Long-acting antibacterial biomass orthopedic material and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a long-acting antibacterial biomass orthopedic material and a preparation method thereof. The nutrient solution is prepared from the following raw materials: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, antibiotics, 2-methylimidazole, sulphobetaine methacrylate and a photoinitiator. A three-layer synergistic structure is constructed through a Ga-doped beta-tricalcium phosphate core / a pH-responsive ZIF-8 and antibiotic shell / a photocuring sulfobetaine (pSBMA) brush layer, and the process adopts three conventional continuous steps of water-phase coprecipitation, room-temperature self-assembly and room-temperature photocuring, so that a high-energy physical deposition or complex surface activation process is avoided; the interlayer compatibility and repeatability are ensured; the finally obtained biomass material realizes triple functions of adhesion barrier resistance, drug burst release and long-term ion antibiosis in a microscopic level, and maintains biocompatibility in a macroscopic level.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a long-acting antibacterial biomass orthopedic material and a preparation method thereof. Background Art

[0002] Biomass orthopedic materials refer to bone defect fillers, scaffolds, or internal fixation devices made from renewable bioresources such as natural bone tissue, animal-derived collagen, chitosan, silk fibroin, alginate, bacterial cellulose, and biodegradable polymers obtained through fermentation or polymerization of bio-based monomers. These materials are prepared through physical and chemical processing or compounding with inorganic bone phases (such as hydroxyapatite and tricalcium phosphate). Because these materials are similar to human components, their chemical composition, pore structure, and biodegradability closely match the natural bone environment. They offer excellent cell adhesion, osteogenic induction, and biodegradability, and have been widely used in conventional fracture treatments, arthrodesis, intervertebral fusion, and alveolar bone reconstruction.

[0003] However, existing biomass orthopedic materials still exhibit the following major drawbacks in clinical applications: Most biomass matrices are hydrophilic and porous, containing amino, polyhydroxyl, or peptide functional groups, which readily adsorb plasma proteins and form a "conditioning film" that facilitates bacterial adhesion. Bacteria can colonize and rapidly form a biofilm within 24-48 hours of implantation. Once formed, the biofilm becomes resistant to conventional antibiotics and host immunity, leading to difficult-to-treat infections and revision surgeries. Existing antimicrobial solutions have short efficacy or significant biosafety risks. Directly incorporating antibiotics into bone cement or biomass scaffolds only produces peak release within the first 24-72 hours after implantation, after which antibiotic concentrations rapidly drop to subinhibitory levels, making it difficult to cover the high-risk infection period of 2-4 weeks post-operatively. While coatings with metal ions such as silver and copper can prolong the antibacterial period, excessive metal ions can easily induce cytotoxicity. Furthermore, metal deposition methods often require advanced equipment such as vacuum evaporation and micro-arc oxidation, resulting in high process costs and barriers to entry. Hydrophilic polymers or hydrophobic coatings can reduce initial adhesion, but lack bactericidal function; once bacteria break through the surface barrier, they may still reproduce in the internal pores. A single passive anti-adhesion strategy is difficult to coordinate with active bactericidal treatment, resulting in limited overall anti-infection capabilities. Complex processes such as vacuum deposition, plasma modification, or laser sintering do not meet the equipment conditions of most hospital scientific research platforms, which also increases costs and limits the promotion of technology. Therefore, based on the above problems, it is extremely necessary to develop a long-lasting antibacterial biomass orthopedic material that can achieve early and efficient bactericidal treatment, sustained antibacterial treatment in the later stage, and take into account osteogenic integration in a clinical environment. Summary of the Invention

[0004] In view of the defects of the prior art, the present invention aims to provide a long-lasting antibacterial biomass orthopedic material and its preparation method. In order to solve the problems of too short early antibacterial window, high toxicity of metal ions, and easy adhesion of bacteria to form biofilm on the surface of biomaterials for fracture repair, the present invention proposes to add gallium ions into β-tricalcium phosphate and use its iron ion mimicry antibacterial mechanism to give the material long-lasting antibacterial effect; deposit ZIF-8 porous metal-organic framework on the surface of gallium-doped core by room temperature self-assembly and encapsulate antibiotics, and use Zn 2+ -The imidazole coordination bond constructs a pH-sensitive shell layer, which enables it to quickly disintegrate in the acidified environment caused by infection and release antibiotics on demand, while stably releasing under physiological pH conditions, achieving the technical effects of high-efficiency sterilization in the early stage and controlled endurance in the subsequent stage; through one-time UV polymerization, the sulfobetaine methacrylate brush layer is firmly bonded to the outer surface of the ZIF-8 shell to form a highly hydrophilic anti-adhesion interface, which significantly reduces protein adsorption and initial bacterial colonization, while delaying the loss of antibiotics, achieving the technical effects of long-term surface anti-fouling and synergistically extending the overall antibacterial cycle.

[0005] The technical effects described in the present invention are achieved through the following technical solution: a long-acting antibacterial biomass orthopedic material, whose constituent raw materials include: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, antibiotics, 2-methylimidazole, sulfobetaine methacrylate and a photoinitiator.

