc oxide nanoparticles, and mixing the prepared defect-engineered modified zinc oxide nanoparticles with a hydrogel solution to form a hydrogel dispersion for perfusing the bone implant scaffold.

[0015] In combination with the third aspect, the present invention provides a first implementation of the third aspect, and the specific steps are as follows:

[0016] First, weigh raw materials of zinc salt, heteroatom metal salt and alkaline substance, mix the zinc salt and heteroatom metal salt to form a metal salt mixture, and add deionized water to the metal salt mixture to prepare a metal salt mixed solution;

[0017] Then, deionized water is added to the prepared alkaline substance to prepare an alkaline solution, the alkaline solution is added dropwise to the metal salt mixed solution to prepare a reaction solution, and deionized water or anhydrous ethanol is added to the reaction solution to form a liquid system containing defect engineering modified zinc oxide through a hydrothermal method;

[0018] Then, the liquid system containing the defect-engineered modified zinc oxide is subjected to alternating centrifugal washing with anhydrous ethanol or deionized water to remove impurity ions, and then dried to obtain defect-engineered modified zinc oxide nanoparticles constructed by defect engineering;

[0019] Finally, the hydrogel is dissolved in a PBS buffer solution in a light-proof water bath and a photoinitiator is added to prepare a hydrogel solution. The prepared defect-engineered modified zinc oxide nanoparticles are added to the hydrogel solution and mixed to prepare a defect-engineered modified zinc oxide hydrogel dispersion.

[0020] In combination with the first embodiment of the third aspect, the present invention provides a second embodiment of the third aspect, wherein the mixing steps are all performed by ultrasonic mixing.

[0021] In combination with the third aspect or several embodiments of the third aspect, the present invention provides a third embodiment of the third aspect, wherein the zinc salt includes zinc acetate, zinc sulfate or zinc chloride, the heteroatom metal salt includes ammonium molybdate, sodium molybdate, copper chloride, copper nitrate, copper sulfate, copper citrate, copper acetate or silver nitrate, and the alkaline substance is sodium hydroxide, ammonia monohydrate or sodium carbonate.

[0022] In combination with the third aspect or several embodiments of the third aspect, the present invention provides a fourth embodiment of the third aspect, wherein the volume ratio of the mixture of the zinc salt and the heteroatom metal salt and the deionized water added to the alkaline substance is in the range of 1-10:1;

[0023] The pH of the reaction solution is 8-12, and the volume ratio of the deionized water or anhydrous ethanol added to the reaction solution to the original reaction solution is in the range of 0.05-20:1;

[0024] Deionized water or anhydrous ethanol is added to the reaction solution to react by a hydrothermal method at a temperature of 100-200° C. and a reaction time of 8-24 hours.

[0025] In combination with the third aspect or several embodiments of the third aspect, the present invention provides a fifth embodiment of the third aspect, wherein the hydrogel is one of methacryloyl gelatin, methacryloyl chitosan, and methacryloyl hyaluronic acid;

[0026] The photoinitiator is one of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, and 2,2'-azo (2-methyl-N-(2-hydroxyethyl)propionamide);

[0027] When forming the hydrogel PBS buffer, the amount of the hydrogel added is 5-20% of the mass of the PBS buffer, and the amount of the photoinitiator added is 0.1-1% of the mass of the PBS buffer.

[0028] When preparing a hydrogel dispersion of defect-engineered modified zinc oxide, 0.01-2 mg of defect-engineered modified zinc oxide is added to 1 mL of the hydrogel solution.

[0029] The beneficial effects of the present invention are:

[0030] 1. The present invention provides a material that has both bone repair and high-efficiency and rapid antibacterial capabilities, thereby increasing the types of bone repair materials. The use of this material can achieve bone repair and postoperative anti-infection;

[0031] 2. The bone implant scaffold structure formed by the implantation method of the present invention can achieve a better antibacterial effect by adopting the perfusion ratio and perfusion method specified in the present invention;

[0032] 3. The scaffold material prepared by the method provided by the present invention is loaded with defect engineering modified zinc oxide-based nanoparticles. Experiments show that the modified zinc oxide has excellent catalytic antibacterial effect enhanced by ultrasonic excitation, thereby reducing the dependence of traditional bone repair materials on antibiotics;

[0033] 4. The hydrogel-loaded defect engineering modified zinc oxide-based nanoparticle-infused 3D printed bone implant scaffold material prepared by the method of the present invention can be adjusted by adjusting the doped heteroatom metal salt, hydrogel and 3D printed bone implant scaffold, so it is easy to adjust according to clinical needs, and is expected to be expanded to clinical fields such as tumor bone defects;

