Long-acting antibacterial biomass orthopedic material and preparation method thereof

Abstract

The application 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 raw materials of the application comprise calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, an antibiotic, 2-methylimidazole, sulfobetaine methacrylate and a photoinitiator. The application constructs a three-level synergistic structure through Ga-doped beta-tricalcium phosphate core / pH-responsive ZIF-8@antibiotic shell / photocured sulfobetaine (pSBMA) brush layer. The process adopts three continuous steps of aqueous coprecipitation, room temperature self-assembly and room temperature photocuring, avoids high-energy physical deposition or a complex surface activation process, and guarantees the compatibility and repeatability between layers. The final obtained biomass material realizes the triple functions of anti-adhesion barrier, drug burst release and long-term ion antibacterial on the micro level, and maintains biocompatibility on the macro level.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a long-acting antibacterial biomass orthopedic material and a preparation method thereof. BACKGROUND

[0002] The biomass orthopedic material refers to a bone defect filler, a scaffold or an internal fixation member prepared by using natural bone tissue, animal-derived collagen, chitosan, silk fibroin, alginate, bacterial cellulose and degradable polymers obtained by polymerization of fermentation or bio-based monomers as raw materials, and then performing physical-chemical treatment or compounding with inorganic bone phases (hydroxyapatite, tricalcium phosphate and the like).

[0003] However, the existing biomass orthopedic material still has the following main defects in clinical application: most of the biomass matrix has a hydrophilic porous structure, contains amino, polyhydroxy or peptide chain functional groups, and is easy to adsorb plasma proteins and form a "conditioning film" conducive to bacterial adhesion. Bacteria can be colonized and quickly form a biofilm within 24-48 hours after implantation, and once the biofilm is formed, it will resist conventional antibiotics and host immunity, leading to refractory infection and revision surgery. The existing antibacterial solutions are short in time or have great biological safety hazards. Direct incorporation of antibiotics into bone cement or biomass scaffolds can only produce a peak release within 24-72 hours after implantation, and then the antibiotic concentration rapidly decreases to a sub-inhibitory level, which is difficult to cover the high-risk period of infection 2-4 weeks after surgery. Although silver, copper and other metal ion coatings can prolong the antibacterial time, excessive metal ions can easily cause cytotoxicity, and the metal deposition method often requires high-end equipment such as vacuum evaporation, micro-arc oxidation, which has high process cost and threshold. Hydrophilic polymers or hydrophobic coating layers can reduce initial adhesion, but lack bactericidal function; once bacteria break through the surface barrier, they can still reproduce in the internal pores. A single passive anti-adhesion strategy is difficult to coordinate with active sterilization, resulting in limited overall anti-infection ability. Complex processes such as vacuum deposition, plasma modification or laser sintering do not meet the equipment conditions of most hospital research platforms, and also increase the cost, limiting the popularization of the technology. Therefore, based on the above problems, it is extremely necessary to develop a long-acting antibacterial biomass orthopedic material that can achieve early and efficient sterilization, sustained antibacterial activity in the later period, and bone integration. SUMMARY

[0004] In view of the defects of the prior art, the purpose of the present application is to provide a long-acting antibacterial biomass orthopedic material and a preparation method thereof. In order to solve the problems of short early antibacterial window, high metal ion toxicity and easy bacterial adhesion to form biofilm of the biomaterial for fracture repair, the present application proposes to incorporate gallium ions into beta-tricalcium phosphate by heterovalent doping, utilize the bacteriostatic mechanism of iron ion mimic state to endow the material with long-acting antibacterial property; deposit ZIF-8 porous metal-organic framework and load antibiotics on the surface of the gallium-doped core by room temperature self-assembly, utilize the Zn 2+ The imidazole coordination bond constructs a pH-sensitive shell layer, which rapidly disintegrates under the acidification environment caused by infection to release antibiotics on demand, and is stable and slow-releasing under physiological pH conditions, achieving the technical effects of efficient sterilization in the early stage and subsequent controlled cruising; the sulfobetaine methacrylate brush layer is firmly bonded to the outer surface of the ZIF-8 shell by one-time ultraviolet polymerization, forming a highly hydrophilic anti-adhesion interface, significantly reducing protein adsorption and initial colonization of bacteria, and delaying the loss of antibiotics, achieving the technical effects of long-term anti-staining on the surface and synergistically prolonging the overall antibacterial period.

