CN117398526B - Bionic composite material containing zinc oxide nanowires and preparation method and application thereof - Google Patents
Bionic composite material containing zinc oxide nanowires and preparation method and application thereof Download PDFInfo
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- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 title claims abstract description 183
- 239000002131 composite material Substances 0.000 title claims abstract description 77
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
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Classifications
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- A—HUMAN NECESSITIES
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A—HUMAN NECESSITIES
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- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Abstract
The invention relates to the technical field of bone defect repair, in particular to a bionic composite material containing zinc oxide nanowires, and a preparation method and application thereof. The bionic composite material consists of collagen fibers, nano hydroxyapatite and zinc oxide nanowires woven in the collagen fibers in a penetrating manner, and is in a spongy loose porous three-dimensional bracket shape. The invention adopts the co-assembly technology to carry out the co-assembly of the inorganic nanowires on the organic collagen and further carries out biomimetic mineralization, thereby greatly improving the bone guiding property and the antibacterial property of the collagen material and promoting the clinical application transformation of the organic-inorganic hybrid composite material in the field of the infectious oral and maxillofacial bone defect repair.
Description
Technical Field
The invention relates to the technical field of bone defect repair, in particular to a bionic composite material with anti-infection and osteogenesis dual functions and containing zinc oxide nanowires, a preparation method and application thereof.
Background
The oromaxillofacial region is in a bacterial environment, and caries, periodontitis, osteomyelitis or peri-implant inflammation caused by bacterial infection can cause defects of jawbone and alveolar bone to different degrees, thereby seriously threatening the oral health of human beings.
Currently, traditional treatments for infectious bone defects have the following bottlenecks:
(1) The antibiotic has a narrow antibacterial spectrum, and is difficult to cope with complex infections such as multiple drug-resistant bacteria;
(2) The administration mode of antibiotics is usually systemic administration, and the concentration of local damaged medicines is reduced due to lack of specific targets, so that drug resistance is easy to generate;
(3) The local delivery mode has unstable curative effect and is easy to generate complications such as ectopic ossification, hypercalcemia and the like;
(4) The traditional bone repair material mainly containing bone powder is not matched with natural bone in the aspects of mechanical property, degradation rate, biological activity and the like, can not reconstruct defective wound surface rapidly, and is easy to cause repeated infection;
(5) The use of antibiotics destroys the organism's microorganisms and immune environment, thus inhibiting the osteoinductive properties of the material, resulting in "double transfusion" of anti-infection and bone repair treatments.
Therefore, in the field of repair and regeneration of infectious maxillofacial bone defects, there is an urgent need to develop a novel bone grafting material having both good osteoinductive and antibacterial properties.
Disclosure of Invention
Aiming at the defects of the traditional bone substitute material in the aspect of infectious oral and maxillofacial bone defect treatment, the invention aims to provide a bionic composite material containing zinc oxide nanowires, and a preparation method and application thereof.
In order to solve the technical problems, the application provides the following technical scheme:
a bionic composite material containing zinc oxide nanowires is composed of collagen fibers, nano hydroxyapatite and zinc oxide nanowires woven in the collagen fibers in an interpenetration mode, and is in a spongy loose porous three-dimensional support shape. Wherein the mass ratio of the zinc oxide nanowire to the type I collagen is 2.5% -20%.