[0034] 5. The 3D printed bone implant scaffold material prepared in the present invention and infused with defect-engineered modified zinc oxide-based nanoparticles can quickly and directionally dissolve and release defect-engineered modified zinc oxide nanoparticles for antibacterial purposes against Gram-positive bacteria, and has high specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the XRD spectrum of the molybdenum-doped modified zinc oxide prepared in the embodiment of the present invention;

[0036] Figure 2The zinc oxide prepared in the embodiment of the present invention and the zinc oxide modified by doping with molybdenum with a concentration of 0.4 mg / mL were dispersed at a power of 1.0 W / cm 2 Singlet oxygen production diagram under ultrasonic excitation for 8 minutes;

[0037] Figure 3 The zinc oxide prepared in the embodiment of the present invention and the zinc oxide modified by doping with molybdenum with a concentration of 0.4 mg / mL were dispersed at a power of 1.0 W / cm 2 The hydroxyl radical production diagram under ultrasonic excitation for 8 minutes;

[0038] Figure 4 The zinc oxide prepared in the embodiment of the present invention and the zinc oxide modified by doping with molybdenum with a concentration of 0.4 mg / mL were dispersed at a power of 1.0 W / cm 2 The superoxide production diagram under ultrasonic excitation for 8 minutes;

[0039] Figure 5 The figure is a comparative diagram of the bactericidal effects of the zinc oxide dispersion prepared in the embodiment of the present invention, the molybdenum-doped modified zinc oxide dispersion and the PBS solution on Staphylococcus aureus; the left one is the bactericidal effect of the PBS group, the middle and right ones are the bactericidal effects of the zinc oxide group and the molybdenum-doped modified zinc oxide group at 1.0 W / cm 2 The bactericidal effect under ultrasonic stimulation;

[0040] Figure 6 The figure is a comparative diagram of the bactericidal effect of the dispersion prepared by the molybdenum-doped modified zinc oxide prepared in the embodiment of the present invention and the PBS solution on Escherichia coli; the left one is the bactericidal effect of the PBS group, and the right one is the bactericidal effect of the molybdenum-doped modified zinc oxide group at 1.0 W / cm 2 The bactericidal effect under ultrasonic stimulation;

[0041] Figure 7 The figure is a comparative diagram of the bactericidal effect of the dispersion prepared by the molybdenum-doped modified zinc oxide prepared in the embodiment of the present invention and the PBS solution on Staphylococcus aureus; the left one is the bactericidal effect of the PBS group, and the right one is the bactericidal effect of the molybdenum-doped modified zinc oxide group at 1.0 W / cm 2 The bactericidal effect under ultrasonic stimulation;

[0042] Figure 8 The figure is a comparative diagram of the bactericidal effects of the dispersion prepared by zinc oxide, the dispersion prepared by molybdenum-doped modified zinc oxide and PBS solution on Staphylococcus aureus biofilm prepared in the embodiment of the present invention; the left one is the bactericidal effect of the PBS group, the middle and right ones are the bactericidal effects of the zinc oxide group and the molybdenum-doped modified zinc oxide group at 1.0 W / cm 2 The bactericidal effect under ultrasonic stimulation;

[0043] Fig. 9This is a photograph of the compatibility test results of the dispersion prepared by molybdenum-doped modified zinc oxide and PBS solution on MC3T3-E1 mouse embryonic osteoblasts prepared in the embodiment of the present invention;

[0044] Fig.10 This is a wound coating image of a 3D-printed polyetheretherketone scaffold perfused with methacrylated gelatin hydrogel-wrapped zinc oxide, molybdenum-doped zinc oxide, and PBS solution prepared in an embodiment of the present invention, after one week of in vivo treatment of a femoral bone defect infection model in SD rats;

[0045] Fig.11 This is a three-dimensional bone reconstruction image of the defect after eight weeks of in vivo treatment of the femoral bone defect infection model of SD rats using a 3D-printed polyetheretherketone scaffold perfused with methacryloyl gelatin hydrogel-wrapped zinc oxide, molybdenum-doped zinc oxide, and PBS solution prepared in an embodiment of the present invention. DETAILED DESCRIPTION

[0046] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments.

[0047] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

[0048] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application for which protection is sought, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0049] Embodiment 1:

[0050] This embodiment discloses a modified zinc oxide-based bone implant scaffold material, which is used to be injected into a 3D printed scaffold to form a complete bone implant scaffold structure, and is used as a replacement material in bone replacement surgeries such as primary hip and knee replacements.

[0051] The priming material contains defect-engineered modified zinc oxide nanoparticles supported by a hydrogel.