[0005] The technical effects of the present application are achieved by the following technical scheme: a long-acting antibacterial biomass orthopedic material, which comprises the following raw materials: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, antibiotic, 2-methyl imidazole, sulfobetaine methacrylate and photoinitiator.

[0006] Preferably, the antibiotic is one of vancomycin hydrochloride and cefuroxime sodium; preferably vancomycin hydrochloride;

[0007] Preferably, the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone;

[0008] Preferably, another aspect of the present application provides a preparation method of a biomass orthopedic material, which specifically comprises the following steps:

[0009] S1: Dissolve calcium nitrate tetrahydrate and gallium nitrate in deionized water, stir to dissolve uniformly, preheat to 60 DEG C, and obtain a mixed solution A; dissolve ammonium dihydrogen phosphate in deionized water, stir to dissolve uniformly, preheat to 60 DEG C, and obtain a mixed solution B;

[0010] S2: Slowly add the mixed solution B in step S1 to the mixed solution A of equal volume at a speed of 2-3 mL / min, keep the stirring speed at 400-600 rpm during the adding process, and adjust the pH to 9.1-9.3 with 1M ammonia water; continue stirring for 2-3 h after the adding is completed to precipitate and age;

[0011] S3: After the operation of step S2 is completed, 0.45 μm PES filter membrane suction filtration, deionized water repeated washing 3 times, 80℃ vacuum drying 10-16h, then heated to 550-600℃ at a rate of 5℃ / min, constant temperature calcination 2-3h, natural cooling to room temperature, over 100 mesh sieve, Ga-doped β-tricalcium phosphate powder is obtained;

[0012] S4: Fish gelatin is added to deionized water, stirred and dissolved uniformly to obtain a 1wt% collagen slurry; Ga-doped β-tricalcium phosphate powder of step S3 is added to the collagen slurry in three times, 300rpm stirring and defoaming treatment for 10-20min, then poured into Φ20mm×10mm PTFE mold, vacuum cooling to-40℃ at a rate of 1℃ / min, treatment for 2-3h, then vacuum heating to 20℃ at a rate of 1-2℃ / min, treatment for 10-12h, a porous carrier is obtained;

[0013] S5: Zinc nitrate hexahydrate and antibiotics are added to deionized water, stirred and dissolved uniformly to obtain a mixed solution C; 2-methylimidazole is dissolved in deionized water, stirred and dissolved uniformly to obtain a mixed solution D; the porous carrier of step S4 is placed in the mixed solution C, under-0.08MPa reduced pressure treatment for 5min, 3 cycles are performed, then poured into the mixed solution D, light shaking to ensure uniform infiltration, room temperature standing for 30-50min, deionized water repeated washing 3 times, 40℃ air drying for 2h, ZIF-8@in-situ self-assembly is obtained;

[0014] S6: Sulfobetaine methacrylate and photoinitiator are dissolved in 70wt% ethanol solution, stirred and dissolved uniformly to obtain a mixed solution E; ZIF-8@in-situ self-assembly of step S5 is immersed in 50wt% ethanol solution for 2min, then immersed in the mixed solution E, after treatment for 5-8min, the hanging piece is taken out and drained for 1-2min, then placed under 365nm LED ultraviolet lamp, the turntable rotates at a uniform speed of 10rpm, irradiation for 5-10min, 40℃ vacuum drying for 4-6h, vacuum drying until the solvent content is less than 1%, biomass orthopedic material is obtained;

[0015] Preferably, in step S1, the ratio of the use amount of calcium nitrate tetrahydrate, gallium nitrate and deionized water is 25-30mmol:1.2-2mmol:100mL;

[0016] Preferably, in step S1, the ratio of the use amount of ammonium dihydrogen phosphate and deionized water is 25-30mmol:100mL;

[0017] Preferably, in step S4, the ratio of the use amount of Ga-doped β-tricalcium phosphate powder and collagen slurry is 1g:8-12mL;

[0018] Preferably, in step S5, the ratio of the amounts of zinc nitrate hexahydrate, antibiotic and deionized water is 4-6 mmol: 0.06-0.1 mmol: 100 mL;

[0019] Preferably, in step S5, the ratio of the amounts of 2-methylimidazole and deionized water is 8-12 mmol: 100 mL;

[0020] Preferably, in step S6, the ratio of the amounts of sulfobetaine methacrylate, photoinitiator and ethanol solution is 1.5-2 g: 0.1-0.12 g: 20 mL;

[0021] Preferably, in step S6, the power density of the ultraviolet lamp is 20 mW / cm 2 .

