Green chemical method for growing carbonate film on surface of magnesium alloy

Abstract

The invention discloses a green chemical method for growing a carbonate film on the surface of a magnesium alloy. The green chemical method comprises the following steps: step 1, cleaning the surface of a magnesium material; 2, immersing the magnesium material into a carbonate buffer solution for surface treatment; step 3, adjusting the pH value; and 4, deionized water cleaning and drying are carried out. The method has the beneficial effects that the carbonate coating prepared on the surface of the pure magnesium or magnesium alloy material is environment-friendly, green, pollution-free, convenient and easy to obtain and obvious in corrosion-resistant effect, and the corrosion time can be adjusted as required; the composite material can be fused with a biological material or an organic material for coating, such as nucleic acid, drugs and the like, for slow release, so that the purposes of treating and regulating metabolism are achieved; the carbonate coating not only can be used for magnesium materials, but also can be used for corrosion-resistant surface treatment of other metal or organic materials and the like.

Description

Technical Field

[0001] The invention relates to the technical field of magnesium alloy surface processing, in particular to a green chemical method for growing a carbonate film on the surface of a magnesium alloy. Background Art

[0002] Mg is an intrinsic component of organisms and a trace element required by the human body. It can be naturally absorbed and excreted without any toxic effects. It is stored in bones and participates in the metabolic activities of various enzymes and coenzymes. Its mechanical strength and elastic modulus are close to those of human bones. Therefore, Mg implants can avoid the "stress shielding" effect that causes a decrease in bone density around the implant. As early as 1907, magnesium was used as a surgical implant. However, since metallic magnesium is easily dissolved in the body, this approach is usually limited to absorbable applications, such as absorbable cardiovascular stents.

[0003] Therefore, controlling the degradation rate of Mg-based biomaterials to apply them to implants remains a challenge. The most important issue is to control the degradation rate. The rapid degradation of Mg materials in physiological environments has been delayed and remedied through different surface modification methods, such as microelectrode oxidation, anodization, phosphate, electrodeposition or biomimetic deposition of calcium phosphate, fluoride chemical conversion and alkaline heat treatment; however, these methods and means still cannot meet clinical needs and requirements;

[0004] Ca 2 CO 3 It is a very important mineral compound in geological and environmental sciences. It is the most abundant biomineral and is found in geological sediments and marine sediments, as well as in many organisms and biominerals. The biomineralization of calcium carbonate is a natural process used by many marine organisms (such as corals) to grow skeletons and shells; such as pearls or mollusk shells, its functions include structural support (i.e. skeleton) and protection (i.e. shell). The natural formation of coral reefs is due to the slow biologically mediated precipitation of calcium carbonate in seawater; the calcium carbonate coating of artificial coral reefs promotes the attachment and growth of coral larvae and photosynthesis on these surfaces.

[0005] Ca 2 CO 3 Precipitation is not a simple process, as it occurs as an amorphous calcium carbonate (ACC) phase, different hydrated metastable phases, and three anhydrous crystalline polymorphs (calcite, aragonite, and wattstone); the mechanism of carbonate coating formation on magnesium surfaces involves the substitution of carbonate groups for hydroxyl groups on the Mg surface, as well as the formation of Ca 2 CO 3 Nucleation and growth of Ca 2 CO 3 The controlled mineralization of the ore requires further study;

[0006] Carbonate-coated areas can be effectively protected from corrosion attack in solution; therefore, carbonates have great potential for controlling the degradation rate of medical Mg devices in physiological fluids; however, research on the application of carbonate coatings in the surface modification of Mg alloys for medical applications is lacking;

[0007] A simple green conversion method has been used at room temperature to generate 2+ Growth of protective magnesium calcite coatings on pure Mg in carbonated water; we have achieved the growth of uniform and dense carbonate coatings in aqueous solution; but this is a complex problem due to (i) the almost instantaneous evolution of hydrogen bubbles, originating from the natural oxidation of Mg in water at atmospheric pressure, and (ii) the complex and difficult to predict growth due to the rapid formation of a complex mixture of oxides and hydroxides. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a green chemical method for growing a carbonate film on the surface of a magnesium alloy, comprising the following steps:

[0009] Step 1: Clean the surface of the magnesium material;

[0010] Step 2: The magnesium material is immersed in a carbonate buffer solution for surface treatment;

[0011] Step 3: Adjust the pH;

[0012] Step 4: Rinse with deionized water and dry.

[0013] As a further supplement to the technical solution, the magnesium material is pure magnesium or a magnesium alloy material.

[0014] As a further supplement to the technical solution, the working environment of step 2 is: immersing the magnesium material in the following carbonate buffer solution under standard laboratory conditions of 25°C and 1 atmosphere of pressure.

[0015] To further supplement this technical solution, the Ca in the carbonate buffer is 2+ The concentration is 5.7-7.7mmol / L, HCO3 － 23.1-31.0mmol / L, Mg 2+ 2.7-4.7mmol / L, Na + 1.6-3.6mmol / L, K + 0.21-0.41mmol / L,Cl － 2.5-3.5mmol / L; HPO4 － 0.3-0.4mmol / L and CO 2 36.8-56.8mmol / L.

[0016] To further supplement the technical solution, the surface cleaning in step 1 is to clean the magnesium material with acetone and anhydrous ethanol under ultrasound for 10 minutes each, then clean it with double distilled deionized water for 5 times, and dry it at 60°C for 1 hour.