[0052] The method for forming a bone implant scaffold using this material is as follows: defect-engineered modified zinc oxide nanoparticles after heteroatoms are introduced through defect engineering are dispersed in a hydrogel material, wherein the hydrogel is one of methacrylated gelatin, methacrylated chitosan, and methacrylated hyaluronic acid.

[0053] The hydrogel is dissolved in a PBS buffer solution in a light-proof water bath and a photoinitiator is added to prepare a hydrogel solution. The photoinitiator is one of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, and 2,2'-azo (2-methyl-N-(2-hydroxyethyl)propionamide).

[0054] When forming a hydrogel PBS buffer, the amount of hydrogel added is 5-20% of the mass of the PBS buffer, and the amount of the photoinitiator added is 0.1-1% of the mass of the PBS buffer. Then the prepared defect-engineered zinc oxide is added to the above hydrogel solution, and a hydrogel dispersion of defect-engineered zinc oxide is prepared by ultrasonic mixing. When preparing the hydrogel dispersion of defect-engineered zinc oxide, 0.01-2 mg of defect-engineered zinc oxide is added to 1 mL of the hydrogel solution.

[0055] Then, a bone frame is 3D printed according to the shape and structure of the designed bone implant scaffold, and the material of the 3D printed bone frame is selected from polyetheretherketone scaffold or hydroxyapatite scaffold. The perfusion material is poured into the bone frame along the groove direction, and after the groove surface is completely filled and overflowed, it is cured and dried by ultraviolet light irradiation, and the curing time of ultraviolet light is 10-60s. After drying, the surface is smoothed according to the design requirements of the bone implant scaffold to form a perfused and cured 3D printed bone implant scaffold.

[0056] Furthermore, for defect engineering modified zinc oxide nanoparticles, this embodiment provides a preparation method, which is as follows:

[0057] First, weigh raw materials of zinc salt, heteroatom metal salt and alkaline substance, mix the zinc salt and heteroatom metal salt to form a metal salt mixture, add deionized water to the metal salt mixture to prepare a metal salt mixed solution, wherein the volume ratio of the added deionized water is in the range of 1-10:1;

[0058] Then, deionized water is added to the prepared alkaline substance to prepare an alkaline solution, and the volume ratio of the deionized water added is also in the range of 1-10:1;

[0059] Alkali solution is then added dropwise to the metal salt mixed solution to prepare a reaction solution, and deionized water or anhydrous ethanol is added to the reaction solution to form a liquid system containing defect engineering modified zinc oxide through a hydrothermal method; wherein the solution pH of the reaction solution is 8-12, and the volume ratio of the deionized water or anhydrous ethanol added to the reaction solution to the original reaction solution is in the range of 0.05-20:1; when deionized water or anhydrous ethanol is added to the reaction solution through a hydrothermal method, the temperature is 100-200°C, and the reaction time is 8-24h.

[0060] Then, the liquid system containing the defect-engineered modified zinc oxide is subjected to alternating centrifugal washing with anhydrous ethanol or deionized water to remove impurity ions, and then dried to obtain the defect-engineered zinc oxide nanoparticles;

[0061] Finally, the hydrogel is dissolved in a PBS buffer solution in a light-proof water bath and a photoinitiator is added to prepare a hydrogel solution. The prepared defect-engineered modified zinc oxide nanoparticles are added to the hydrogel solution and mixed to prepare a defect-engineered modified zinc oxide hydrogel dispersion.

[0062] Furthermore, the zinc salts described in this embodiment include zinc acetate, zinc sulfate and zinc chloride, the heteroatom metal salts include ammonium molybdate, sodium molybdate, copper chloride, copper nitrate, copper sulfate, copper citrate, copper acetate, silver nitrate, and the alkaline substance is sodium hydroxide, ammonia monohydrate and sodium carbonate.

[0063] As an embodiment, a specific material preparation method is provided with reference to the materials provided above.