[0006] Preferably, the antibiotic is one of vancomycin hydrochloride and cefuroxime sodium; preferably vancomycin hydrochloride; Preferably, the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone; Preferably, another aspect of the present invention provides a method for preparing a biomass orthopedic material, which specifically comprises the following steps: S1: dissolving calcium nitrate tetrahydrate and gallium nitrate in deionized water, stirring and dissolving uniformly, and preheating to 60°C to obtain a mixed solution A; dissolving ammonium dihydrogen phosphate in deionized water, stirring and dissolving uniformly, and preheating to 60°C to obtain a mixed solution B; S2: Slowly add the mixed solution B in step S1 to the equal volume of mixed solution A at a rate of 2-3 mL / min, maintaining a rotation speed of 400-600 rpm during the addition process, and adjust the pH to 9.1-9.3 with 1M ammonia water. After the addition is completed, continue stirring for 2-3 hours to allow precipitation and aging; S3: After completing step S2, filter through a 0.45 μm PES filter, wash three times with deionized water, and vacuum dry at 80°C for 10-16 hours. Then, heat to 550-600°C at a rate of 5°C / min, calcine at this constant temperature for 2-3 hours, cool naturally to room temperature, and sieve through a 100-mesh filter to obtain Ga-doped β-tricalcium phosphate powder. S4: Add fish-derived gelatin to deionized water and stir to dissolve evenly to obtain a 1 wt% collagen slurry; add the Ga-doped β-tricalcium phosphate powder of step S3 to the collagen slurry three times, stir and defoam at 300 rpm for 10 to 20 minutes, pour into a Φ20 mm × 10 mm PTFE mold, cool to -40°C in a vacuum at a rate of 1°C / min, treat for 2 to 3 hours, then heat to 20°C in a vacuum at a rate of 1 to 2°C / min, and treat for 10 to 12 hours to obtain a porous carrier; S5: zinc nitrate hexahydrate and antibiotics were added to deionized water, stirred and dissolved uniformly to obtain a mixed solution C; 2-methylimidazole was dissolved in deionized water, stirred and dissolved uniformly to obtain a mixed solution D; the porous support of step S4 was placed in the mixed solution C, and reduced pressure treatment was carried out at -0.08 MPa for 5 minutes, and three cycles were performed, and then poured into the mixed solution D, gently shaken to ensure uniform infiltration, and allowed to stand at room temperature for 30 to 50 minutes, repeatedly washed with deionized water three times, and air-dried at 40°C for 2 hours to obtain ZIF-8@in situ self-assembly; S6: dissolving sulfobetaine methacrylate and a photoinitiator in a 70 wt % ethanol solution, stirring and dissolving uniformly to obtain a mixed solution E; immersing the ZIF-8@ in situ self-assembled product of step S5 in a 50 wt % ethanol solution for 2 minutes, and then immersing the product in the mixed solution E. After treating for 5 to 8 minutes, the coupon was removed and drained for 1 to 2 minutes, and then placed under a 365 nm LED UV lamp with a turntable at a constant speed of 10 rpm for 5 to 10 minutes, and vacuum dried at 40° C. for 4 to 6 hours, and vacuum dried until the solvent content is less than 1%, thereby obtaining a biomass orthopedic material; Preferably, in step S1, the ratio of the amount of calcium nitrate tetrahydrate, gallium nitrate and deionized water is 25-30 mmol:1.2-2 mmol:100 mL; Preferably, in step S1, the ratio of the amount of ammonium dihydrogen phosphate to deionized water is 25-30 mmol:100 mL; Preferably, in step S4, the ratio of the amount of the Ga-doped β-tricalcium phosphate powder to the amount of the collagen slurry is 1 g: 8-12 mL; Preferably, in step S5, the ratio of the zinc nitrate hexahydrate, antibiotics and deionized water is 4-6 mmol:0.06-0.1 mmol:100 mL; Preferably, in step S5, the ratio of the amount of 2-methylimidazole to deionized water is 8-12 mmol:100 mL; Preferably, in step S6, the ratio of the amount of the sulfobetaine methacrylate, the photoinitiator and the ethanol solution is 1.5-2 g: 0.1-0.12 g: 20 mL; Preferably, in step S6, the power density of the UV lamp is 20 mW / cm 2 .

