CN114306741B - Method for constructing metal-organic molecule compound and inorganic phase hybrid functional coating on surface of degradable metal - Google Patents
Method for constructing metal-organic molecule compound and inorganic phase hybrid functional coating on surface of degradable metal Download PDFInfo
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Abstract
The invention discloses a method for constructing a metal-organic molecular compound and inorganic phase hybrid functional coating on a degradable metal surface, which comprises the steps of firstly generating a rough and stable inorganic zinc phosphate coating on an untreated degradable metal surface by a liquid phase chemical deposition method; and then circularly and alternately evaporating and depositing the degradable metal with the surface being the inorganic zinc phosphate coating in the collagen/ferulic acid/hydroxyethylidene diphosphonic acid mixed solution and the metal ion solution to obtain the metal-organic molecular compound and inorganic phase hybrid functional coating. According to the invention, through the chemical coordination effect among organic functional molecules, metal ions and inorganic components, a compact and uniform metal-organic molecule compound and inorganic phase hybrid functional coating with high hydrophilicity and surface energy is formed by composite hybridization, and the coating has good osseointegration, bone regeneration, vascular regeneration, antibacterial and anti-inflammatory biological functions while considering degradable metal corrosion degradation control and biocompatibility.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a method for constructing a metal-organic molecular compound and inorganic phase hybrid functional coating on the surface of degradable metal.
Background
Compared with the traditional metal implant material, the degradable metal is used as a temporary implant, and has the remarkable advantages of avoiding secondary operation, accelerating the healing process and the like, so that the risk is greatly reduced. Based on the advantages, the degradable zinc material gradually enters the visual field of people, and the wide attention is attracted. So far, degradable metals are mainly classified into magnesium, iron and zinc. The degradable metal zinc is expected to be used for treating orthopedic diseases and cardiovascular diseases due to the relatively moderate corrosion rate and potential biological functionality of the degradable metal zinc relative to magnesium and iron. But the further application of the degradable zinc-based metal is limited due to the lack of good corrosion degradation mode and biological functionality. In view of these shortcomings, the problems can be solved to a great extent by modifying the surface of the zinc-based metal.
Currently, in the research on the surface modification of zinc-based metals, a single inorganic coating is mainly used, the inorganic coating can improve the corrosion behavior of degradable zinc-based metals to a certain extent, but cannot improve the biocompatibility of the degradable zinc-based metals, and the zinc modified by the inorganic coating cannot meet the comprehensive biological functionality required by the materials of implants, such as: osseointegration, bone regeneration, angiogenesis, antibacterial and anti-inflammatory functions, and the like.
Disclosure of Invention
Aiming at the problems, the invention provides a method for constructing a metal-organic molecule compound and inorganic phase hybrid functional coating on the surface of degradable metal, so as to solve the problems that the corrosion behavior regulation and the biocompatibility improvement of zinc-based degradable metal cannot be considered at the same time, and the biological functionality is insufficient in the existing surface modification technology.
In order to achieve the purpose, the invention adopts the technical scheme that: the method for constructing the metal-organic molecule compound and inorganic phase hybrid functional coating on the surface of the degradable metal comprises the following steps:
(1) placing the pretreated degradable metal in zinc phosphate deposition solution, heating and reacting at 40-60 ℃ for 1.5-2.5 h, taking out, cleaning, and drying until the surface liquid volatilizes;
(2) mixing ferulic acid, hydroxyethylidene diphosphate and water according to the material-to-liquid ratio of 1-5 mg: 0.5-2 mg:1ml, stirring at 60-80 ℃ until the materials are fully dissolved, and then cooling to 35-40 ℃ to obtain a mixed solution; then mixing the mixed solution with collagen according to the liquid-material ratio of 1 ml: 8-12 mg of the mixture is mixed, stirred for 20-40 min at the temperature of 35-40 ℃, and then the pH of the solution is adjusted to 5.5-6.5, so as to obtain a mixed solution A;
(3) putting the degradable metal treated in the step (1) into the mixed solution A, taking out the degradable metal after 4-6 seconds, and drying until the surface liquid volatilizes;
(4) soaking the degradable metal treated in the step (3) in a coordination ion solution with the concentration of 5-12 mg/ml and the pH of 5.5-6.5 for 4-6 min, and then taking out and drying until the surface liquid volatilizes;
(5) repeating the steps (3) and (4) for 3-6 times.