[0022] The beneficial effects of the present application are as follows:

[0023] The present application proposes to construct a three-level synergistic structure by Ga-doped β-tricalcium phosphate core / pH-responsive ZIF-8@antibiotic shell / photocured sulfobetaine (pSBMA) brush layer. Specifically, the innermost layer of gallium-doped tricalcium phosphate bone phase assumes the dual roles of mechanical support and ion regulation, and gallium ions gradually enter the surrounding body fluid in a slow-release manner in the bone-like microenvironment, on the one hand, disrupting the iron metabolism pathway on which bacteria survive, and long-term inhibiting biofilm formation; on the other hand, they can be taken up by osteogenic precursor cells and activate their differentiation signaling pathways, continuously driving the bone formation process from the inside of the material. The lattice of the doped material is stable and has moderate solubility, so that the antibacterial and bone-promoting effects naturally extend in time without excessive concentration. The middle layer is a metal-organic framework shell formed on the surface of the bone phase by room temperature self-assembly, and a commonly used clinical antibiotic is loaded in the framework pores. The shell is sensitive to environmental pH: under physiological conditions, the structure remains intact, and the antibiotic is released in a slow-diffusion-framework hydrolysis manner; when the local environment is acidified due to bacterial activity, the coordination bond rapidly dissociates, the shell collapses, and the release of antibiotics and zinc ions is accelerated. It provides concentrated sterilization in the early stage of potential infection, avoids unnecessary drug consumption in the absence of infection, and simultaneously utilizes zinc ions to synergistically promote mineralization. The outer sulfobetaine brush layer formed by photocuring is extremely hydrophilic and electrically neutral, which can greatly weaken protein adsorption and initial bacterial adhesion. The brush layer not only directly reduces the probability of surface colonization, but also does not excessively block the body fluid due to its permeability, so that the internal framework can still sense the environmental pH and respond as needed, reducing the adhesion load to indirectly extend the antibacterial window; at the same time, the weak interaction between the brush layer and the framework improves the stability of the overall interface and reduces the risk of wear and delamination. There are multiple couplings between the three layers in structure and chemistry: the porous bone phase provides deep pores, allowing the framework to be uniformly deposited; the zinc-imidazole framework of the framework and the sulfogroup of the brush layer are closely attached through hydrogen bonding and electrostatic interaction, and the hydrophilic interface of the brush layer in turn adjusts the framework disintegration release rate; gallium and zinc jointly participate in osteogenic molecules, making the antibacterial and bone-promoting signals parallel rather than mutually exclusive. The overall preparation process of the present application adopts three consecutive steps of aqueous coprecipitation, room temperature self-assembly and room temperature photocuring, avoiding high-energy physical deposition or complex surface activation processes, ensuring layer compatibility and repeatability; ultimately, the biological material obtained realizes the triple functions of anti-adhesion barrier, antibiotic burst release and long-term ion antibacterial on the microscale, and maintains biocompatibility on the macroscale. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only illustrate the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1 The mechanical property test results of the biomass material prepared in Example 1-3 and Comparative Example 1-3 of the present application are shown in the following figures:

[0026] Figure 2 The staphylococcus aureus inhibition test results of the biomass material prepared in Example 1 and Comparative Example 1-3 of the present application are shown in the following figures:

[0027] Figure 3 The escherichia coli inhibition test results of the biomass material prepared in Example 1 and Comparative Example 1-3 of the present application are shown in the following figures:

[0028] Figure 4 The Zn2+ release results of the biomass orthopedic material prepared in Example 1 and Comparative Example 1, 3 of the present application under pH 6.0 acidic conditions are shown in the following figures: 2+

[0029] The Zn2+ release results of the biomass orthopedic material prepared in Example 1 and Comparative Example 1, 3 of the present application under pH 7.4 neutral conditions are shown in the following figures: 2+ Figure 5 The biocompatibility test results of the biomass material prepared in Example 1 and Comparative Example 1-3 of the present application are shown in the following figures:

[0030] Figure 6 The SEM scanning electron microscope graph of the biomass material prepared in Example 1 of the present application before acid treatment is shown in the following figure:

[0031] Figure 7 The SEM scanning electron microscope graph of the biomass material prepared in Example 1 of the present application after acid treatment is shown in the following figure.