[0007] The beneficial effects of the present invention are as follows: This paper proposes a three-layered synergistic structure constructed by combining a Ga-doped β-tricalcium phosphate core, a pH-responsive ZIF-8@antibiotic shell, and a photocurable sulfobetaine (pSBMA) brush layer. Specifically, the innermost Ga-doped tricalcium phosphate bone phase serves the dual roles of mechanical support and ion regulation. Within the bone-like microenvironment, Ga ions are gradually released into the surrounding body fluids via a sustained release mechanism. This disrupts the iron metabolism pathways essential for bacterial survival, leading to long-term inhibition of biofilm formation. Furthermore, Ga ions are taken up by osteoblast precursor cells and activate their differentiation signaling pathways, continuously driving bone formation from within the material. The doped lattice maintains stability and moderate solubility, allowing for a naturally extended antibacterial and bone-promoting effect without over-concentration. The intermediate layer self-assembles onto the bone phase surface through room-temperature self-assembly, forming a metal-organic framework shell. Commonly used clinical antibiotics are encapsulated within the framework's pores. This shell is sensitive to ambient pH: under physiological conditions, it maintains structural integrity, allowing for a gradual release of antibiotics via diffusion and gradual hydrolysis of the framework. However, when localized acidification occurs due to bacterial activity, coordination bonds rapidly dissociate, causing the shell to disintegrate, accelerating the release of antibiotics and zinc ions. This provides concentrated sterilization in the early stages of potential infection while avoiding unnecessary drug consumption in the absence of infection. It also utilizes zinc ions to synergistically promote mineralization. The external, photocured sulfobetaine brush layer is extremely hydrophilic and electrically neutral, significantly reducing protein adsorption and initial bacterial adhesion. This brush layer not only directly reduces the probability of surface colonization, but also, due to its permeability, does not excessively block body fluids, allowing the internal framework to still sense environmental pH and respond as needed. This reduced adhesion load delays the framework's dissolution trigger point, indirectly extending the antibacterial window. Simultaneously, the weak interaction between the brush layer and the framework enhances the overall interface stability and reduces the risk of wear and delamination. Multiple structural and chemical couplings exist between the three layers: the porous bone phase provides deep channels, enabling uniform deposition of the framework; the framework's zinc-imidazole backbone and the sulfonyl groups of the brush layer adhere tightly to each other through hydrogen bonds and electrostatic interactions; the brush layer's hydrophilic interface, in turn, regulates the framework's rate of disintegration and release; and gallium and zinc co-operate in osteogenic molecules, enabling parallel, rather than mutually exclusive, antibacterial and bone-promoting signals. The overall preparation process of the present invention adopts three relatively conventional consecutive steps of aqueous co-precipitation, room temperature self-assembly and room temperature photocuring, avoiding high-energy physical deposition or complex surface activation processes, ensuring interlayer compatibility and repeatability; the biomass material finally obtained realizes the triple functions of anti-adhesion barrier, antibiotic burst release and long-term ionic antibacterial at the microscopic level, and maintains biocompatibility at the macroscopic level. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0009] Figure 1 The mechanical properties test results of the biomass materials prepared in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 are shown; Figure 2 This is a graph showing the antibacterial test results of Staphylococcus aureus on the biomass materials prepared in Example 1 and Comparative Examples 1 to 3 of the present invention; Figure 3 This is a graph showing the Escherichia coli antibacterial test results of the biomass materials prepared in Example 1 and Comparative Examples 1 to 3 of the present invention; Figure 4 Zn content of the biomass orthopedic materials prepared in Example 1 and Comparative Examples 1 and 3 under acidic conditions of pH 6.0 2+ Release result graph; Figure 5 Zn content of the biomass orthopedic materials prepared in Example 1 and Comparative Examples 1 and 3 under neutral conditions of pH 7.4 2+ Release result graph; Figure 6 The graph shows the biocompatibility test results of the biomass materials prepared in Example 1 and Comparative Examples 1 to 3 of the present invention; Figure 7 This is a SEM image of the biomass material prepared in Example 1 of the present invention before acid treatment; Figure 8 This is a SEM scanning electron microscope image of the biomass material prepared in Example 1 of the present invention after acid treatment. DETAILED DESCRIPTION

[0010] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. It should be noted that unless otherwise specified, the raw materials involved in the present invention were purchased through conventional commercial channels.

[0011] Example 1: A long-acting antibacterial biomass orthopedic material, the raw materials of which include: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, vancomycin hydrochloride, 2-methylimidazole, sulfobetaine methacrylate and a photoinitiator.