The preparation method of the bionic composite material containing the zinc oxide nanowires comprises the following steps:
(1) Adding zinc acetate into urea aqueous solution, uniformly mixing and sealing in an autoclave, heating, cooling to room temperature, centrifuging the precipitate, washing with deionized water and alcohol, drying, and calcining in a muffle furnace to obtain ZnO nanowire powder; weighing zinc oxide nanowire powder with certain mass, adding distilled water, and performing ultrasonic dispersion to prepare ZnO nanowire dispersion liquid;
(2) Dialyzing and concentrating the type I collagen solution, dropwise adding the ZnO nanowire dispersion liquid prepared in the step (1) into the concentrated collagen solution, and stirring and uniformly mixing to obtain a mixed dispersion system;
(3) Placing the mixed dispersion system prepared in the step (2) in a mould, pre-freezing and freeze-drying to obtain an initial collagen/nano zinc oxide composite material;
(4) Placing the initial collagen/nano zinc oxide composite material prepared in the step (3) into a solvent containing 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, performing light-shielding crosslinking, soaking with glycine solution, washing with double distilled water, and further performing freeze-drying to obtain a bioactive collagen/nano zinc oxide composite material;
(5) Immersing the bioactive collagen/nano zinc oxide composite material obtained in the step (4) into mineralization liquid for mineralization to obtain a mineralized material;
(6) And (3) fully washing the mineralized material obtained in the step (5) in deionized water, pre-freezing and freeze-drying to obtain the mineralized collagen/nano zinc oxide composite material.
Wherein, in the step (1), zinc acetate is added into urea aqueous solution, the stirring speed is 500-800 rpm/min, the obtained mixture is sealed in an autoclave lined with polytetrafluoroethylene, and the mixture is heated for 6-7 hours at 95 ℃. The concentration of the ZnO nanowire dispersion is 5-20mg/mL.
Wherein, in the step (2), the concentration of the type I collagen solution after dialysis concentration is 3.0-5.0mg/mL; a dialysis bag with a molecular weight of 3000-5000 is used. The zinc oxide nano wire accounts for 2.5 to 20 percent of the mass ratio of the type I collagen; and adding the ZnO nanowire dispersion liquid into the concentrated collagen solution, and stirring for 0.5-1 hour.
Wherein in the step (3), the pre-freezing temperature is-40 ℃ and the pre-freezing time is 24-48 hours.
Wherein in the step (4), the solvent is ethanol, and the concentration of the 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride in the ethanol is 1wt.%; glycine was dissolved at a concentration of 1wt.% in deionized water.
Wherein the mineralizing liquid in the step (5) comprises 3.08 mM Na 3 N、2.7 mM KCl、8.3 mM Na 2 HPO 4 、136.9 mM NaCl、1.25 mM K 2 HPO 4 ·3H 2 O, 0.2g/mL white Portland cement and 0.25 mM polyacrylic acid.
The bionic composite material containing the zinc oxide nanowire can be used for preparing medical products for promoting the repair of infectious oral and maxillofacial bone defects.
The bionic composite material containing the zinc oxide nanowires, which is prepared by the invention, is detected by adopting the following test method:
(1) Surface morphology and elemental composition of mineralized collagen/nano zinc oxide composite: detection is carried out by a scanning electron microscope and an energy spectrometer.
(2) Biocompatibility of mineralized collagen/nano zinc oxide composite: after co-culturing the material with human bone marrow mesenchymal stem cells (hBMMSCs), the activity of the cells was examined with CCK 8.
(3) Antibacterial properties of mineralized collagen/nano zinc oxide composite: after co-culturing the material with streptococcus mutans or porphyromonas gingivalis, detecting the number of bacteria by a plating method, and detecting the distribution and morphology of the bacteria by a scanning electron microscope.
(4) Effects of mineralized collagen/nano zinc oxide composite on osteoblasts: the material is co-cultured with hBMMSCs and subjected to osteogenic induction differentiation, and the expression of osteogenic genes is detected by PCR.
Compared with the prior art, the bionic composite material containing the zinc oxide nanowire and the preparation method and application thereof have at least the following beneficial effects:
the bionic composite material consists of collagen fibers, nano hydroxyapatite and zinc oxide nanowires woven in the collagen fibers in a penetrating manner, and is in a spongy loose porous three-dimensional bracket shape. The invention adopts the co-assembly technology to carry out the co-assembly of the inorganic nanowires on the organic collagen, further carries out biomimetic mineralization, presents a bone-like nano structure, greatly improves the bone guiding property and the antibacterial property of the collagen material, and promotes the clinical application transformation of the organic-inorganic hybrid composite material in the field of the infectious oral and maxillofacial bone defect repair.