[0032] DETAILED DESCRIPTION Figure 8 The technical solutions of the present application will be described in detail below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. It should be noted that, unless otherwise specified, the raw materials involved in the present application are purchased through conventional commercial channels.

[0033] Example 1: A long-acting antibacterial biomass orthopedic material, which is composed of the following raw materials: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, vancomycin hydrochloride, 2-methylimidazole, sulfobetaine methacrylate, and photoinitiator.

[0034] The preparation of the biomass orthopedic material includes the following steps:

[0035] The preparation of the biomass orthopedic material includes the following steps:

[0036] ​S1: 80 mmol of calcium nitrate tetrahydrate and 4.5 mmol of gallium nitrate were dissolved in deionized water, stirred to dissolve uniformly, made up to 300 mL, preheated to 60°C, and a mixed solution A was obtained; 80 mmol of ammonium dihydrogen phosphate was dissolved in deionized water, stirred to dissolve uniformly, made up to 300 mL, preheated to 60°C, and a mixed solution B was obtained;

[0037] S2: The mixed solution B in step S1 was slowly added to an equal volume of mixed solution A at a rate of 2.5 mL / min, the stirring speed was maintained at 500 rpm during the addition, and the pH was adjusted to 9.2 with 1M ammonia water. After the addition was completed, the stirring was continued for 2.5 h for precipitation aging;

[0038] S3: After the completion of step S2, 0.45 μm PES filter membrane was used for suction filtration, deionized water was used for repeated washing 3 times, and 80°C vacuum drying was performed for 12 h. Then, the temperature was increased to 600°C at a rate of 5°C / min, and constant temperature calcination was performed for 2 h. The natural cooling was performed to room temperature, and 100 mesh screening was performed, and Ga-doped β-tricalcium phosphate powder was obtained;

[0039] S4: 10 g of fish-derived gelatin was added to deionized water, stirred to dissolve uniformly, made up to 100 mL, and a 1 wt% collagen slurry was obtained. 10 g of Ga-doped β-tricalcium phosphate powder in step S3 was added to 100 mL of collagen slurry in three times within 10 min, and 300 rpm stirring was performed for 15 min for bubble removal. Then, it was poured into a Φ20 mm x 10 mm PTFE mold, vacuum cooling was performed to -40°C at a rate of 1°C / min, and treatment was performed for 2.5 h. Then, vacuum warming was performed to 20°C at a rate of 1.5°C / min, and treatment was performed for 11 h, and a porous carrier was obtained;

[0040] S5: 5 mmol of zinc nitrate hexahydrate and 0.08 mmol of vancomycin hydrochloride were added to deionized water, stirred to dissolve uniformly, made up to 100 mL, and a mixed solution C was obtained. 10 mmol of 2-methylimidazole was dissolved in 100 mL of deionized water, stirred to dissolve uniformly, and a mixed solution D was obtained. 10 g of the porous carrier in step S4 was placed in the mixed solution C, and -0.08 MPa reduced pressure treatment was performed for 5 min for 3 cycles. Then, it was poured into the mixed solution D, light shaking was performed to ensure uniform immersion, and room temperature standing was performed for 40 min. Deionized water was used for repeated washing 3 times, and 40°C air drying was performed for 2 h, and ZIF-8@in-situ self-assembly was obtained;

[0041] S6: 9 g sulfobetaine methacrylate and 0.55 g 2-hydroxy-2-methyl-1-phenyl-1- propanone were dissolved in a 70 wt% ethanol solution, stirred to dissolve uniformly, and diluted to 100 mL to obtain a mixed solution E; the ZIF-8@in-situ self-assembly of step S5 was immersed in a 50 wt% ethanol solution for 2 min, then immersed in the mixed solution E, and after 6 min of treatment, the hanging piece was drained for 1.5 min, then placed under a 365 nm LED ultraviolet lamp, with a power density of 20 mW / cm 2 , the turntable was uniformly rotated at 10 rpm, irradiated for 8 min, and vacuum dried at 40°C for 5 h to obtain a biomass orthopedic material with a solvent content of less than 1%.

[0042] Example 2: A long-acting antibacterial biomass orthopedic material, which is composed of the following raw materials: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, vancomycin hydrochloride, 2-methylimidazole, sulfobetaine methacrylate, and a photoinitiator.