[0012] The preparation of biomass orthopedic materials includes the following steps: S1: Dissolve 80 mmol of calcium nitrate tetrahydrate and 4.5 mmol of gallium nitrate in deionized water, stir and dissolve uniformly, dilute to 300 mL, and preheat to 60°C to obtain a mixed solution A; dissolve 80 mmol of ammonium dihydrogen phosphate in deionized water, stir and dissolve uniformly, dilute to 300 mL, and preheat to 60°C to obtain a mixed solution B; S2: Slowly add the mixed solution B in step S1 to the equal volume of mixed solution A at a rate of 2.5 mL / min, maintaining a rotation speed of 500 rpm during the addition process, and adjust the pH to 9.2 with 1 M ammonia water. After the addition is completed, continue stirring for 2.5 hours to allow precipitation and aging; S3: After completing step S2, the product was filtered through a 0.45 μm PES filter membrane, washed three times with deionized water, and dried under vacuum at 80°C for 12 h. The product was then heated to 600°C at a rate of 5°C / min and calcined at this temperature for 2 h. The product was naturally cooled to room temperature and sieved through a 100-mesh filter to obtain Ga-doped β-tricalcium phosphate powder. S4: 10 g of fish-derived gelatin was added to deionized water, stirred and dissolved evenly, and the volume was adjusted to 100 mL to obtain a 1 wt% collagen slurry; 10 g of Ga-doped β-tricalcium phosphate powder from step S3 was added to 100 mL of collagen slurry three times within 10 minutes, stirred and defoamed at 300 rpm for 15 minutes, poured into a Φ20 mm × 10 mm PTFE mold, and vacuum-cooled to -40°C at a rate of 1°C / min for 2.5 hours, then vacuum-heated to 20°C at a rate of 1.5°C / min for 11 hours to obtain a porous carrier; S5: Add 5 mmol of zinc nitrate hexahydrate and 0.08 mmol of vancomycin hydrochloride to deionized water, stir and dissolve evenly, and dilute to 100 mL to obtain a mixed solution C; dissolve 10 mmol of 2-methylimidazole in 100 mL of deionized water, stir and dissolve evenly to obtain a mixed solution D; place 10 g of the porous support from step S4 in the mixed solution C, treat under reduced pressure at -0.08 MPa for 5 min, perform 3 cycles, then pour into the mixed solution D, shake gently to ensure uniform infiltration, let stand at room temperature for 40 min, repeatedly wash with deionized water 3 times, and air-dry at 40°C for 2 h to obtain ZIF-8@in situ self-assembly; S6: 9 g of sulfobetaine methacrylate and 0.55 g of 2-hydroxy-2-methyl-1-phenyl-1-propanone were dissolved in 70 wt% ethanol solution, stirred and dissolved evenly, and the volume was adjusted to 100 mL to obtain a mixed solution E; the ZIF-8@ in situ self-assembled product of step S5 was immersed in a 50 wt% ethanol solution for 2 min, and then immersed in the mixed solution E. After treating for 6 min, the coupon was removed and drained for 1.5 min, and then placed under a 365 nm LED UV lamp with a power density of 20 mW / cm2 , the turntable was rotated at a constant speed of 10 rpm, irradiated for 8 minutes, and vacuum dried at 40°C for 5 hours. Vacuum dried until the solvent content was less than 1% to obtain biomass orthopedic materials.

[0013] Example 2: A long-acting antibacterial biomass orthopedic material, the raw materials of which include: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, vancomycin hydrochloride, 2-methylimidazole, sulfobetaine methacrylate and a photoinitiator.

[0014] The preparation of biomass orthopedic materials includes the following steps: S1: Dissolve 75 mmol of calcium nitrate tetrahydrate and 3.6 mmol of gallium nitrate in deionized water, stir and dissolve uniformly, dilute to 300 mL, and preheat to 60°C to obtain a mixed solution A; dissolve 75 mmol of ammonium dihydrogen phosphate in deionized water, stir and dissolve uniformly, dilute to 300 mL, and preheat to 60°C to obtain a mixed solution B; S2: Slowly add the mixed solution B in step S1 to the equal volume of mixed solution A at a rate of 2 mL / min, maintaining a rotation speed of 400 rpm during the addition process, and adjust the pH to 9.3 with 1 M ammonia water. After the addition is completed, continue stirring for 3 hours to allow precipitation and aging; S3: After completing step S2, the product was filtered through a 0.45 μm PES filter membrane, washed three times with deionized water, and dried under vacuum at 80°C for 10 h. The product was then heated to 550°C at a rate of 5°C / min and calcined at this temperature for 3 h. The product was naturally cooled to room temperature and sieved through a 100-mesh filter to obtain Ga-doped β-tricalcium phosphate powder. S4: 10 g of fish-derived gelatin was added to deionized water, stirred and dissolved evenly, and the volume was adjusted to 100 mL to obtain a 1 wt% collagen slurry; 10 g of Ga-doped β-tricalcium phosphate powder from step S3 was added to 80 mL of collagen slurry three times within 10 minutes, stirred and defoamed at 300 rpm for 10 minutes, poured into a Φ20 mm × 10 mm PTFE mold, and vacuum-cooled to -40°C at a rate of 1°C / min for 2 hours, then vacuum-heated to 20°C at a rate of 1°C / min for 10 hours to obtain a porous carrier; S5: 4 mmol of zinc nitrate hexahydrate and 0.06 mmol of cefuroxime sodium were added to deionized water, stirred and dissolved uniformly, and the volume was adjusted to 100 mL to obtain a mixed solution C; 8 mmol of 2-methylimidazole was dissolved in deionized water, stirred and dissolved uniformly, and the volume was adjusted to 100 mL to obtain a mixed solution D; 10 g of the porous support from step S4 was placed in the mixed solution C, and the solution was treated at -0.08 MPa under reduced pressure for 5 min, and three cycles were performed. The solution was then poured into the mixed solution D, gently shaken to ensure uniform infiltration, and allowed to stand at room temperature for 30 min. The solution was repeatedly washed with deionized water three times and air-dried at 40°C for 2 h to obtain ZIF-8@in situ self-assembly; S6: Dissolve 7.5 g of sulfobetaine methacrylate and 0.5 g of 2-hydroxy-2-methyl-1-phenyl-1-propanone in 70 wt % ethanol solution, stir and dissolve evenly, and dilute to 100 mL to obtain a mixed solution E; immerse the ZIF-8@ in situ self-assembled sample from step S5 in a 50 wt % ethanol solution for 2 min, and then immerse it in the mixed solution E. After treating for 5 min, remove the coupon and drain for 1 min, then place it under a 365 nm LED UV lamp with a power density of 20 mW / cm 2 , the turntable was rotated at a constant speed of 10 rpm, irradiated for 5 minutes, and vacuum dried at 40°C for 4 hours. Vacuum dried until the solvent content was less than 1% to obtain biomass orthopedic materials.