The bionic composite material containing the zinc oxide nanowire, the preparation method and the application thereof are further described below with reference to the accompanying drawings.
Drawings
Fig. 1 and 2 are physical and chemical property detection results of a bionic composite material containing zinc oxide nanowires.
FIG. 3 is a graph showing the proliferation potency of CCK-8 assay cells on biomimetic composites containing zinc oxide nanowires.
FIG. 4 is a graph showing the results of immunofluorescence detection of cell extension and adhesion on biomimetic composites containing zinc oxide nanowires.
Fig. 5 and 6 are diagrams showing the osteoinductive properties of biomimetic composites with zinc oxide nanowires tested by PCR.
Fig. 7 and 8 are broad spectrum antibacterial ability test results of biomimetic composites containing zinc oxide nanowires.
Fig. 9 is a result of repairing an infectious bone defect with a biomimetic composite material containing zinc oxide nanowires.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
The invention is further illustratively described below with reference to the accompanying drawings, which are intended to be illustrative of the present disclosure in detail. It should be noted that the examples are only some examples of a preparation method of a bionic composite material containing zinc oxide nanowires in the present invention, and do not represent all the examples of the present invention. All adjustments and modifications made according to the patent scope of the invention are within the scope of the invention.
Example 1
(1) 1.0975g of zinc acetate solid is weighed and dissolved in 25mL of deionized water to prepare a zinc source solution of 0.2M; 0.6006g of urea was then weighed into 25mL of deionized water to prepare a 0.4M precipitant solution. The prepared zinc source solution is dropwise added into the precipitant solution, the stirring speed is controlled to be 500rpm/min in the dripping process, the solution is sealed in a 100mL beaker after the dripping is finished, and the solution is placed in an oven and heated for 6h at 95 ℃. After the reaction, the beaker was cooled to room temperature, the supernatant was removed, and the lower white solid was taken out, washed with deionized water and ethanol 3 to 4 times, respectively, and then dried at 80℃for 12 hours. And placing the obtained precursor solid powder in a crucible, calcining for 0.5h at 400 ℃ in a muffle furnace, setting the heating speed of the muffle furnace to be 1 DEG/min, and collecting a solid sample for standby to obtain the zinc oxide necklace-shaped nanowire. Weighing zinc oxide nanowire powder with certain mass, adding distilled water, and performing ultrasonic dispersion to prepare the zinc oxide nanowire powder with the concentration of 10 mg/mL.
(2) The purified type I collagen solution (Corning) extracted from rat tail was concentrated by dialysis (PEG 3500 Da) to a collagen concentration of 4mg/mL. And (3) dropwise adding the zinc oxide nanowire solution prepared in the step (1) (the zinc oxide nanowire accounts for 5-10% of the type I collagen in percentage by mass) into the concentrated collagen solution, and stirring for half an hour at 37 ℃ to fully and uniformly mix the solution to obtain a mixed dispersion system.
(3) And (3) placing the mixed dispersion system in the step (2) in a mould, pre-freezing for more than 24 hours at the temperature of minus 40 ℃, and then freeze-drying to obtain the initial collagen/nano zinc oxide composite material.
(4) Placing the initial collagen/nano zinc oxide composite material in the step (3) in an ethanol solvent containing 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, wherein the concentration of the (1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride in ethanol is 1 wt.%), crosslinking for 4 hours in a dark place, soaking in glycine solution with the mass percent of 1%, flushing with double distilled water, and further freeze-drying to obtain the bioactive collagen/nano zinc oxide composite material.
(5) Preparation of a solution containing 3.08 mM Na 3 N、2.7 mM KCl、8.3 mM Na 2 HPO 4 、136.9 mM NaCl、1.25 mM K 2 HPO 4 ·3H 2 Mineralizing solution of O, 0.2. 0.2g/mL white Portland cement and 0.25. 0.25 mM polyacrylic acid. Immersing the bioactive collagen/nano zinc oxide composite material obtained in the step (4) into mineralization liquid, and mineralizing for 7 days.