[0043] The preparation of the biomass orthopedic material includes the following steps:

[0044] S1: 75 mmol of calcium nitrate tetrahydrate and 3.6 mmol of gallium nitrate were dissolved in deionized water, stirred to dissolve uniformly, and diluted to 300 mL, and preheated to 60°C to obtain a mixed solution A; 75 mmol of ammonium dihydrogen phosphate was dissolved in deionized water, stirred to dissolve uniformly, and diluted to 300 mL, and preheated to 60°C to obtain a mixed solution B;

[0045] S2: The mixed solution B in step S1 was slowly added to an equal volume of mixed solution A at a rate of 2 mL / min, the stirring speed was maintained at 400 rpm during the addition, and the pH was adjusted to 9.3 with 1M ammonia water, and after the addition was completed, the stirring was continued for 3 h for precipitation aging;

[0046] S3: After the completion of step S2, the 0.45 μm PES filter membrane was suction filtered, washed with deionized water for 3 times, vacuum dried at 80°C for 10 h, then heated to 550°C at a rate of 5°C / min, and kept at a constant temperature for 3 h, then naturally cooled to room temperature, and sieved through a 100 mesh sieve to obtain Ga-doped β-tricalcium phosphate powder;

[0047] S4: 10 g of fish-derived gelatin was added to deionized water, stirred to dissolve uniformly, and diluted to 100 mL to obtain a 1 wt% collagen slurry; 10 g of Ga-doped β-tricalcium phosphate powder of step S3 was added to 80 mL of collagen slurry in three portions within 10 min, and after 10 min of stirring at 300 rpm to remove bubbles, it was poured into a Φ20 mm x 10 mm PTFE mold, vacuum cooled to -40°C at a rate of 1°C / min for 2 h, then vacuum heated to 20°C at a rate of 1°C / min for 10 h to obtain a porous carrier.

[0048] S5: 4 mmol of zinc nitrate hexahydrate and 0.06 mmol of cefuroxime sodium were added to deionized water, stirred and dissolved uniformly, and diluted to 100 mL to obtain a mixed solution C; 8 mmol of 2-methylimidazole was dissolved in deionized water, stirred and dissolved uniformly, and diluted to 100 mL to obtain a mixed solution D; 10 g of the porous carrier of step S4 was placed in the mixed solution C, treated under reduced pressure of -0.08 MPa for 5 min, 3 cycles were performed, then poured into the mixed solution D, gently shaken to ensure uniform immersion, and placed at room temperature for 30 min; deionized water was repeatedly washed for 3 times, and air-dried at 40°C for 2 h to obtain ZIF-8@in-situ self-assembly;

[0049] S6: 7.5 g of sulfobetaine methacrylate and 0.5 g of 2-hydroxy-2-methyl-1-phenyl-1-propanone were dissolved in a 70 wt% ethanol solution, stirred and dissolved uniformly, and diluted to 100 mL to obtain a mixed solution E; the ZIF-8@in-situ self-assembly of step S5 was immersed in a 50 wt% ethanol solution for 2 min, then immersed in the mixed solution E, treated for 5 min, taken out and drained for 1 min, then placed under a 365 nm LED ultraviolet lamp, the power density was 20 mW / cm 2 , the turntable was uniformly rotated at 10 rpm, irradiated for 5 min, and vacuum dried at 40°C for 4 h to obtain a biomass orthopedic material with a solvent content of less than 1%.

[0050] Example 3: A long-acting antibacterial biomass orthopedic material, which is composed of the following raw materials: calcium nitrate tetrahydrate, ammonium dihydrogen phosphate, gallium nitrate, zinc nitrate hexahydrate, vancomycin hydrochloride, 2-methylimidazole, sulfobetaine methacrylate, and a photoinitiator.

[0051] The preparation of the biomass orthopedic material includes the following steps:

[0052] S1: 90 mmol of calcium nitrate tetrahydrate and 6 mmol of gallium nitrate were dissolved in deionized water, stirred and dissolved uniformly, and diluted to 300 mL, and preheated to 60°C to obtain a mixed solution A; 90 mmol of ammonium dihydrogen phosphate was dissolved in deionized water, stirred and dissolved uniformly, and diluted to 300 mL, and preheated to 60°C to obtain a mixed solution B;

[0053] S2: The mixed solution B in step S1 was slowly added to an equal volume of the mixed solution A at a rate of 3 mL / min, the stirring speed was maintained at 600 rpm during the addition, and the pH was adjusted to 9.1 with 1M ammonia water; after the addition was completed, the stirring was continued for 2 h for precipitation aging;