Generating a layer of rough and stable inorganic zinc phosphate coating on the surface of the degradable metal by a chemical liquid deposition method; and then performing multiple circulating alternate evaporation deposition on the degradable metal with the surface being the inorganic zinc phosphate coating in the collagen/ferulic acid/hydroxyethylidene diphosphonic acid mixed solution and the solution rich in magnesium ions, calcium ions, ferrous ions, copper ions, zinc ions, strontium ions and chromium ions to form the metal-organic molecular compound and inorganic phase hybrid functional coating. In the mixed solution, ferulic acid and hydroxyethylidene diphosphonic acid are combined with hydrophilic groups in collagen by intermolecular forces such as hydrogen bonds and the like to form a collagen/ferulic acid/hydroxyethylidene diphosphonic acid compound, and then the collagen/ferulic acid/hydroxyethylidene diphosphonic acid compound is evaporated and deposited on the surface of zinc phosphate by the physical adsorption and chemical coordination action with a zinc phosphate layer, and further the collagen/ferulic acid/hydroxyethylidene diphosphonic acid compound is chemically coordinated with a solution rich in magnesium ions, calcium ions, ferrous ions, copper ions, zinc ions, strontium ions and chromium ions to increase the crosslinking degree among all organic components of the coating, improve the overall quality of the coating, form a metal-organic molecular compound, and are alternately deposited by circulation for several times, finally, the collagen/ferulic acid/hydroxyethylidene diphosphonic acid compound, a zinc phosphate layer and metal ions are subjected to composite hybridization to form a uniform, compact and stable metal-organic molecular compound and inorganic phase hybridization functional coating.
Further, the pretreatment process of the degradable metal in the step (1) comprises the following steps: polishing the degradable metal, and then putting the metal into absolute ethyl alcohol for ultrasonic cleaning for 10-20 min.
Further, the zinc phosphate deposition solution in the step (1) is prepared by the following steps: mixing 6-9 mg/ml sodium dihydrogen phosphate solution and 1-3 mg/ml zinc nitrate solution according to the volume ratio of 5: 2, or mixing 10-15 mg/ml phosphoric acid solution and 10-15 mg/ml zinc nitrate solution according to a volume ratio of 5: 2 and mixing.
Further, the ferulic acid, the hydroxy ethylidene diphosphate and the water in the step (2) are mixed according to the material-liquid ratio of 3mg to 1.5mg to 1 ml.
Further, in the step (2), the mixed solution and the collagen are mixed according to the liquid-material ratio of 1 ml: 10mg are mixed.
Further, the complex ion solution in the step (4) is a magnesium ion solution, a calcium ion solution, a ferrous ion solution, a copper ion solution, a zinc ion solution, a strontium ion solution, or a chromium ion solution.
Further, the magnesium ion solution is prepared by the following steps: dissolving anhydrous magnesium sulfate in water at 37 ℃, and stirring at 37 ℃ until the anhydrous magnesium sulfate is dissolved to prepare a magnesium ion solution of 5-12 mg/ml.
Further, the drying method is drying at 37 ℃ until the surface liquid volatilizes.
The invention also provides application of the degradable metal with the surface built with the metal-organic molecule compound and inorganic phase hybrid functional coating in preparing bone repair materials or cardiovascular disease prevention and treatment materials.
The invention has the beneficial effects that:
1. the invention provides a method for constructing a metal-organic molecule compound and an inorganic phase hybrid functional layer on a degradable metal surface. Firstly, generating a layer of rough and stable inorganic zinc phosphate coating on the surface of untreated degradable metal by a liquid phase chemical deposition method; and then circularly and alternately evaporating and depositing the degradable metal with the surface being the inorganic zinc phosphate coating in the collagen/ferulic acid/hydroxyethylidene diphosphonic acid mixed solution and the metal ion solution to finally obtain the metal-organic molecular compound and inorganic phase hybrid functional coating. The functional coating constructed by the invention has better biological functions of osseointegration, bone regeneration, blood vessel regeneration, antibiosis and anti-inflammation while giving consideration to the corrosion degradation control and biocompatibility of the degradable metal.
2. The functional layer constructed by the method has the biological functions of good osseointegration, bone regeneration, angiogenesis, antibiosis and anti-inflammation while considering both degradable metal corrosion degradation control and biocompatibility; on one hand, a metal-organic molecular compound and an inorganic phase hybrid functional layer are formed by the composite hybridization of metal ions, collagen/ferulic acid/hydroxyethylidene diphosphate organic components and an inorganic zinc phosphate layer, the structure is uniform and compact, the property is stable and firm, and good corrosion protection is provided for degradable metals; the metal-organic molecule compound in the functional layer can chelate calcium ions in the implant environment, induce the deposition of active calcium phosphate and further regulate and control the corrosivity of zinc-based metal and the bone integration capability; on the other hand, the collagen of the functional layer is the natural tissue of the human body, so that the biocompatibility of the coating is ensured. The ferulic acid coating can well promote the proliferation and adhesion of endothelial cells and has a potential angiogenesis function, and meanwhile, the ferulic acid is a natural phenolic acid and has excellent antibacterial and anti-inflammatory capabilities. The hydroxyethylidene diphosphate is not only a good corrosion inhibitor, but also can promote the adhesion and proliferation of osteocytes and has the potential osteogenesis effect. Finally, the addition of the metal ions not only increases the overall crosslinking degree of the coating to form a more stable coating structure, but also endows the coating with different biological functions. The functional coating endows the degradable metal with corresponding biological functionality through the combined action of the components, and has potential application value in preparing bone repair materials or cardiovascular disease implant materials.