[0015] Example 3: A long-acting antibacterial biomass orthopedic material, the raw materials of which include: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, vancomycin hydrochloride, 2-methylimidazole, sulfobetaine methacrylate and a photoinitiator.

[0016] The preparation of biomass orthopedic materials includes the following steps: S1: Dissolve 90 mmol of calcium nitrate tetrahydrate and 6 mmol of gallium nitrate in deionized water, stir and dissolve uniformly, dilute to 300 mL, and preheat to 60°C to obtain a mixed solution A; dissolve 90 mmol of ammonium dihydrogen phosphate in deionized water, stir and dissolve uniformly, dilute to 300 mL, and preheat to 60°C to obtain a mixed solution B; S2: Slowly add the mixed solution B in step S1 to the equal volume of mixed solution A at a rate of 3 mL / min, maintaining a rotation speed of 600 rpm during the addition process, and adjust the pH to 9.1 with 1 M ammonia water. After the addition is completed, continue stirring for 2 hours to allow precipitation and aging; S3: After completing step S2, the product was filtered through a 0.45 μm PES filter membrane, washed three times with deionized water, and dried under vacuum at 80°C for 16 h. The product was then heated to 580°C at a rate of 5°C / min and calcined at this temperature for 2.5 h. The product was naturally cooled to room temperature and sieved through a 100-mesh filter to obtain Ga-doped β-tricalcium phosphate powder. S4: 15 g of fish-derived gelatin was added to deionized water, stirred and dissolved evenly, and the volume was adjusted to 150 mL to obtain a 1 wt% collagen slurry; 10 g of Ga-doped β-tricalcium phosphate powder from step S3 was added to 120 mL of collagen slurry three times within 10 minutes, stirred and defoamed at 300 rpm for 20 minutes, poured into a Φ20 mm × 10 mm PTFE mold, and vacuum-cooled to -40°C at a rate of 1°C / min for 3 hours, then vacuum-heated to 20°C at 2°C / min for 12 hours to obtain a porous carrier; S5: 6 mmol zinc nitrate hexahydrate and 0.1 mmol vancomycin hydrochloride were added to deionized water, stirred and dissolved uniformly, and the volume was adjusted to 100 mL to obtain a mixed solution C; 12 mmol 2-methylimidazole was dissolved in deionized water, stirred and dissolved uniformly, and the volume was adjusted to 100 mL to obtain a mixed solution D; 10 g of the porous support from step S4 was placed in the mixed solution C, and the solution was treated at -0.08 MPa under reduced pressure for 5 min, and three cycles were performed. The solution was then poured into the mixed solution D, gently shaken to ensure uniform infiltration, and allowed to stand at room temperature for 50 min. The solution was repeatedly washed with deionized water three times and air-dried at 40°C for 2 h to obtain ZIF-8@in situ self-assembly; S6: Dissolve 10 g of sulfobetaine methacrylate and 0.6 g of 2-hydroxy-2-methyl-1-phenyl-1-propanone in 70 wt % ethanol solution, stir and dissolve evenly, and dilute to 100 mL to obtain a mixed solution E; immerse the ZIF-8@ in situ self-assembled sample from step S5 in a 50 wt % ethanol solution for 2 min, and then immerse it in the mixed solution E. After treating for 8 min, remove the coupon and drain for 2 min, and then place it under a 365 nm LED UV lamp with a power density of 20 mW / cm 2 , the turntable was rotated at a constant speed of 10 rpm, irradiated for 10 min, and vacuum dried at 40°C for 6 h. Vacuum dried until the solvent content was less than 1% to obtain biomass orthopedic materials.

[0017] Comparative Example 1: The operating process parameters of Comparative Example 1 and Example 1 are basically the same. The main difference is that Comparative Example 1 removes the sulfobetaine methacrylate brush layer, that is, the operation of step S6 is removed, and the rest of the process remains unchanged.

[0018] Comparative Example 2: The operating process parameters of Comparative Example 2 and Example 1 are basically the same. The main difference is that the ZIF-8@ in situ self-assembly is removed in Comparative Example 2, that is, the operation of step S5 is removed and the operation of S6 is directly performed. The rest of the process remains unchanged.

[0019] Comparative Example 3: The operating process parameters of Comparative Example 3 and Example 1 are basically the same, the main difference is that gallium doping is removed in Comparative Example 3, that is, gallium nitrate in step S1 is removed, and the rest of the process remains unchanged.

[0020] Performance testing: Mechanical properties test: The biomass orthopedic material samples prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were tested for compressive strength (MPa) and elastic modulus (GPa) according to ISO 5833. Figure 1 shown.