[0064] (a) Preparation of defect engineering modified zinc oxide

[0065] Weigh 0.408g of zinc chloride dihydrate, 0.256g of copper chloride dihydrate and 5mL of ammonia monohydrate respectively, mix zinc chloride and copper chloride dihydrate, then add 3mL of deionized water to the mixture of zinc chloride and copper chloride dihydrate, and form a mixed solution of zinc chloride and copper chloride after ultrasonic mixing for 30 minutes; then drop ammonia monohydrate into the mixed solution of zinc chloride and copper chloride to generate a reaction solution, stir and react for 30 minutes at room temperature, then add 80mL of anhydrous ethanol to the reaction solution and transfer the mixed solution to a polytetrafluoroethylene reactor, and hydrothermally heat at 180°C for 13h to obtain a liquid system of modified zinc oxide doped with copper. The liquid system of modified zinc oxide doped with copper is washed by alternating centrifugation with anhydrous ethanol and deionized water to remove impurity ions, and then vacuum dried at 65°C to obtain powdered modified zinc oxide nanoparticles doped with copper;

[0066] (b) Preparation of 3D printed bone implant scaffolds infused with hydrogel-loaded defect-engineered modified zinc oxide-based nanoparticles

[0067] 0.6 g of methacryloyl chitosan was dissolved in 5 mL of PBS buffer solution in a 50° C. water bath in the dark, and then 0.02 g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate was added as a photoinitiator to prepare a hydrogel solution; 1.5 mg of the defect-engineered copper-doped zinc oxide prepared in step (1) was weighed and added to the above hydrogel solution, and ultrasonically mixed for 10 min to prepare a copper-doped zinc oxide hydrogel dispersion; the copper-doped zinc oxide hydrogel dispersion was infused into a 3D-printed hydroxyapatite scaffold, cured under ultraviolet light for 15 s, and then freeze-dried for 12 h to obtain a 3D-printed bone implant scaffold infused with hydrogel-loaded defect-engineered modified zinc oxide-based nanoparticles.

[0068] As another embodiment, the process steps are as follows:

[0069] (a) Preparation of defect engineering modified zinc oxide

[0070] Weigh 0.27g of zinc sulfate monohydrate, 0.255g of silver nitrate and 0.6g of sodium hydroxide respectively, mix the zinc sulfate monohydrate and the silver nitrate, then add 3mL of deionized water to the mixture of zinc sulfate monohydrate and silver nitrate and the sodium hydroxide respectively, and form a mixed solution of zinc sulfate and silver nitrate and a sodium hydroxide solution after ultrasonic mixing for 30 minutes; then drop the sodium hydroxide solution into the mixed solution of zinc sulfate and silver nitrate to generate a reaction solution, stir and react for 30 minutes at room temperature, then add 60mL of deionized water to the reaction solution and transfer the mixed solution to a polytetrafluoroethylene reactor, and hydrothermally heat at 120°C for 10 hours to obtain a liquid system of modified zinc oxide doped with silver. The liquid system of modified zinc oxide doped with silver is washed by alternating centrifugation with anhydrous ethanol and deionized water to remove impurity ions, and then vacuum dried at 65°C to obtain powdered modified zinc oxide nanoparticles doped with silver;

[0071] (b) Preparation of 3D printed bone implant scaffolds infused with hydrogel-loaded defect-engineered modified zinc oxide-based nanoparticles

[0072] 1.6 g of methacryloyl hyaluronic acid was dissolved in 20 mL of PBS buffer solution in a 60° C. water bath in the dark, and then 0.1 g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate was added as a photoinitiator to prepare a hydrogel solution; 2 mg of the defect-engineered silver-doped zinc oxide prepared in step (1) was weighed and added to the above hydrogel solution, and ultrasonically mixed for 10 min to prepare a silver-doped zinc oxide hydrogel dispersion; the silver-doped zinc oxide hydrogel dispersion was infused into a 3D-printed hydroxyapatite scaffold, cured under ultraviolet light for 25 s, and then freeze-dried for 12 h to obtain a 3D-printed bone implant scaffold infused with hydrogel-loaded defect-engineered modified zinc oxide-based nanoparticles.

[0073] Furthermore, in order to verify the performance of multiple materials, a specific material and its preparation method are provided, and multiple experiments are performed to verify the performance.

[0074] First, prepare the sample, weigh 0.66g of zinc acetate dihydrate, 0.726g of sodium molybdate dihydrate and 1.2g of sodium hydroxide, mix the zinc acetate dihydrate and sodium molybdate dihydrate, then add 6mL of deionized water to the mixture of zinc acetate dihydrate and sodium molybdate dihydrate and sodium hydroxide, respectively, and form a mixed solution of zinc acetate and sodium molybdate and a sodium hydroxide solution after ultrasonic mixing for 30min.

[0075] Then, a sodium hydroxide solution was dropped into a mixed solution of zinc acetate and sodium molybdate to generate a reaction solution, and the reaction was stirred at room temperature for 30 minutes. 130 mL of anhydrous ethanol was added to the reaction solution, and the mixed solution was transferred to a polytetrafluoroethylene reactor and hydrothermally heated at 100° C. for 13 hours to obtain a modified zinc oxide liquid system containing doped molybdenum.