Drawings
FIG. 1 is an SEM photograph of a metal-organic molecule composite and an inorganic phase hybrid functional layer constructed on the surface of a zinc sheet;
FIG. 2 is an infrared spectrum of the metal-organic molecular composite and inorganic phase hybrid functional layer and components used for construction;
FIG. 3 shows the electrochemical characterization results before and after the metal-organic molecular composite and inorganic hybrid functional layer are constructed on the surface of pure zinc;
FIG. 4 shows the water contact angle and surface energy tests before and after the modified metal-organic molecular composite constructed on the surface of pure zinc and the inorganic phase hybridization functional layer;
FIG. 5 shows the deposition results of calcium and phosphorus salts on the front and back surfaces of a metal-organic molecular composite and an inorganic phase hybrid functional layer constructed on the surface of pure zinc;
FIG. 6 is a direct osteoblast culture fluorescence staining diagram before and after constructing a metal-organic molecular compound and an inorganic phase hybridization functional layer on the surface of pure zinc;
FIG. 7 shows the statistics of adherent cell count in osteoblast direct culture;
FIG. 8 is a direct endothelial cell culture fluorescence staining diagram before and after constructing a metal-organic molecular compound and an inorganic phase hybrid functional layer on the surface of pure zinc;
FIG. 9 shows statistics of endothelial cell adhesion count in direct culture;
FIG. 10 is a direct macrophage-culturing fluorescence staining diagram before and after a metal-organic molecule compound and an inorganic phase hybridization functional layer are constructed on the surface of pure zinc;
FIG. 11 shows the statistics of the number of adherent cells cultured directly on macrophages;
FIG. 12 shows the concentrations of inflammatory factor TNF-alpha before and after the metal-organic molecular composite and inorganic phase hybrid functional layer are constructed on the surface of pure zinc;
FIG. 13 shows the colony counts on the surface of Staphylococcus aureus and Escherichia coli samples before and after hybridization of metal-organic molecule composite on pure zinc surface with inorganic phase hybridization functional layer;
FIG. 14 shows the antibacterial rate of Staphylococcus aureus in bacterial suspension before and after hybridization of pure zinc surface metal-organic molecule composite with inorganic phase hybrid functional layer;
FIG. 15 shows the antibacterial ratio of Escherichia coli in bacterial suspension before and after hybridization of pure zinc surface metal-organic molecule composite and inorganic phase hybrid functional layer.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1
A method for constructing a metal-organic molecule compound and inorganic phase hybrid functional coating on a degradable metal surface comprises the following steps:
(1) pre-treatment: polishing and flattening pure zinc wafer with 600#, 1200# and 2000# abrasive paper in sequence, cleaning with anhydrous ethanol under ultrasound for 3 times, 5 minutes each time, and drying at room temperature until surface liquid volatilizes;
(2) preparation of zinc phosphate primer layer: adding 7.5mg/ml NaH 2 PO 4 And 2mg/ml Zn (NO) 3 ) 2 ·6H 2 O solution according to the volume ratio of 5: 2, mixing, placing in a 50 ℃ water bath for reaction for 2h, then placing the zinc sheet dried in the step (1) in the water bath, heating at 50 ℃ for reaction for 2h, taking out, washing with water, and then placing in a 37 ℃ constant-temperature drying oven for drying until the surface liquid volatilizes;
(3) uniformly mixing ferulic acid, hydroxyethylidene diphosphate and water according to the material-liquid ratio of 3mg:1.5mg:1ml, stirring at 70 ℃ until the mixture is fully dissolved, and then cooling to 37 ℃ to obtain a mixed solution; then mixing the mixed solution with collagen according to the liquid-material ratio of 1 ml: mixing 10mg of the mixture evenly, stirring the mixture for 30min at 37 ℃, and then adjusting the pH of the solution to 6 by using 0.1mol/L NaOH solution to obtain a mixed solution A;
(4) putting the dried sample obtained in the step (2) into the mixed solution A, taking out the sample after 5 seconds, and putting the sample into a constant-temperature drying oven at 37 ℃ until the liquid on the dried surface volatilizes;
(5) 12mg/ml of MgSO 4 Adjusting the pH of the solution to 6 by using 0.1mol/L NaOH solution, then putting the dried sample zinc obtained in the step (4) into the solution to be soaked for 5min, taking out the sample, and placing the sample in a constant-temperature drying oven at 37 ℃ until the surface liquid volatilizes;
(6) repeating the steps (4) and (5) for 5 times.