[0021] based on Figure 1The results show that the biomass orthopedic material prepared in the embodiment of the present invention has relatively conventional and stable mechanical properties, and is mainly suitable for filling and repairing bone defects in non-load-bearing or low-load-bearing parts or as a bone tissue engineering scaffold; based on the results of comparative example 1 and embodiment 1, after removing the brush layer, the bonding strength between the ZIF-8 shell and the Ga-doped β-tricalcium phosphate matrix is ​​weakened, and microcracks or debonding are easily generated at the interface under pressure, which may lead to a slight decrease in mechanical properties; based on the results of comparative example 2 and embodiment 1, the ZIF-8 shell serves as an intermediate layer, and its metal-organic framework structure is in the Ga-doped β-tricalcium phosphate bone phase. A layer of nano / micrometer-scale reinforcing phase is formed on the surface. The absence of this layer not only loses its own reinforcing effect, but also destroys the weak interaction between the brush layer and the framework, further weakening the mechanical properties. In addition, in the absence of a ZIF-8 layer, the hydrophilic pSBMA brush layer is directly coated on the hydrophobic Ga-doped β-tricalcium phosphate surface, and the binding force may be weaker, resulting in reduced mechanical properties. Based on the results of Comparative Example 3 and Example 1, the solubility of undoped pure β-tricalcium phosphate is relatively high, and more obvious particle dissolution-reprecipitation may occur during the preparation process, resulting in adverse changes in the microstructure and indirectly weakening the strength.

[0022] Antibacterial test: The biomass orthopedic material samples (10 mm × 5 mm) prepared in Example 1 and Comparative Examples 1 to 3 were immersed in 10 mL of sterile PBS buffer and shaken at 60 rpm at 37 ° C for 28 days. The PBS buffer was replaced every 24 h for the first 14 days and the old immersion solution was collected and frozen (-20 ° C) for later use. The samples were taken out on the 0th day, the 7th day and the 14th day, and gently washed once with PBS. The samples were transferred to a new bacterial suspension (1×10 Staphylococcus aureus and 1×10 Escherichia coli) and the samples were washed with PBS. 6 CFU / mL suspension), record the sterilization rate (%), the sterilization rate results of Staphylococcus aureus and Escherichia coli are as follows Figure 2 and Figure 3 From the 14th to the 28th day, the PBS buffer was replaced every 48 hours and the old immersion solution was collected and frozen (-20°C) for later use. On the 21st and 28th day, the samples were taken out, gently washed once with PBS, and transferred to the new bacterial suspension (1×10 Staphylococcus aureus and 1×10 Escherichia coli). 6 CFU / mL suspension), repeat the 24h contact sterilization process, record the sterilization rate (%), each batch of samples was tested three times independently, and the test results were averaged. The results for Staphylococcus aureus and Escherichia coli were as follows: Figure 2 and Figure 3 As shown; The specific test steps for the sterilization rate are as follows: take Staphylococcus aureus and Escherichia coli and suspend them in TSB medium to 1×10 6CFU / mL, and a suspension was obtained for later use; the biomass orthopedic material samples (10 mm × 5 mm) prepared in Example 1 and Comparative Examples 1 to 3 were placed in a 36-well plate; 1 mL of inoculum was added to each well and the plates were allowed to stand at 37°C for 24 h; 100 μL of the mixture was serially diluted and applied to a TSA plate, the CFU before inoculation was recorded, and the sterilization rate (%) was calculated as (bacterial CFU before inoculation - bacterial CFU after inoculation) / bacterial CFU before inoculation × 100%.

[0023] based on Figure 2 and Figure 3 The results showed that the biomass orthopedic material prepared in the embodiment of the present invention exerted excellent antibacterial properties through multi-level synergistic effects, and maintained a sterilization rate of more than 95% during long-term use for 28 days. Based on the results of Comparative Example 1 and Example 1, the early sterilization rate of Comparative Example 1 was excellent, but it declined rapidly from the 7th to the 14th day; there was a significant attenuation in the middle and late stages. This may be due to the absence of the pSBMA brush layer, the surface of the material may be quickly contaminated by proteins and bacterial exocytic polymers, the initial amount of adherent bacteria is high, the biofilm matures earlier, and the protein fouling film will partially adsorb / intercept the antibiotics released in the early stage, reducing their effective diffusion. The tight matrix of the mature biofilm will also affect the subsequent Ga 3+ / Zn 2+ The extravasation forms a diffusion barrier. Due to the faster accumulation of bacteria, local acidification is more likely to occur, and ZIF-8 is disintegrated in large quantities in advance. The antibiotics may be quickly consumed in the early stage. After losing the sustained-release effect of the brush layer, the risk of rapid release of antibiotics in the 0-14th day increases significantly. During the 14-28th day, the material basically relies on the lower speed of Ga 3+ play an effect; however, the aforementioned biofilm barrier in turn weakens Ga 3+ The spread of the virus has a significant impact on its later effects.