[0076] The liquid system containing the modified zinc oxide doped with molybdenum is washed by alternating centrifugation with anhydrous ethanol and deionized water to remove impurity ions, and then vacuum dried at 65° C. to obtain powdered modified oxide nanoparticles doped with molybdenum.

[0077] 1 g of methacrylated gelatin was dissolved in 10 mL of PBS buffer solution in a 55°C water bath in the dark, and then 0.03 g of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone was added as a photoinitiator to prepare a hydrogel solution; 2 mg of the prepared defect-engineered molybdenum-doped zinc oxide was weighed and added to the above hydrogel solution, and ultrasonically mixed for 10 minutes to prepare a molybdenum-doped zinc oxide hydrogel dispersion.

[0078] The hydrogel dispersion of molybdenum-doped zinc oxide was infused into the 3D-printed polyetheretherketone scaffold, cured under ultraviolet light for 20 seconds, and then freeze-dried for 12 hours to obtain a 3D-printed bone implant scaffold infused with hydrogel-loaded defect-engineered modified zinc oxide-based nanoparticles.

[0079] For sample testing, the prepared Mo-doped ZnO (Mo-ZnO) was first subjected to XRD analysis, and its XRD spectrum is shown in Figure 1 .

[0080] Conclusion: From Figure 1 It can be seen that the Mo-doped ZnO prepared in this example corresponds well to the PDF card of ZnO and no other impurity peaks are detected, which proves that Mo is highly dispersedly doped into ZnO, indicating that the doping of impurities can be effectively achieved by the above method.

[0081] The Mo-doped ZnO (Mo-ZnO) prepared in this example and the ZnO prepared by the method described in this example were tested for ultrasonic catalytic performance.

[0082] The ZnO is prepared by dissolving 0.66 g of zinc acetate dihydrate and 1.2 g of sodium hydroxide in 6 mL of deionized water to prepare a zinc acetate solution and a sodium hydroxide solution, respectively. The zinc acetate solution and the sodium hydroxide solution are then reacted and 130 mL of anhydrous ethanol is added, and the ZnO is obtained by hydroheating at 100°C for 13 hours.

[0083] Singlet oxygen production test: Deionized water was used to prepare a 400 µg / mL zinc oxide dispersion and a molybdenum-doped zinc oxide dispersion, respectively. The purchased SOSG fluorescent probe was prepared into a 5 mM SOSG storage solution with dimethyl sulfoxide. 1 mL of zinc oxide dispersion and 1 mL of molybdenum-doped zinc oxide dispersion were taken, and 1 µL of SOSG storage solution was added. Ultrasonic therapy was used at a power of 1.0 W / cm 2 The samples were treated under ultrasonic excitation for 8 minutes, and finally the peak intensity at 525 nm under 480 nm excitation was tested by fluorescence spectrometer. The test results are shown in Figure 2 .

[0084] Conclusion: From Figure 2 It can be seen that under the same concentration and ultrasonic excitation energy, the singlet oxygen yield of the Mo-doped ZnO dispersion within 8 min was significantly higher than that of the ZnO dispersion, indicating that Mo-doped ZnO has better ultrasonic catalytic efficiency.

[0085] Hydroxyl radical production test: Deionized water was used to prepare 400 µg / mL zinc oxide dispersion and molybdenum-doped zinc oxide dispersion, respectively. The purchased HPF fluorescent probe was prepared into 500 µM HPF storage solution with dimethyl sulfoxide. 1 mL of zinc oxide dispersion and 1 mL of molybdenum-doped zinc oxide dispersion were taken, and 20 µL of HPF storage solution was added. Ultrasonic therapy was used at a power of 1.0 W / cm 2 The samples were treated under ultrasonic excitation for 8 minutes, and finally the peak intensity at 515 nm under 492 nm excitation was tested by fluorescence spectrometer. The test results are shown in Figure 3 .

[0086] Conclusion: From Figure 3 It can be seen that under the same concentration and ultrasonic excitation energy, the hydroxyl radical yield of the molybdenum-doped zinc oxide dispersion was significantly higher than that of the zinc oxide dispersion within 8 min, indicating that the molybdenum-doped zinc oxide has a better ultrasonic catalytic efficiency.

[0087] Superoxide production test: Deionized water was used to prepare 400µg / mL zinc oxide dispersion and molybdenum-doped zinc oxide dispersion, respectively. The purchased DHE fluorescent probe was prepared into 25mg / mL DHE storage solution with dimethyl sulfoxide, and DNA was prepared into 5mg / mL DNA solution. 1mL zinc oxide dispersion and 1mL molybdenum-doped zinc oxide dispersion were taken, and 10µL DHE storage solution and 150µL DNA solution were added. Ultrasonic therapy was used at a power of 1.0W / cm 2 The samples were treated under ultrasonic excitation for 8 minutes, and finally the peak intensity at 590nm under 490nm excitation was tested by fluorescence spectrometer. The test results are shown in Figure 4 .