Example 2
A method for constructing a metal-organic molecule compound and inorganic phase hybrid functional coating on a degradable metal surface comprises the following steps:
(1) pre-treatment: sequentially polishing and flattening a pure magnesium sample by using 600#, 1200# and 2000# abrasive paper, cleaning for 3 times by using absolute ethyl alcohol under ultrasonic waves for 4 minutes each time, and drying at room temperature until surface liquid volatilizes;
(2) preparation of zinc phosphate primer layer: mixing 6mg/ml Na 2 HPO 4 And 1mg/ml Zn (NO) 3 )·6H 2 O solution according to the volume ratio of 5: 2, mixing, placing the mixture in a 50 ℃ water bath kettle for reaction for 2h, then placing the dried sample in the step (1) in the water bath kettle, heating the mixture at 60 ℃ for reaction for 1.5h, taking out the mixture, washing the mixture with water, and drying the mixture at room temperature until the surface liquid volatilizes;
(3) uniformly mixing ferulic acid, hydroxyethylidene diphosphate and water according to the material-liquid ratio of 1.5mg:1mg:1ml, stirring at 60 ℃ until the mixture is fully dissolved, and then cooling to 35 ℃ to obtain a mixed solution; then mixing the mixed solution with collagen according to the liquid-material ratio of 1 ml: 9mg, stirring the mixture for 40min at the temperature of 35 ℃, and then adjusting the pH of the solution to 6.5 by using 0.1mol/L NaOH solution to obtain a mixed solution A;
(4) putting the dried sample obtained in the step (2) into the mixed solution A, taking out after 6 seconds, putting the sample into a constant-temperature drying oven at 35 ℃ until the liquid on the dried surface volatilizes;
(5) 5mg/ml of MgSO 4 Adjusting the pH of the solution to 6.5 by using 0.1mol/L NaOH solution, then soaking the dried sample magnesium obtained in the step (4) in the solution for 6min, taking out the sample magnesium, and placing the sample magnesium in a constant-temperature drying oven at 35 ℃ until the surface liquid volatilizes;
(6) repeating the steps (4) and (5) for 4 times.
Example 3
A method for constructing a metal-organic molecule compound and inorganic phase hybrid functional coating on a degradable metal surface comprises the following steps:
(1) pre-treatment: polishing a pure iron sample to remove a surface oxide layer so as to expose pure iron, cleaning the pure iron sample for 3 times under ultrasound by using absolute ethyl alcohol for 6 minutes each time, and drying the pure iron sample at room temperature until surface liquid volatilizes;
(2) preparation of zinc phosphate primer layer: adding 9mg/ml Na 2 HPO 4 And 3mg/ml Zn (NO) 3 )·6H 2 O solution according to the volume ratio of 5: 2, placing the mixture in a water bath kettle at 50 ℃ for reaction for 2 hours, then placing the pure magnesium sample dried in the step (1) in the water bath kettle, heating the mixture at 40 ℃ for reaction for 2.5 hours, taking out the mixture, and washing the mixture with waterThen drying at room temperature until the surface liquid volatilizes;
(3) uniformly mixing ferulic acid, hydroxyethylidene diphosphate and water according to the material-liquid ratio of 5mg:2mg:1ml, stirring at 60 ℃ until the mixture is fully dissolved, and then cooling to 35 ℃ to obtain a mixed solution; then mixing the mixed solution with collagen according to the liquid-material ratio of 1 ml: mixing 12mg, stirring at 40 deg.C for 20min, and adjusting pH to 5.5 to obtain mixed solution A;
(4) putting the dried sample obtained in the step (2) into the mixed solution A, taking out the sample after 4 seconds, and putting the sample into a constant-temperature drying oven at 40 ℃ until the liquid on the dried surface volatilizes;
(5) adding 7mg/ml CuSO 4 Adjusting the pH of the solution to 5.5 by using 0.1mol/L NaOH solution, then soaking the dried sample iron obtained in the step (4) in the solution for 4min, taking out the sample iron, and placing the sample iron in a constant-temperature drying oven at 40 ℃ until the surface liquid volatilizes;
(6) repeating the steps (4) and (5) for 6 times.