[0054] S3: After the operation of step S2 is completed, 0.45 μm PES filter membrane is used for suction filtration, deionized water is used for repeated washing for 3 times, 80℃ vacuum drying is performed for 16h, then the temperature is increased to 580℃ at a speed of 5℃ / min, constant temperature calcination is performed for 2.5h, natural cooling to room temperature, 100 mesh screening, Ga-doped β-tricalcium phosphate powder is obtained;

[0055] S4: 15g fish gelatin is added into deionized water, stirred and dissolved uniformly, and then diluted to 150mL to obtain a 1wt% collagen slurry; 10g Ga-doped β-tricalcium phosphate powder of step S3 is added into 120mL collagen slurry in three times within 10min, 300rpm stirring is performed for 20min to remove bubbles, then poured into a Φ20mm×10mm PTFE mold, vacuum cooling to-40℃ at a speed of 1℃ / min, processing for 3h, then vacuum heating to 20℃ at a speed of 2℃ / min, processing for 12h, and a porous carrier is obtained;

[0056] S5: 6mmol zinc nitrate hexahydrate and 0.1mmol vancomycin hydrochloride are added into deionized water, stirred and dissolved uniformly, and then diluted to 100mL to obtain a mixed solution C; 12mmol 2-methylimidazole is dissolved in deionized water, stirred and dissolved uniformly, and then diluted to 100mL to obtain a mixed solution D; 10g porous carrier of step S4 is placed in the mixed solution C, and a reduced pressure of-0.08MPa is applied for 5min for 3 cycles, then poured into the mixed solution D, and light shaking is performed to ensure uniform immersion, and then placed at room temperature for 50min, deionized water is used for repeated washing for 3 times, and 40℃ air drying is performed for 2h to obtain ZIF-8@in-situ self-assembly;

[0057] S6: 10g sulfobetaine methacrylate and 0.6g 2-hydroxy-2-methyl-1-phenyl-1-propanone are dissolved in a 70wt% ethanol solution, stirred and dissolved uniformly, and then diluted to 100mL to obtain a mixed solution E; the ZIF-8@in-situ self-assembly of step S5 is immersed in a 50wt% ethanol solution for 2min, then immersed in the mixed solution E, treated for 8min, then taken out and drained for 2min, and then placed under a 365nm LED ultraviolet lamp, a power density of 20mW / cm 2 , a turntable is uniformly rotated at 10rpm, irradiated for 10min, and then vacuum dried at 40℃ for 6h to obtain a biomass orthopedic material.

[0058] Comparative Example 1: The operation process parameters of comparative example 1 and example 1 are basically the same, the main difference is that the sulfobetaine methacrylate brush layer is removed in comparative example 1, that is, the operation of step S6 is removed, and the rest of the process remains unchanged.

[0059] 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.

[0060] 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.

[0061] Performance testing:

[0062] 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.

[0063] based on Figure 1 The 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.

[0064] 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. 6The bactericidal rate (%) was recorded, and the results of the bactericidal rate of S. aureus and E. coli are shown in Figs. 2 and 3, respectively. Figure 2 and Figure 3 From the 14th day to the 28th day, the PBS buffer replacement frequency was adjusted to 48 h replacement, and the old immersion solution was collected and stored at -20°C for standby. On the 21st day and the 28th day, the samples were taken out, washed once with PBS, and transferred to a new bacterial suspension (S. aureus and E. coli 1×10 6 CFU / mL suspension), the bactericidal process was repeated for 24 h, the bactericidal rate (%) was recorded, each batch of samples was tested three times independently, and the test results were averaged. The results of S. aureus and E. coli are shown in Figs. 2 and 3, respectively. Figure 2 and Figure 3

[0065] The specific test steps of the bactericidal rate are as follows: S. aureus and E. coli were suspended in TSB medium to 1×10 6 CFU / mL to obtain a suspension for standby; the biomass orthopedic material samples (10 mm×5 mm) prepared in Example 1 and Comparative Examples 1-3 were placed in a 36-well plate; 1 mL of inoculum was added to each well, and the plate was incubated at 37°C for 24 h; 100 µL of the mixed solution was serially diluted and plated on TSA plates, the CFU before inoculation was recorded, and the bactericidal rate (%) was calculated as (bacterial CFU before inoculation-bacterial CFU after inoculation) / bacterial CFU before inoculation×100%.