(6) And (3) fully washing the composite material obtained in the step (5) in deionized water, pre-freezing for 24 hours at the temperature of minus 40 ℃, and further freeze-drying to obtain the mineralized collagen/nano zinc oxide composite material.
Example 2
(1) 1.0975g of zinc acetate solid is weighed and dissolved in 25mL of deionized water to prepare a zinc source solution of 0.2M; 0.6006g of urea was then weighed into 25mL of deionized water to prepare a 0.4M precipitant solution. The prepared zinc source solution is dropwise added into the precipitant solution, the stirring speed is controlled to be 500rpm/min in the dripping process, the solution is sealed in a 100mL beaker after the dripping is finished, and the solution is placed in an oven and heated for 6h at 95 ℃. After the reaction, the beaker was cooled to room temperature, the supernatant was removed, and the lower white solid was taken out, washed with deionized water and ethanol 3 to 4 times, respectively, and then dried at 80℃for 12 hours. And placing the obtained precursor solid powder in a crucible, calcining for 0.5h at 400 ℃ in a muffle furnace, setting the heating speed of the muffle furnace to be 1 DEG/min, and collecting a solid sample for standby to obtain the zinc oxide necklace-shaped nanowire. Weighing zinc oxide nanowire powder with a certain mass, adding distilled water, and performing ultrasonic dispersion to prepare the zinc oxide nanowire powder with the concentration of 5 mg/mL.
(2) The purified type I collagen solution (Corning) extracted from rat tail was concentrated by dialysis (PEG 3500 Da) to a collagen concentration of 3.0. 3.0 mg/mL. And (3) dropwise adding the zinc oxide nanowire solution prepared in the step (1) (the zinc oxide nanowire accounts for 5% of the type I collagen in percentage by mass) into the concentrated collagen solution, and stirring for one hour at 37 ℃ to fully and uniformly mix the solution to obtain a mixed dispersion system.
(3) And (3) placing the mixed dispersion system in the step (2) in a mould, pre-freezing for 24 hours at the temperature of minus 40 ℃, and then freeze-drying to obtain the initial collagen/nano zinc oxide composite material.
(4) Placing the initial collagen/nano zinc oxide composite material in the step (3) in an ethanol solvent containing 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, wherein the concentration of the (1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride in ethanol is 1 wt.%), crosslinking for 4 hours in a dark place, soaking in glycine solution with the mass percent of 1%, flushing with double distilled water, and further freeze-drying to obtain the bioactive collagen/nano zinc oxide composite material.
(5) Preparation of a solution containing 3.08 mM Na 3 N、2.7 mM KCl、8.3 mM Na 2 HPO 4 、136.9 mM NaCl、1.25 mM K 2 HPO 4 ·3H 2 Mineralizing solution of O, 0.2. 0.2g/mL white Portland cement and 0.25. 0.25 mM polyacrylic acid. Immersing the bioactive collagen/nano zinc oxide composite material obtained in the step (4) into mineralization liquid, and mineralizing for 7 days.
(6) And (3) fully washing the composite material obtained in the step (5) in deionized water, pre-freezing for 24 hours at the temperature of minus 40 ℃, and further freeze-drying to obtain the mineralized collagen/nano zinc oxide composite material.