[0024] Based on the results of Comparative Example 2 and Example 1, the early bactericidal rate of Comparative Example 2 was slightly lower than that of Example 1, and the overall bactericidal rate decreased linearly; this may be because the pSBMA brush layer was retained, which could still effectively reduce the initial adhesion amount and protein fouling film, and promote the continuous diffusion and action of ions, but the antibiotic layer was missing. The entire system lacked an on-demand burst release mechanism, had no sustained release of antibiotics, and the antibacterial effect depended on Ga 3+ Steady-state release and physical load reduction of the brush layer, while Ga 3+ It is difficult to achieve the instantaneous concentration required for high-intensity sterilization within 24 hours, resulting in a low sterilization rate on day 0.

[0025] Based on the results of Comparative Example 3 and Example 1, the sterilization rate of Comparative Example 3 is relatively excellent in the early and middle stages, but the sterilization rate in the middle and late stages shows a cliff-like decline; this may be due to the lack of Ga 3+ The steady-state long-term effect, when the antibiotics are gradually exhausted, the remaining antibacterial factors in the system are mainly Zn2+ (from ZIF-8 framework) and the anti-adhesion effect of pSBMA; Zn 2+ The concentration and persistence of Ga 3+ , and its sustained antibacterial effect on certain bacteria, especially Staphylococcus aureus, is even worse.

[0026] Triggered sustained-release test: The biomass orthopedic material samples (100 mg) prepared in Example 1 and Comparative Examples 1 and 3 (Comparative Example 2 does not contain zinc ions and is not tested) were placed in PBS buffer at pH 7.4 and MES buffer at pH 6.0, respectively. Both buffers contained 0.1% Tween-80. The samples were shaken at 60 rpm at 37°C. 0.5 mL of the immersion solution was extracted at 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h, and an equal amount of new buffer was added to calculate the Zn 2+ The percentage sustained-release curve was obtained by performing three independent tests on each batch of samples and taking the average value of the test results. Figure 4 and Figure 5 shown.

[0027] based on Figure 4 and Figure 5 The results showed that the biomass orthopedic material prepared in the embodiment of the present invention exhibited obvious pH-responsive drug release characteristics, showing sustained release under a physiological neutral environment (pH 7.4), and triggering a burst release of antibiotics under acidic microenvironment conditions (pH 6.0); based on the results of Comparative Example 1 and Example 1, the lack of the pSBMA brush layer in Comparative Example 1 resulted in direct exposure of ZIF-8 to the solution after acidification, resulting in faster disintegration and a steeper front curve; based on the results of Comparative Example 3 and Example 1, the effect of gallium doping in Comparative Example 3 on the acid disintegration kinetics of ZIF-8 itself was small, and the difference in burst release effect from Example 1 was small.

[0028] Biocompatibility test: Take the frozen old extracts extracted on the 1st, 3rd, 7th, 14th and 28th day in the antibacterial test, filter out the suspended particles, and obtain sterile clarified extracts. Then, these extracts were added to the cell culture system, and human bone marrow stromal cells were used for 48h CCK-8 cell viability detection and fluorescent live-dead staining. The control was TSB medium without extraction (negative control) and TSB medium containing 10% dimethyl sulfoxide (positive control). The survival rate (%) was calculated as follows: (OD value of the example - OD value of the negative control group) / (OD value of the negative control group - OD value of the positive control group) × 100%. Each batch of samples was tested three times independently, and the test results were averaged. The results are shown in the figure. Figure 6 shown.

[0029] based on Figure 6The results showed that the biomass orthopedic materials prepared in the examples of the present invention showed relatively excellent biocompatibility and could ensure long-term effective implantation in the human body. The cell activity of the biomass orthopedic materials prepared in the examples was close to that of the negative control group in the early stage, while it was slightly higher than that of the negative control group in the later stage. This may be because ZIF-8 only slowly released vancomycin and Zn in neutral PBS. 2+ The concentration is mostly below the osteoblast toxicity threshold; the pSBMA brush layer does not directly enter the extract and has no adverse effects on cells; Ga 3+ Steady-state low-dose extravasation resulted in overall cell viability close to that of the negative control; in the middle and late stages, antibiotic concentrations were further reduced, and Ga 3+ / Zn 2+ The osteogenic signal may gradually appear, and the cell activity is slightly better than that of the negative control group; based on the analysis of the results of Comparative Example 1 and Example 1, the absence of the brush layer makes the early antibiotics and Zn 2+ The extravasation is faster. Although disintegration is not triggered under neutral conditions, the diffusion resistance is significantly reduced. The instantaneous peak of the dissolved matter in the extract may be slightly higher. The cell viability is most likely to decrease slightly in the early stage. In the middle and late stages, with the rapid release of soluble components, the toxicity burden of the late extract is reduced, and the activity is significantly improved compared with the early stage. Based on the results of Comparative Example 2 and Example 1, there is a lack of sudden release of antibiotics. The extract is mainly composed of low-dose Ga 3+ , Ga 3+ In the low dose range, it has a certain effect on promoting proliferation / differentiation of MSCs. Therefore, the cell activity throughout the whole process is close to or slightly higher than that of the negative control. Based on the results of Comparative Example 3 and Example 1, the lack of Ga 3+ The osteogenic effect of the neutral PBS still contains a small amount of vancomycin and Zn 2+ , the cell activity slightly decreased in the early stage; after the antibiotics decayed in the middle and late stages, Zn 2 + The mild osteogenic effect of β-catenin may slightly enhance cell activity.