[0088] Conclusion: From Figure 4 It can be seen that under the same concentration and ultrasonic excitation energy, the superoxide production of the Mo-doped ZnO dispersion was significantly higher than that of the ZnO dispersion within 8 min, indicating that Mo-doped ZnO has better ultrasonic catalytic efficiency.

[0089] Then the molybdenum-doped zinc oxide prepared in this example was used to sterilize Escherichia coli and Staphylococcus aureus: the Escherichia coli (ATCC25922) used in the experiment was purchased from Shanghai Yaji Biotechnology Co., Ltd., and the Staphylococcus aureus (ATCC25923) was purchased from Shenzhen Zike Biotechnology Co., Ltd. 5 CFU of Staphylococcus aureus was mixed with 0.4 mg / mL zinc oxide dispersion, molybdenum-doped zinc oxide dispersion and PBS solution in equal volumes to form zinc oxide group, molybdenum-doped zinc oxide group and PBS group, each with three wells. The zinc oxide group and molybdenum-doped zinc oxide group were heated at 1.0 W / cm 2 After each group was left to stand for 5 minutes, 100 μL of the mixture was taken on the LB solid medium, spread evenly, and placed in a 37°C oven for two days, and then photographed. The photographs taken are shown in Figure 5 .

[0090] Conclusion: From Figure 5 It can be seen that compared with PBS and pure zinc oxide groups, the molybdenum-doped zinc oxide group has a 2 It has enhanced bactericidal ability against Staphylococcus aureus under ultrasonic stimulation.

[0091] The concentration was 2×10 5CFU of Escherichia coli and Staphylococcus aureus were inoculated into two 48-well plates, and bacterial slides were placed in the well plates in advance; after incubation for 24 hours, the bacterial solution was aspirated, and 0.5 mL of 0.2 mg / mL molybdenum-doped zinc oxide dispersion and PBS solution were added to different wells to form a molybdenum-doped zinc oxide group and a PBS group, respectively. Each group of each bacteria had three wells, and the molybdenum-doped zinc oxide group must be heated at 1.0 W / cm 2 After the material was removed, it was washed three times with PBS, then stained with the LIVE / DEAD™ BacLight™ Bacterial Viability Detection Kit for 30 minutes, the dye solution was removed, and then washed three times with PBS, and then placed under an inverted fluorescence microscope for observation and photography. The photographs taken are shown in Figure 6 , Figure 7 .

[0092] Conclusion: From Figure 6 , Figure 7 It can be seen that compared with the PBS group, the Mo-doped ZnO group has a 2 It has excellent bactericidal ability against Escherichia coli and Staphylococcus aureus under ultrasonic stimulation.

[0093] The concentration was 1×10 7 CFU of Staphylococcus aureus was inoculated into a 48-well plate, and a bacterial slide was placed in the well plate in advance; the liquid was changed every day thereafter, and after each group grew a uniform biofilm, the bacterial liquid was aspirated, and 0.5 mL of 0.2 mg / mL zinc oxide dispersion, molybdenum-doped zinc oxide dispersion, and PBS solution were added to different wells to form a zinc oxide group, a molybdenum-doped zinc oxide group, and a PBS group, with three wells in each group, and the zinc oxide group and the molybdenum-doped zinc oxide group must be heated at 1.0 W / cm 2 After all groups were processed, the materials were discarded, and the excess materials were washed three times with PBS. Then, the slides were fixed with 4% paraformaldehyde solution for 30 minutes. After fixation, the paraformaldehyde was aspirated, and a crystal violet solution with a concentration of 1g / L was added to stain for 1 hour in the dark. After washing once with PBS, the slides were placed in a 50°C oven to dry, and the images were scanned after drying. The scanned images are shown in Figure 8 .

[0094] Conclusion: From Figure 8 It can be seen that compared with PBS and pure zinc oxide groups, the molybdenum-doped zinc oxide group has a 2 It also has enhanced bactericidal ability against biofilm formed by Staphylococcus aureus under ultrasonic stimulation.