Example 4
A method for constructing a metal-organic molecule compound and inorganic phase hybrid functional coating on a degradable metal surface comprises the following steps:
(1) pre-treatment: polishing and flattening pure zinc wafer with 600#, 1200# and 2000# abrasive paper in sequence, cleaning with anhydrous ethanol under ultrasound for 3 times and 5 minutes each time, and drying at room temperature until surface liquid volatilizes;
(2) preparation of zinc phosphate primer layer: mixing a 12mg/ml phosphoric acid solution and a 12mg/ml zinc nitrate solution according to a volume ratio of 5: 2, mixing, placing in a 50 ℃ water bath for reaction for 2h, then placing the zinc sheet dried in the step (1) in the water bath, heating at 50 ℃ for reaction for 2h, taking out, washing with water, and then placing in a 37 ℃ constant-temperature drying oven for drying until the surface liquid volatilizes;
(3) uniformly mixing ferulic acid, hydroxyethylidene diphosphate and water according to the material-liquid ratio of 1mg:0.5mg:1ml, stirring at 70 ℃ until the mixture is fully dissolved, and then cooling to 37 ℃ to obtain a mixed solution; then mixing the mixed solution with collagen according to the liquid-material ratio of 1 ml: 8mg, stirring the mixture for 30min at 37 ℃, and then adjusting the pH of the solution to 6 by using 0.1mol/L NaOH solution to adjust the pH of the solution to 6, thus obtaining a mixed solution A;
(4) putting the dried sample obtained in the step (2) into the mixed solution A, taking out the sample after 5 seconds, and putting the sample into a constant-temperature drying oven at 37 ℃ until the liquid on the dried surface volatilizes;
(5) mixing 6mg/ml ZnSO 4 Adjusting the pH of the solution to 6 by using 0.1mol/L NaOH solution, then putting the dried sample obtained in the step (4) into the solution to be soaked for 5min, taking out the sample, and placing the sample in a constant-temperature drying oven at 37 ℃ until the surface liquid volatilizes;
(6) repeating the steps (4) and (5) for 3 times.
The surface topography of the sample obtained in example 1 was observed by SEM as follows:
the prepared coating is shown in figure 1, and the surface of a zinc sheet polished by sand paper is flat and has scratches; the surface of the zinc is constructed with a metal-organic molecule compound and an inorganic phase hybridization functional layer, the surface is completely covered by collagen, and a complex formed by the coordination of collagen, ferulic acid, hydroxyl ethylidene diphosphate and magnesium ions can be seen on the surface of the collagen.
The infrared spectrum test was carried out by the tabletting method for collagen, ferulic acid and hydroxyethylidene diphosphate powder, and the infrared spectrum test by the reflection method for the zinc sheet obtained in example 1 having a surface built with a metal-organic molecular composite and an inorganic phase hybrid functional layer:
infrared spectroscopy (1469 cm) as shown in FIG. 2 -1 And 1646cm -1 The stretching vibration peaks of amide I bond and amide II bond appear at 1523cm from amino group in collagen -1 And 1686cm -1 The peaks respectively show characteristic peaks of C ═ O and C ═ C, which are derived from carboxyl and aromatic carbon-carbon double bond in ferulic acid, and are in 1193cm -1 The characteristic peak of P ═ O is shown and is derived from the phosphate group in hydroxyethylidene diphosphonic acid, which indicates that the metal-organic molecular compound and the inorganic phase hybridization functional layer have been successfully prepared on the surface of pure zinc.
The zinc sheet obtained in example 1 and having a surface built with a metal-organic molecular composite and an inorganic phase hybrid functional layer was subjected to potentiodynamic polarization curve test using an IM6 electrochemical workstation as follows:
and connecting the zinc sheet and the sample with the polished back surfaces to a lead by using conductive silver adhesive, and sealing the sample by using silicon rubber to expose the surface of the picture. And (3) putting the sample into a three-way pipe taking Hank's as electrolyte, wherein a saturated calomel electrode is taken as a reference electrode, metal platinum is taken as a contrast electrode, the sample is taken as a working electrode, and an IM6 electrochemical workstation is utilized to carry out potentiodynamic polarization curve test on the sample.
The polarization curve of the sample is shown in FIG. 3, and it can be seen that the zinc surface modified by the metal-organic molecular composite and the inorganic phase hybrid functional layer has a more positive self-corrosion potential E relative to pure zinc corr Meanwhile, the self-corrosion current is reduced, which shows that the modified coating can slow down the corrosion of the zinc substrate and control the corrosion mode.