[0030] Spectrum test: Scanning electron microscopy was used to characterize the surface morphology of the biomass orthopedic material prepared in Example 1 before and after acid treatment in the triggered sustained release test. The microstructural changes were as follows: Figure 7 and Figure 8 shown.

[0031] based on Figure 7 and Figure 8 The results analysis, Figure 7 This is the SEM of the sample without acid treatment, showing uniformly distributed polyhedral particles on the surface, which are identified as ZIF-8 crystals, proving that the intermediate layer was successfully deposited in situ; Figure 8This is the SEM image after treatment at pH 6.0 for 24 hours. The original crystal particles basically disappeared and etched residues appeared, indicating that the ZIF-8 shell quickly disintegrated in an acidic environment and released guest molecules.

[0032] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A long-lasting antibacterial biomass orthopedic material, characterized in that: The raw materials include: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, antibiotics, 2-methylimidazole, sulfobetaine methacrylate and photoinitiator.

2. The long-lasting antibacterial biomass orthopedic material according to claim 1, characterized in that: The photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone.

3. The long-lasting antibacterial biomass orthopedic material according to claim 2, characterized in that: The antibiotic is one of vancomycin hydrochloride and cefuroxime sodium.

4. A method for preparing a long-acting antibacterial biomass orthopedic material according to any one of claims 1 to 3, characterized in that: The specific steps include: S1: dissolving calcium nitrate tetrahydrate and gallium nitrate in deionized water, stirring and dissolving them uniformly, and preheating them to obtain a mixed solution A; dissolving ammonium dihydrogen phosphate in deionized water, stirring and dissolving them uniformly, and preheating them to obtain a mixed solution B; S2: slowly adding the mixed solution B in step S1 dropwise to the mixed solution A, stirring continuously during the addition process, and adjusting the pH with aqueous ammonia. After the addition is complete, stirring is continued for precipitation and aging treatment; S3: After completing step S2, filter the mixture through a membrane, repeatedly wash with deionized water, and vacuum dry. Then, heat the mixture, calcine the mixture at a constant temperature, cool the mixture naturally to room temperature, and screen the mixture to obtain Ga-doped β-tricalcium phosphate powder. S4: adding fish-derived gelatin into deionized water, stirring and dissolving uniformly to obtain a collagen slurry; adding the Ga-doped β-tricalcium phosphate powder of step S3 into the collagen slurry three times, stirring and removing bubbles, pouring into a mold, slowly cooling and freezing, and then slowly heating in a vacuum to obtain a porous carrier; S5: zinc nitrate hexahydrate and antibiotics were added to deionized water and stirred to dissolve uniformly to obtain a mixed solution C; 2-methylimidazole was dissolved in deionized water and stirred to dissolve uniformly to obtain a mixed solution D; the porous support in step S4 was placed in the mixed solution C, decompressed for 3 cycles, and then poured into the mixed solution D, gently shaken to ensure uniform infiltration, allowed to stand at room temperature, repeatedly washed with deionized water, and air-dried to obtain ZIF-8@in situ self-assembly; S6: Dissolve sulfobetaine methacrylate and a photoinitiator in an ethanol solution, stir and dissolve evenly to obtain a mixed solution E; immerse the ZIF-8@ in situ self-assembled product of step S5 in the ethanol solution, and then immerse it in the mixed solution E. After treatment, remove the hanging piece and drain the liquid, then place it under an LED ultraviolet lamp, rotate the turntable at a constant speed, irradiate and cure, and vacuum dry to obtain a biomass orthopedic material.

5. A method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 4, characterized in that: In step S1, the ratio of the calcium nitrate tetrahydrate, gallium nitrate and deionized water is 25-30 mmol:1.2-2 mmol:100 mL.

6. A method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 5, characterized in that: In step S1, the ratio of the amount of ammonium dihydrogen phosphate to deionized water is 25-30 mmol:100 mL.

7. A method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 6, characterized in that: In step S4, the ratio of the Ga-doped β-tricalcium phosphate powder to the collagen slurry is 1 g:8-12 mL.

8. A method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 7, characterized in that: In step S5, the ratio of the zinc nitrate hexahydrate, the antibiotic and the deionized water is 4-6 mmol:0.06-0.1 mmol:100 mL.

9. A method for preparing a long-acting antibacterial biomass orthopedic material according to claim 8, characterized in that: In step S5, the ratio of the amount of 2-methylimidazole to deionized water is 8-12 mmol:100 mL.

10. A method for preparing the long-acting antibacterial biomass orthopedic material according to claim 9, characterized in that: In step S6, the ratio of the amount of sulfobetaine methacrylate, photoinitiator and ethanol solution is 1.5-2g:0.1-0.12g:20mL; the power density of the ultraviolet lamp is 20mW / cm 2 .

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