Example 3
(1) 1.0975g of zinc acetate solid is weighed and dissolved in 25mL of deionized water to prepare a zinc source solution of 0.2M; 0.6006g of urea was then weighed into 25mL of deionized water to prepare a 0.4M precipitant solution. The prepared zinc source solution is dropwise added into the precipitant solution, the stirring speed is controlled to be 500rpm/min in the dripping process, the solution is sealed in a 100mL beaker after the dripping is finished, and the solution is placed in an oven and heated for 6h at 95 ℃. After the reaction, the beaker was cooled to room temperature, the supernatant was removed, and the lower white solid was taken out, washed with deionized water and ethanol 3 to 4 times, respectively, and then dried at 80℃for 12 hours. And placing the obtained precursor solid powder in a crucible, calcining for 0.5h at 400 ℃ in a muffle furnace, setting the heating speed of the muffle furnace to be 1 DEG/min, and collecting a solid sample for standby to obtain the zinc oxide necklace-shaped nanowire. Weighing zinc oxide nanowire powder with certain mass, adding distilled water, and performing ultrasonic dispersion to prepare the zinc oxide nanowire powder with the concentration of 10 mg/mL.
(2) The purified type I collagen solution (Corning) extracted from rat tail was concentrated by dialysis (PEG 3500 Da) to give a collagen concentration of 5.0. 5.0 mg/mL. And (3) dropwise adding the zinc oxide nanowire solution prepared in the step (1) (the zinc oxide nanowire accounts for 10% of the type I collagen in percentage by mass) into the concentrated collagen solution, and stirring at 37 ℃ for half an hour to one hour to fully and uniformly mix the mixture to obtain a mixed dispersion system.
(3) And (3) placing the mixed dispersion system in the step (2) in a mould, pre-freezing for 48 hours at the temperature of minus 40 ℃, and then freeze-drying to obtain the initial collagen/nano zinc oxide composite material.
(4) Placing the initial collagen/nano zinc oxide composite material in the step (3) in an ethanol solvent containing 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, wherein the concentration of the (1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride in ethanol is 1 wt.%), crosslinking for 4 hours in a dark place, soaking in glycine solution with the mass percent of 1%, flushing with double distilled water, and further freeze-drying to obtain the bioactive collagen/nano zinc oxide composite material.
(5) Preparation of a solution containing 3.08 mM Na 3 N、2.7 mM KCl、8.3 mM Na 2 HPO 4 、136.9 mM NaCl、1.25 mM K 2 HPO 4 ·3H 2 Mineralizing solution of O, 0.2. 0.2g/mL white Portland cement and 0.25. 0.25 mM polyacrylic acid. Immersing the bioactive collagen/nano zinc oxide composite material obtained in the step (4) into mineralization liquid, and mineralizing for 7 days.
(6) And (3) fully washing the composite material obtained in the step (5) in deionized water, pre-freezing for 48 hours at the temperature of minus 40 ℃, and further freeze-drying to obtain the mineralized collagen/nano zinc oxide composite material.
The following experiments were performed on mineralized collagen/nano zinc oxide nanowire (IMC/ZnO) multifunctional composite materials prepared in examples 1-3, and the results are shown in fig. 1-5.
(1) Bionic composite material surface physical and chemical property detection (fig. 1-2):
the material prepared in example 1 was subjected to surface scanning analysis for element distribution by an X-ray photoelectron spectrometer, by observing the surface morphology and the nanostructure of the material by a scanning electron microscope and a transmission electron microscope.
The results of fig. 1 show that: (a) The figures are respectively transmission electron microscope images of pure collagen, znO nanowires and IMC/ZnO composite materials: znO nanowires (diameter 89.35 +/-8.43 nm) are uniformly distributed in mineralized collagen to form an IMC/ZnO (diameter 154.94 +/-58.26 nm) composite fiber material with a bone-like nanostructure; (b) The graph shows the energy spectrum of IMC/ZnO, and the calcium, phosphorus and zinc are uniformly distributed in the IMC/ZnO.