[0066] Based on the results of Figure 2 and Figure 3 analysis, the biomass orthopedic material prepared in the present application has excellent antibacterial performance through the synergistic effect of multiple levels, and still maintains a bactericidal rate of more than 95% in long-term use of 28 days; based on the results analysis of Comparative Example 1 and Example 1, the bactericidal rate of Comparative Example 1 is excellent in the early stage, but it decreases rapidly from the 7th-14th day; it significantly decays in the middle and late stages; this may be due to the absence of the pSBMA brush layer, the material surface may be quickly contaminated with protein and bacterial exopolymer, the initial adhesion of bacteria is high, the biofilm matures earlier, the protein contaminated film may partially adsorb / intercept the antibiotics released in the early stage, reducing its effective diffusion; the tight matrix of the mature biofilm also forms a diffusion barrier to the exosmosis of Ga 3+ / Zn 2+ , as the bacteria accumulate faster, local acidification is more likely to occur, ZIF-8 is disintegrated in large quantities in advance, antibiotics may be rapidly consumed in the early stage, and the risk of rapid release of antibiotics in the 0th-14th day increases significantly after the loss of the release effect of the brush layer, and the material basically relies on the slower Ga 3+ in the 14th-28th day to play an effect; but the aforementioned biofilm barrier in turn weakens the diffusion of Ga 3+ , and its late effect is significantly affected. ​

[0067] Based on the analysis of 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 showed a linear downward trend; this may be due to the retention of the pSBMA brush layer, which can still effectively reduce the initial adhesion amount and protein fouling film, promote the continuous diffusion and action of ions, but the lack of antibiotic layer, the whole system lacks on-demand burst mechanism, and the antibiotic release is slow, and the antibacterial effect depends on Ga 3+ The steady release and physical unloading of the brush layer, and Ga 3+ It is difficult to achieve the instantaneous concentration required for high-intensity sterilization within 24 hours, resulting in a low bactericidal rate on the 0th day.

[0068] Based on the analysis of the results of Comparative Example 3 and Example 1, the bactericidal rate of Comparative Example 3 was excellent in the early and middle stages, but showed a cliff-like decline in the middle and late stages; this may be due to the lack of Ga 3+ The steady release and physical unloading of the brush layer, and Ga 2+ The steady release and physical unloading of the brush layer, and Ga 2+ The concentration and persistence of Zn 3+ are weaker than those of Ga 3+ , and the sustained antibacterial effect on some bacteria, especially Staphylococcus aureus, is poorer.

[0069] Triggered release test: Biomass orthopedic material samples (100 mg) prepared by Example 1 and Comparative Examples 1, 3 (Comparative Example 2 does not contain zinc ions and is not involved in the test) were placed in PBS buffer with pH 7.4 and MES buffer with pH 6.0, both containing 0.1% Tween-80, and shaken at 37°C and 60 rpm. 0.5 mL of the extract was taken at 0.5h, 1h, 2h, 4h, 8h, 24h, 48h and 72h, and an equal amount of new buffer was added. The percentage release curve of Zn 2+ was calculated, and each batch of samples was tested three times independently, and the average value was taken. The results are shown in Figure 4 and Figure 5 .

[0070] Based on the analysis of the results of Figure 4 and Figure 5 , the biomass orthopedic material prepared in the present application showed obvious pH-responsive drug release characteristics. It showed sustained release in a physiological neutral environment (pH 7.4), and triggered antibiotic burst release under acidic conditions (pH 6.0). Based on the analysis of the results of Comparative Example 1 and Example 1, the lack of pSBMA brush layer in Comparative Example 1 caused ZIF-8 to be directly exposed to the solution after acidification, resulting in faster disintegration and a steeper curve in the early stage. Based on the analysis of the results of Comparative Example 3 and Example 1, the doping of gallium in Comparative Example 3 had little effect on the acid disintegration kinetics of ZIF-8 itself, and the burst release effect was similar to that of Example 1.