[0095] The defect-engineered zinc oxide prepared in this example was subjected to a compatibility test on MC3T3-E1 mouse embryonic osteoblasts: The MC3T3-E1 mouse embryonic osteoblasts used in the experiment were purchased from Wuhan Shann Biotechnology Co., Ltd. MC3T3-E1 mouse embryonic osteoblasts were cultured in an α-MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin by mass, and the standard cell culture conditions were 37°C and 5% CO2 by volume. All samples and facilities used were sterilized by autoclave and irradiated with ultraviolet light for more than 0.5h.

[0096] MC3T3-E1 mouse embryonic osteoblasts were placed in a 48-well plate, and cell slides were placed in the well plate in advance. The cell density was 5×10 3 After incubation for 24 hours, 0.2 mg / mL of molybdenum-modified zinc oxide solution and PBS buffer were added, and each group had three wells. The molybdenum-modified zinc oxide group was heated at 1.0 W / cm 2 Ultrasonication was performed for 2 minutes. After the end, the material was aspirated, washed three times with PBS, fixed with 4% paraformaldehyde solution for 4 hours, washed three times with PBS, and then dehydrated with 30%, 50%, 70%, 90%, 95%, and 100% ethanol aqueous solutions for 10 minutes respectively. After natural air drying, it was placed under a field emission scanning electron microscope for observation and photography.

[0097] Conclusion: See the photos taken Fig. 9 The left picture shows the compatibility test results of PBS group on MC3T3-E1 cells, and the right picture shows the Mo-doped ZnO group at 1.0W / cm 2 The photo of the compatibility test results of MC3T3-E1 cells under ultrasonic excitation was obtained by field emission scanning electron microscopy. Figure 7 It can be seen that the molybdenum-doped zinc oxide group has good compatibility with MC3T3-E1 mouse embryonic osteoblasts.

[0098] Eight-week-old SD male rats were evenly and randomly divided into different groups. The rats were anesthetized under sterile conditions, and a non-penetrating defect with a diameter of 3 mm and a length of 5 mm was created in their femurs. Then, the zinc oxide, molybdenum-doped zinc oxide, and PBS solution prepared in this example were wrapped in methacrylated gelatin and perfused into a 3D-printed polyetheretherketone scaffold with a diameter of 3 mm and a length of 5 mm. The prepared scaffold was implanted into the femoral defect of the rats and 20 μL of a 10 7 The rats were then bandaged. The zinc oxide group and the molybdenum-doped zinc oxide group were exposed to 1.0 W / cm 2The rats were stimulated under ultrasound for 5 minutes. After that, the rats in each group continued to be fed. After one week of feeding, some rats were killed, and the wounds were dipped with sterile cotton swabs and soaked in sterile LB liquid culture medium. The collected bacterial culture was diluted to an appropriate concentration for plating, and then the solid culture medium was placed in a 37°C oven for two days and photographed. The photos taken are shown in Fig.10 .

[0099] Conclusion: From Fig.10 It can be seen that compared with PBS and pure zinc oxide groups, the molybdenum-doped zinc oxide group has a 2 It still has enhanced bactericidal ability against Staphylococcus aureus in vivo under ultrasonic stimulation.

[0100] After eight weeks of feeding, the remaining rats were killed, and the femurs of the rats were fixed and scanned with CT for imaging. The imaging results are shown in Fig.11 .

[0101] Conclusion: From Fig.11 It can be seen that compared with PBS and pure zinc oxide groups, the molybdenum-doped zinc oxide group has a 2 Ultrasonic stimulation can promote bone defect repair in rats in vivo.

[0102] At the same time, in the antibacterial experiments on the above samples, it was found that in addition to the original defect-engineered modified zinc oxide material having good antibacterial effect, the coated methacryloyl gelatin hydrogel material can also be degraded by a gelatinase secreted by Gram-positive bacteria (Staphylococcus aureus, MRSA, etc.), and then the defect-engineered modified zinc oxide material can be quickly released against the Gram-positive bacteria, thereby achieving the effect of on-demand release and minimizing damage to normal tissues.

[0103] Specifically, at 37°C, 150 µL of photocured 10% methacrylated gelatin hydrogel was immersed in 3 mL of immersion solution. When the immersion solution was a gelatinase solution with a concentration of 50 µg / mL, the hydrogel was completely degraded in less than one day; when the immersion solution was a gelatinase solution with a concentration of 5 µg / mL, the hydrogel was completely degraded within two days; when the immersion solution was a gelatinase solution with a starting concentration of 1×10 7 When the immersion liquid was a solution of Staphylococcus aureus with a concentration of CFU, the hydrogel was completely degraded within three days; when the immersion liquid was a gelatinase solution with a concentration of 1µg / mL, the hydrogel was completely degraded within six days; and when the immersion liquid was a PBS buffer solution with a concentration of 1µg / mL, the hydrogel showed no obvious degradation within seven days.