The results of fig. 2 show that: (a) three-dimensional IMC/ZnO overview. (b) three-dimensional IMC/ZnO scanning electron microscope image: after freeze drying, a three-dimensional porous IMC/ZnO composite scaffold (pore diameter, 111-200 μm) is formed, which is similar to the structure of the cancellous bone (pore diameter, 125-220 μm). (c) ICP-MS showed that IMC/ZnO can release Zn continuously under neutral pH and acidic pH conditions 2+ 。
(2) Effect of IMC/ZnO composite on cell proliferation, adhesion, osteogenic differentiation (fig. 3-6):
FIG. 3 shows the proliferation capacity of hBMMSCs on composite material with CCK-8 detecting reagent. After hBMMSCs are co-cultured with IMC/ZnO materials with different concentrations for 1, 3 and 5 days, cells are washed by PBS, and the cell viability is detected by CCK8 reagent. The results showed 5% (IMC/ZnO 0.05 ) And 10% (IMC/ZnO) 0.1 ) The ZnO nanowire composite mineralized collagen material can promote proliferation of hBMMSCs.。
FIG. 4 shows immunofluorescence results showing that cells were in IMC/ZnO compared to IMC 0.1 The upper spreading is most obvious, and the adhesive force is higher.
In FIG. 5, PCR shows that under normal conditions, IMC/ZnO 0.1 Compared with IMC, the hBMMSCs can promote osteogenic differentiation, and high-express alkaline phosphatase and bone morphogenetic protein-2.。
In FIG. 6, PCR shows that under lipopolysaccharide-induced inflammatory conditions, IMC/ZnO 0.1 Compared with IMC, the hBMMSCs can promote osteogenic differentiation and express bone morphogenetic protein-2 and osteocalcin.。
(3) Broad spectrum antimicrobial capability detection of biomimetic composites (fig. 7-8):
respectively 1×10 8 CFU/mL gram-negative bacterium Porphyromonas gingivalis (W83, FIG. 7), 1X 10 7 CFU/mL gram-positive bacteria Streptococcus mutans (UA 159, FIG. 8) were co-cultured with the bionic complex for 24h. In fig. 7, (a) is a bacterial plaque, and the results show that: IMC/ZnO 0.05 The antibacterial rate against Porphyromonas gingivalis (W83) was about 60%, and when the ZnO concentration was increased to 10%, IMC/ZnO 0.1 The antibacterial rate increased to 80% (fig. 7 (a)). FIG. 7 (b) shows the number and morphology of bacteria detected by a scanning electron microscope, showing that the IMC/ZnO is compared with the IMC alone group 0.1 The number of Porphyromonas gingivalis (W83) in the group was significantly reduced, and the bacterial envelope was significantly collapsed and deformed (FIG. 7 (b)). In FIG. 8, the bacterial plating shows IMC/ZnO relative to IMC 0.1 Has good antibacterial performance on gram positive bacteria Streptococcus mutans (UA 159).。
(4) Study of biomimetic composite repair of infectious periodontal defects (fig. 9):
preparation of critical bone defect model of mandible (diameter 3 mm), coating Porphyromonas gingivalis at concentration of 10 μg/mL on defect site to obtain infectious bone defect model, and implanting different scaffold materials (IMC, IMC/ZnO) 0.1 ) Blank no material implantation. FIG. 9 shows micro-CT results, after 4 weeks, relative to IMC and blank, IMC/ZnO 0.1 Obviously promote the regeneration and repair of the infectious bone defect.。
According to the experimental results, the bionic composite material containing the zinc oxide nanowire, which is prepared by the invention, has good biocompatibility, bone guiding property and antibacterial property, and can promote the clinical application transformation of the organic-inorganic hybrid composite material in the field of infectious bone defect repair.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (10)
1. A bionic composite material containing zinc oxide nanowires is characterized in that: the bionic composite material is in a spongy loose porous three-dimensional bracket shape and consists of collagen fibers, nano hydroxyapatite and zinc oxide nanowires woven in the collagen fibers in an interpenetration manner.