[0071] Biocompatibility test: The frozen old immersion liquid extracted in the first day, the third day, the seventh day, the fourteenth day and the twenty-eighth day of the antibacterial test was filtered to remove suspended particles to obtain a sterile and clear extraction liquid, and then the extraction liquid was added to a cell culture system, and a 48h CCK-8 cell viability test and a fluorescent live and dead staining were performed using human bone marrow stromal cells, and a TSB culture medium without extraction (negative control) and a TSB culture medium containing 10% dimethyl sulfoxide (positive control) were used as controls, and the survival rate (%) = (Example OD value - negative control group OD value) / (negative control group OD value - positive control group OD value) x 100%, three independent tests were performed for each batch of samples, and the test results were averaged, and the results are shown in Figure 6 .

[0072] Based on Figure 6 The results show that the biomass bone material prepared in the embodiment has excellent biocompatibility, which can ensure long-term effective implantation in the human body. The cell activity of the biomass bone material prepared in the embodiment is close to that of the negative control group in the early stage, and is slightly higher than that of the negative control group in the later stage. This may be due to the slow release of vancomycin and Zn 2+ in the neutral PBS, and the concentration is much lower than the osteoblast toxicity threshold; the pSBMA brush layer does not directly enter the extraction liquid, and has no adverse effect on the cells; the steady-state low-dose exosmosis leads to a whole cell activity close to the negative control; the antibiotic concentration is further reduced in the middle and late stages, and the Ga 3+ / Zn 3+ osteogenic signal may gradually appear, and the cell activity is slightly better than that of the negative control group; based on the results analysis of Comparative Example 1 and Example 1, the absence of the brush layer makes the exosmosis of antibiotics and Zn 2+ faster, although it does not trigger disintegration under neutral conditions, but the diffusion resistance is obviously reduced, and the instantaneous peak of the dissolved substance in the extraction liquid may be slightly higher, and the cell activity is most likely to decrease slightly in the early stage, and the toxicity burden of the extraction liquid is reduced in the later stage, and the activity is obviously improved compared with the early stage; based on the results analysis of Comparative Example 2 and Example 1, the absence of the burst release component of the antibiotic, the main component in the extraction liquid is low-dose Ga 2+ , Ga 3+ has a certain effect on the proliferation / differentiation of MSCs in the low-dose range, so the whole cell activity is close to or slightly higher than the negative control; based on the results analysis of Comparative Example 3 and Example 1, the absence of the osteogenic promoting effect of Ga 3+ , there is still a small amount of vancomycin and Zn 3+ in the neutral PBS, and the early cell activity decreases slightly; after the antibiotic decays in the middle and late stages, Zn 2+ 2 + ​The mild osteogenic effect of the mild acid treatment can slightly enhance the cell activity.

[0073] Atlas test: The surface morphology of the biomass bone material prepared in Example 1 before and after the acid treatment for triggering the slow-release test was characterized by a scanning electron microscope, and the microstructure changes were as shown in Figure 7 and Figure 8 .

[0074] Based on the results analysis of Figure 7 and Figure 8 , Figure 7 The SEM of the unacidic treatment sample showed that the surface was uniformly distributed with polyhedral particles, which were determined as ZIF-8 crystals, proving that the intermediate layer was successfully deposited in situ. Figure 8 The SEM of the sample after 24h treatment at pH 6.0 showed that the original crystal particles basically disappeared and appeared etched residues, indicating that the ZIF-8 shell layer rapidly disintegrated and released the guest molecules in the acidic environment.

[0075] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a long-lasting antibacterial biomass orthopedic material, 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: Add zinc nitrate hexahydrate and antibiotics to deionized water, stir and dissolve evenly to obtain a mixed solution C; dissolve 2-methylimidazole in deionized water, stir and dissolve evenly to obtain a mixed solution D; place the porous support in step S4 in the mixed solution C, decompress the support for 3 cycles, and then pour it into the mixed solution D, shake it gently to ensure uniform infiltration, let it stand at room temperature, repeatedly wash it with deionized water, and air-dry it 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.

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

4. The method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 3, 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.

5. The method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 4, characterized in that: In step S1, the ratio of the amount of ammonium dihydrogen phosphate to deionized water is 25-30 mmol:100 mL.

6. The method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 5, 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.

7. The method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 6, 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.

8. The method for preparing a long-lasting antibacterial biomass orthopedic material according to claim 7, characterized in that: In step S5, the ratio of the amount of 2-methylimidazole to deionized water is 8-12 mmol:100 mL.

9. The method for preparing a long-acting antibacterial biomass orthopedic material according to claim 8, 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 .

10. A long-lasting antibacterial biomass orthopedic material, characterized in that: Prepared by the method according to any one of claims 1 to 9.

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