[0104] The present invention is not limited to the above optional implementations, and anyone can derive other various forms of products under the enlightenment of the present invention. The above specific implementations should not be understood as limiting the scope of protection of the present invention. The scope of protection of the present invention should be based on the definition in the claims, and the description can be used to interpret the claims.

Claims

1. Modified zinc oxide-based bone implant scaffold material, characterized in that: Contains hydrogel loaded at 1.0W / cm 2 The invention relates to molybdenum-doped defect-engineered modified zinc oxide nanoparticles which are effective under ultrasonic excitation, and the hydrogel is one of methacrylated gelatin, methacrylated chitosan and methacrylated hyaluronic acid.

2. A method for forming a bone implant scaffold material, using the modified zinc oxide-based bone implant scaffold material of claim 1, characterized in that: The perfusion material composed of defect-engineered modified zinc oxide nanoparticles loaded by hydrogel is perfused into the 3D printed bone implant scaffold, cured under ultraviolet light, and then dried to obtain a perfused and cured 3D printed bone implant scaffold.

3. A method for forming a bone implant scaffold material according to claim 2, characterized in that: The curing time of the ultraviolet light is 10-60s.

4. A method for forming a bone implant scaffold material according to claim 2, characterized in that: The 3D printed bone implant scaffold is a polyetheretherketone scaffold or a hydroxyapatite scaffold.

5. A method for preparing a modified zinc oxide-based bone implant scaffold material, used for preparing the modified zinc oxide-based bone implant scaffold material according to claim 1, characterized in that: First, alkaline solution is added to a mixed solution of zinc salt and molybdenum atom metal salt to form a reaction solution. The reaction solution is then treated by a hydrothermal method to remove impurity ions and then dried to obtain defect-engineered modified zinc oxide nanoparticles. The prepared defect-engineered modified zinc oxide nanoparticles are mixed with a hydrogel solution to form a hydrogel dispersion for perfusing bone implant scaffolds.

6. The method for preparing a modified zinc oxide-based bone implant scaffold material according to claim 5, characterized in that: The specific steps are as follows: First, weigh raw materials of zinc salt, molybdenum atom metal salt and alkaline substance, mix the zinc salt and the molybdenum atom metal salt to form a metal salt mixture, and add deionized water to the metal salt mixture to prepare a metal salt mixed solution; Then, deionized water is added to the prepared alkaline substance to prepare an alkaline solution, the alkaline solution is added dropwise to the metal salt mixed solution to prepare a reaction solution, and deionized water or anhydrous ethanol is added to the reaction solution to form a liquid system containing defect engineering modified zinc oxide through a hydrothermal method; Then, the liquid system containing the defect-engineered modified zinc oxide is subjected to alternating centrifugal washing with anhydrous ethanol or deionized water to remove impurity ions, and then dried to obtain defect-engineered modified zinc oxide nanoparticles constructed by defect engineering; Finally, the hydrogel is dissolved in a PBS buffer solution in a light-proof water bath and a photoinitiator is added to prepare a hydrogel solution. The prepared defect-engineered modified zinc oxide nanoparticles are added to the hydrogel solution and mixed to prepare a defect-engineered modified zinc oxide hydrogel dispersion.

7. The method for preparing a modified zinc oxide-based bone implant scaffold material according to claim 6, characterized in that: The mixing steps are all carried out by ultrasonic mixing.

8. A method for preparing a modified zinc oxide-based bone implant scaffold material according to any one of claims 5 to 7, characterized in that: in, The volume ratio of the mixture of the zinc salt and the molybdenum atom metal salt and the deionized water added to the alkaline substance is in the range of 1-10:1; The pH of the reaction solution is 8-12, and the volume ratio of the deionized water or anhydrous ethanol added to the reaction solution to the original reaction solution is in the range of 0.05-20:1; Deionized water or anhydrous ethanol is added to the reaction solution to react by a hydrothermal method at a temperature of 100-200° C. and a reaction time of 8-24 hours.

9. A method for preparing a modified zinc oxide-based bone implant scaffold material according to any one of claims 6-7, characterized in that: The photoinitiator is one of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, and 2,2'-azo (2-methyl-N-(2-hydroxyethyl)propionamide); When forming the hydrogel PBS buffer, the amount of the hydrogel added is 5-20% of the mass of the PBS buffer, and the amount of the photoinitiator added is 0.1-1% of the mass of the PBS buffer; When preparing a hydrogel dispersion of defect-engineered modified zinc oxide, 0.01-2 mg of defect-engineered modified zinc oxide is added to 1 mL of the hydrogel solution.

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