2. The method for preparing the bionic composite material containing the zinc oxide nanowire, which is characterized in that: the method comprises the following steps:
(1) Adding zinc acetate into urea aqueous solution, uniformly mixing and sealing in an autoclave, heating, cooling to room temperature, centrifuging the precipitate, washing with deionized water and alcohol, drying, and calcining in a muffle furnace to obtain ZnO nanowire powder; weighing zinc oxide nanowire powder with certain mass, adding distilled water, and performing ultrasonic dispersion to prepare ZnO nanowire dispersion liquid;
(2) Dialyzing and concentrating the type I collagen solution, dropwise adding the ZnO nanowire dispersion liquid prepared in the step (1) into the concentrated collagen solution, and stirring and uniformly mixing to obtain a mixed dispersion system;
(3) Placing the mixed dispersion system prepared in the step (2) in a mould, pre-freezing and freeze-drying to obtain an initial collagen/nano zinc oxide composite material;
(4) Placing the initial collagen/nano zinc oxide composite material prepared in the step (3) into a solvent containing 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride, performing light-shielding crosslinking, soaking with glycine solution, washing with double distilled water, and further performing freeze-drying to obtain a bioactive collagen/nano zinc oxide composite material;
(5) Immersing the bioactive collagen/nano zinc oxide composite material obtained in the step (4) into mineralization liquid for mineralization to obtain a mineralized material;
(6) And (3) fully washing the mineralized material obtained in the step (5) in deionized water, pre-freezing and freeze-drying to obtain the mineralized collagen/nano zinc oxide composite material.
3. The method for preparing the bionic composite material containing the zinc oxide nanowire according to claim 2, which is characterized in that: in the step (1), zinc acetate is added into urea aqueous solution, stirring speed is 500-800 rpm/min, and the obtained mixture is sealed in an autoclave lined with polytetrafluoroethylene and heated at 95 ℃ for 6-7 hours.
4. The method for preparing the bionic composite material containing the zinc oxide nanowire according to claim 2, which is characterized in that: in the step (1), the concentration of the ZnO nanowire dispersion liquid is 5-20mg/mL.
5. The method for preparing the bionic composite material containing the zinc oxide nanowire according to claim 2, which is characterized in that: in the step (2), the concentration of the type I collagen solution after dialysis concentration is 3.0-5.0mg/mL; a dialysis bag with a molecular weight of 3000-5000 is used.
6. The method for preparing the bionic composite material containing the zinc oxide nanowire according to claim 2, which is characterized in that: in the step (2), the zinc oxide nanowire accounts for 2.5-20% of the type I collagen in mass ratio; and adding the ZnO nanowire dispersion liquid into the concentrated collagen solution, and stirring for 0.5-1 hour.
7. The method for preparing the bionic composite material containing the zinc oxide nanowire according to claim 2, which is characterized in that: in the step (3), the pre-freezing temperature is-40 ℃ and the pre-freezing time is 24-48 hours.
8. The method for preparing the bionic composite material containing the zinc oxide nanowire according to claim 2, which is characterized in that: in the step (4), the solvent is ethanol, and the concentration of the 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride in the ethanol is 1wt.%; glycine was dissolved at a concentration of 1wt.% in deionized water.
9. The method for preparing the bionic composite material containing the zinc oxide nanowire according to claim 2, which is characterized in that: the mineralization liquid in the step (5) comprises 3.08 mM Na 3 N、2.7 mM KCl、8.3 mM Na 2 HPO 4 、136.9 mM NaCl、1.25 mM K 2 HPO 4 ·3H 2 O, 0.2g/mL white Portland cement and 0.25 mM polyacrylic acid.
10. Use of the biomimetic composite material comprising zinc oxide nanowires according to claim 1 for the preparation of a medical product for promoting repair of infectious oromaxillofacial bone defects.
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| CN103110980A (en) * | 2013-02-17 | 2013-05-22 | 山东建筑大学 | In-situ growth material for treating bone defect or decayed teeth and preparation method thereof |
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| CN103110980A (en) * | 2013-02-17 | 2013-05-22 | 山东建筑大学 | In-situ growth material for treating bone defect or decayed teeth and preparation method thereof |
| CN111150882A (en) * | 2020-01-10 | 2020-05-15 | 北京大学口腔医学院 | Silver nanowire-mineralized collagen co-assembled bionic scaffold and preparation method and application thereof |
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