The water contact angle and the surface energy of the zinc sheet surface-structured with the metal-organic molecule composite and the inorganic phase hybrid functional layer obtained in example 1 were tested as follows:
1.5 mu L of RO water is dripped on the pure zinc and the surface of a sample with a metal-organic molecular compound and inorganic phase hybridization functional layer constructed on the surface, and the water contact angle is photographed and tested.
2. 1 mu L of diiodomethane is dripped on the pure zinc and the surface of a sample with a metal-organic molecular compound and inorganic phase hybridization functional layer constructed on the surface, and the contact angle is tested.
The measured water contact angle and surface energy are shown in fig. 4, and it can be seen that the zinc water contact angle of the metal-organic molecular composite constructed on the surface and the inorganic phase hybridization functional layer is 12 °, and the surface energy is obviously increased compared with zinc, because collagen, ferulic acid and hydroxy ethylidene diphosphate have a large amount of hydrophilic groups, the surface energy is increased. Meanwhile, the complex formed with magnesium ions enables the surface of zinc to have a multi-scale micro-nano structure, the specific surface area is increased, and the surface energy is increased.
The calcium-phosphorus salt deposition results of the front and back surfaces of the metal-organic molecular composite and the inorganic phase hybrid functional layer constructed on the pure zinc surface in example 1 are tested as follows:
1. preparing a saturated calcium phosphate solution: 3.87mM CaCl 2 、2.32mM NH 4 H 2 PO 4 And 50mM NaCl solution at 25 deg.C, and adjusting pH with 50mM Tris solutionTo 7.2;
2. and (3) putting the sample into the saturated calcium phosphate solution for 10s, taking out, drying in a vacuum drying oven for 30min, repeating the steps for 10 times, putting the sample into the saturated calcium phosphate solution, standing for 12h at 37.5 ℃, cleaning and drying for later use.
The SEM observation of the sample showed that, as shown in fig. 5, the sample surface after the ion-crosslinked modified collagen functional layer was formed was significantly thicker than pure zinc, and showed a crystalline structure compared to the pure zinc control. This indicates that the sample after the surface construction of the metal-organic molecule composite and the inorganic phase hybridization functional layer promotes the deposition of calcium phosphate. The modified coating forms a compact structure, and functional groups in components such as collagen in the coating can better adsorb calcium phosphate.
The biocompatibility of the coating prepared in example 1 was evaluated as follows:
the cells used were mouse preosteoblasts (MC3T3-E1), mouse Endothelial Cells (EC), mouse Macrophages (MA), purchased from Wuhan Severe technologies, Inc. DMEM (seimer feishell instruments ltd, suzhou) supplemented with 10% FBS was used as the cell culture medium. The inoculation experiment can be carried out when the cells are cultured until about 80% of monolayer confluency, and the specific steps are as follows:
preparation of the experiment: sterilizing the consumables and samples required by the experiment (the consumables adopt high temperature and high pressure sterilization, the samples are subjected to ultraviolet sterilization, the front and the back surfaces are 30min respectively)
Taking out a cell culture bottle with a cell monolayer of more than 80% on a superclean workbench, pouring out a culture medium, washing the cell culture bottle with PBS for three times, sucking the cell culture bottle with a straw, uniformly adding 1ml of pancreatin into the cell culture bottle for digestion, adding 3ml of the culture medium after 1 minute to stop digestion and blow beating to prevent cell agglomeration, and finally adding a new culture medium to obtain a cell suspension with the cell density of 10000 cells/ml.
Inoculation: the cell suspension was applied dropwise to the surface of the sample with a pipette at 1ml per well, and then placed under 5% CO 2 Culturing in 37 deg.C constant temperature incubator, wherein osteoblast and endothelial cells are cultured for 6h, 12h, and 24h, and macrophageThe cells were cultured for 6h and 24 h.
Cell staining is carried out by using rhodamine to observe the growth form of cells, and the specific staining steps are as follows:
1. fixing: sucking the culture medium in a 24-pore plate, washing the culture medium with physiological saline for three times, and adding 2.5% glutaraldehyde for fixing for 4 hours;
2. dyeing: sucking out glutaraldehyde fixing solution, washing with normal saline for 3 times, and dripping 100 μ l rhodamine solution in dark condition for soaking for 2 min.
3. The number and state of cell adhesion were observed by fluorescence microscopy.
The specific steps for measuring the concentration of TNF-alpha inflammatory factor by Elisa kit are as follows:
1. setting a standard hole: and respectively adding 5 standard solutions with different concentrations into the ELISA plate for the kit, wherein each well is 50 mu L, and the concentrations of the standard solutions in the TNF-alpha kit are 480, 320, 160, 80 and 40 ng/L.
2. Setting a sample hole: firstly, adding 40 mul of sample diluent into a sample hole to be detected, and then adding 10 mul of sample to be detected;
3. adding an enzyme: adding 50 mul of enzyme-labeled reagent into each hole;
4. and (3) incubation: sealing the enzyme label plate with a sealing film, and putting the enzyme label plate into an incubator at 37 ℃ for incubation for 30 min;
5. reaction: taking out the ELISA plate, sucking out the liquid, adding washing liquid, washing for 5 times, respectively adding 50 μ l of color-developing agent A liquid and color-developing agent B liquid in the Elisa kit, keeping out of the sun, and placing in an incubator for reaction for 15 min;
6. terminating the reaction and measuring; after incubation, 50 mul of stop liquid is added into each hole, and the absorbance value of the solution at 450nm is measured by using an enzyme-linked immunosorbent assay, and the concentration is calculated.
Osteoblast cells were cultured as shown in FIGS. 6-7, and from the fluorescence images, it can be seen that the morphology of osteoblasts on the surface of unmodified pure zinc was shrunk, and the number of osteoblasts gradually decreased with the increase of the culture time. The cell number of the zinc after the metal-organic molecular compound and the inorganic phase hybridization functional layer is obviously more than that of pure zinc, the cells begin to spread after 6 hours of culture, the cell number increases with the increase of the culture time, and the shape is better. The endothelial cells are cultured as shown in fig. 8-9, the unmodified pure zinc surface endothelial cells basically die after being cultured for about 6 hours, the endothelial cells are spherical and do not start to adhere after the sample with the modified collagen-metal ion composite functional layer constructed on the surface is cultured for 6 hours, and the endothelial cells have spindle-shaped forms and are obviously increased in number when being cultured for 12 hours and 24 hours. The macrophage culture is shown in fig. 10-12, the macrophage morphology on the pure zinc surface has certain spreading after 6h of culture, the cell number has certain reduction after 24h of culture, the macrophage of the sample with the modified collagen-metal ion composite functional layer constructed on the surface is reduced after 6h of culture, and the cell number is obviously reduced after 24h, which indicates that the modified sample has more obvious ability of inhibiting the growth of the macrophage. The TNF-alpha content of the sample with the metal-organic molecule compound and the inorganic phase hybrid functional layer constructed on the surface is reduced to a certain extent relative to that of pure zinc, which shows that the coating modified sample can not cause the activation of macrophage to cause serious inflammatory reaction. The collagen has good biocompatibility to osteoblasts and endothelial cells, and the addition of the ferulic acid, the hydroxyethylidene diphosphonic acid and the magnesium ions can further enhance the biocompatibility of the osteoblasts and the endothelial cells on the basis of the collagen and can also inhibit the expression of inflammatory factors.
The antibacterial performance of the sample with the metal-organic molecular compound and the inorganic phase hybrid functional layer constructed on the surface is evaluated as follows:
1. preparation of the experiment: sterilizing consumables and samples required by the experiment (the consumables are sterilized at high temperature and high pressure, the samples are sterilized by ultraviolet, and the front and the back of each consumable are 30 min);
2. and (3) recovering bacteria: drawing lines on an LB plate to recover staphylococcus aureus and escherichia coli;
3. taking a single colony the next day, inoculating the single colony into a liquid culture medium, sucking bacterial liquid, and diluting a fresh bacterial suspension to an optical density of 0.02 (an absorbance value of 600 nm);
4. inoculation: sucking a proper volume of bacterial liquid to ensure that the total concentration of the thalli is 1 x 10 6 CFU/ml, adding bacterial suspension to the surface of sample 1ml per well by pipette, culturing at 37 deg.C for 24 hr, wherein no sample is addedBacterial suspension of the article was used as a negative control; after 24 the absorbance value of 100. mu.l of bacterial suspension from each sample was measured by absorbance value at a wavelength of 600nm and according to the formula AR (%) - (OD) control -OD sample )/OD control And (5) calculating the antibacterial rate. OD control Is the OD value of the stainless steel of the negative control group; OD sample Is the OD value of the sample set.
5. Transferring the sample into a sterile centrifuge tube containing 5ml of a bacterial liquid culture medium, carrying out vortex oscillation for 1min at the maximum rotation speed to remove bacteria on the surface, collecting bacterial suspension, diluting by 100 times, coating the bacterial suspension by using 30 mu l of bacterial liquid per dish, inverting the plate, putting the plate into an incubator at 37 ℃ for culturing for 12h, and counting bacterial colonies to calculate the antibacterial rate.
The results of colony count and antibacterial rate are shown in fig. 13-15, and it can be seen that the number of staphylococcus aureus and escherichia coli adhered to the surface of the sample after the metal-organic molecular composite and the inorganic phase hybrid functional layer are constructed on the surface is greatly reduced compared with the unmodified pure zinc. The result shows that the sample with the metal-organic molecular compound and the inorganic phase hybrid functional layer constructed on the surface has good antibacterial performance.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (7)
1. A method for constructing a metal-organic molecule compound and inorganic phase hybrid functional coating on a degradable metal surface is characterized by comprising the following steps:
(1) placing the pretreated degradable metal in zinc phosphate deposition solution, heating and reacting at 40-60 ℃ for 1.5-2.5 h, taking out, cleaning, and drying until the surface liquid volatilizes;
(2) mixing ferulic acid, hydroxyethylidene diphosphate and water according to the material-to-liquid ratio of 1-5 mg: 0.5-2 mg:1ml, stirring at 60-80 ℃ until the materials are fully dissolved, and then cooling to 35-40 ℃ to obtain a mixed solution; then mixing the mixed solution with collagen according to the liquid-material ratio of 1 ml: 8-12 mg of the mixture is mixed, stirred for 20-40 min at the temperature of 35-40 ℃, and then the pH of the solution is adjusted to 5.5-6.5, so as to obtain a mixed solution A;
(3) putting the degradable metal treated in the step (1) into the mixed solution A, taking out the degradable metal after 4-6 seconds, and drying until the surface liquid volatilizes;
(4) soaking the degradable metal treated in the step (3) in a coordination ion solution with the concentration of 5-12 mg/ml and the pH of 5.5-6.5 for 4-6 min, and then taking out and drying until the surface liquid volatilizes;
(5) repeating the steps (3) and (4) for 3-6 times;
the zinc phosphate deposition solution in the step (1) is prepared by the following steps: mixing 6-9 mg/ml sodium dihydrogen phosphate solution and 1-3 mg/ml zinc nitrate solution according to the volume ratio of 5: 2, or mixing 10-15 mg/ml phosphoric acid solution and 10-15 mg/ml zinc nitrate solution according to a volume ratio of 5: 2, mixing;
in the step (2), ferulic acid, hydroxy ethylidene diphosphate and water are mixed according to the material-liquid ratio of 3mg to 1.5mg to 1 ml;
in the step (2), the mixed solution and the collagen are mixed according to the liquid-material ratio of 1 ml: 10mg are mixed.
2. The construction method according to claim 1, wherein the pretreatment process of the degradable metal in the step (1) comprises the steps of: polishing the degradable metal, and then putting the metal into absolute ethyl alcohol for ultrasonic cleaning for 10-20 min.
3. The construction method according to claim 1, characterized in that: the coordination ion solution in the step (4) is a magnesium ion solution, a calcium ion solution, a ferrous ion solution, a copper ion solution, a zinc ion solution, a strontium ion solution or a chromium ion solution.
4. The construction method according to claim 3, wherein the magnesium ion solution is prepared by the following steps: dissolving anhydrous magnesium sulfate in water at 37 ℃, and stirring at 37 ℃ until the anhydrous magnesium sulfate is dissolved to prepare a magnesium ion solution of 5-12 mg/ml.
5. The construction method according to claim 1, characterized in that: the drying method is drying at 37 ℃ until the surface liquid volatilizes.
6. The degradable metal with the surface built with the metal-organic molecule compound and the inorganic phase hybridization functional coating prepared by the building method of any one of claims 1 to 5.
7. The use of the degradable metal with the surface built with the metal-organic molecule composite and inorganic phase hybrid functional coating according to claim 6 in the preparation of bone repair materials or cardiovascular disease prevention and treatment materials.
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| BR112020000750A2 (en) * | 2017-07-12 | 2020-07-14 | Termir, Inc. | antimicrobial composition, method of treatment and/or prevention of bacterial and/or fungal infection |
| CN110042375B (en) * | 2019-04-28 | 2020-05-19 | 西南交通大学 | Method for constructing organic-inorganic hybrid functional coating on surface of degradable absorption metal base layer |
| CN112826981A (en) * | 2021-01-04 | 2021-05-25 | 西南交通大学 | Method for preparing bone repair and regeneration promoting functional coating on degradable metal surface |
| CN113577383B (en) * | 2021-07-21 | 2022-10-14 | 西南交通大学 | Metal-organic/inorganic hybrid coating for promoting bone regeneration and regulating corrosion on degradable metal surface and preparation method thereof |
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| FR2838640A1 (en) * | 2002-04-19 | 2003-10-24 | Oreal | A method of cosmetic treatment of hair by application of a biotin derivative and a complexing agent where either or both are bonded to a group covalently bonding to hair and either or both to a cosmetically active group |
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