[0017] As a further supplement to the technical solution, the pH in step three is adjusted to be between 7.5 and 11.9.

[0018] In a further supplement to the technical solution, the pH value is adjusted to 8.0.

[0019] Its beneficial effect is that the carbonate coating prepared on the surface of the magnesium material is environmentally friendly, green, pollution-free, easy to obtain, has obvious corrosion resistance, and the corrosion resistance time can be adjusted according to needs; it can also be fused with biological materials or organic materials, such as nucleic acids and drugs, for sustained release to achieve the purpose of treatment and regulation of metabolism, and can also be used for corrosion-resistant surface treatment of other metals or organic materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . Hydrogen evolution of the absorbable pure magnesium stent before and after it enters the carbonate aqueous solution; A: before the stent is immersed; B: 1 hour after the stent is immersed, rows of gas columns are seen on the stent surface, as well as bubbles attached to the stent surface;

[0021] Figure 2 .Optical images showing the evolution of pure Mg cubes during carbonation: A: before immersion and B: after 15 min, C: 1 h, D: 1 week of immersion time;

[0022] Figure 3 .XRD patterns showing the evolution of carbonation time of pure Mg flakes: (a) before immersion and (b) after 1 h, (c) 1 day, and (d) 1 week of immersion time (c: calcite; *: aragonite; m: Mg);

[0023] Figure 4 Raman spectroscopy shows the evolution of hydroxyl (3650 cm-1) and carbonate groups (1086 cm-1) in carbonate coatings formed on pure Mg with immersion time;

[0024] Figure 5 .A: Scanning electron microscope (SEM); B: 2D atomic force microscope (AFM) scan and C: 3D AFM image showing the surface morphology after 20 minutes of immersion, scanned in a 1 μm × 1 μm area;

[0025] Figure 6 .Shows the AFM images of pure Mg squares before immersion in carbonate solution (A); 15 minutes after immersion (B); 1 hour (C); 24 hours after immersion (D), the scale is (8μm×8μm);

[0026] Figure 7 The viability of NCTC mouse fibroblasts and HUVEC human umbilical vein endothelial cells cultured for 72 h in the presence of pure Mg and Mg-ACC for 1 h was evaluated by MTT assay;

[0027] Figure 8 Optical microscopy (Giemsa staining) and fluorescent staining images of mouse fibroblasts (NCTC) and human osteoblasts (SaOS-2): A: Giemsa staining of NCTC cells without Pure-Mg treatment (α), 30 min (β) and 1 h (γ); B: NaHCO staining of SaOS-2 cells 3 Giemsa staining of untreated (α), treated for 30 min (β) and 1 h (γ); C: Fluorescence staining of NCTC cells with Pure-Mg untreated (α), treated for 30 min (β) and 1 h (γ); D: NaHCO in SaOS-2 cells 3 Fluorescence staining of untreated (α), treated for 30 min (β) and 1 h (γ). DETAILED DESCRIPTION

[0028] In order to make the technical solution more clear to those skilled in the art, Figure 1 -8 The technical solution of the present invention is described in detail:

[0029] A green chemical method for growing a carbonate film on a magnesium alloy surface comprises the following steps:

[0030] Step 1: Clean the surface of the magnesium material;

[0031] Step 2: The magnesium material is immersed in a carbonate buffer solution for surface treatment;

[0032] Step 3: Adjust the pH;

[0033] Step 4: Rinse with deionized water and dry.

[0034] The magnesium material is pure magnesium or magnesium alloy material; the working environment of step 2 is: immersing the magnesium material in the following carbonate buffer solution under standard laboratory conditions of 25°C and 1 atmosphere pressure.

[0035] Among them, Ca in carbonate buffer 2+ The concentration is 5.7-7.7mmol / L, HCO3 － 23.1-31.0mmol / L, Mg 2+ 2.7-4.7mmol / L, Na + 1.6-3.6mmol / L, K +

[0036] 0.21-0.41mmol / L,Cl － 2.5-3.5mmol / L; HPO4 － 0.3-0.4mmol / L and CO 2

[0037] 36.8-56.8mmol / L.

[0038] Among them, step 1 surface cleaning is to clean the magnesium material with acetone and anhydrous ethanol under ultrasound for 10 minutes each, then clean it with double distilled deionized water for 5 times, and dry it at 60°C for 1 hour.

[0039] The pH value in step 3 is adjusted between 7.5 and 11.9, preferably, the pH value is adjusted to 8.0. The higher the pH value, the slower and thinner the carbonate coating grows, and the shorter the corrosion resistance time.

[0040] Example

[0041] 1. Surface carbonate modification method

[0042] The pure Mg intravascular stent was immersed in a carbonate buffer solution with the concentrations of Ca 2+ The concentration is 5.7-7.7mmol / L, HCO3 － 23.1-31.0mmol / L, Mg 2+ 2.7-4.7mmol / L, Na+1.6-3.6mmol / L, K + 0.21-0.41mmol / L,Cl － 2.5-3.5mmol / L; HPO4 － 0.3-0.4mmol / L and CO 2 36.8-56.8mmol / L; The concentrations of calcium and dissolved inorganic carbon (DIC) are key parameters of the surface modification method to facilitate the nucleation of calcium carbonate on the surface while avoiding its nucleation in solution; Calcium carbonate precipitates from the solution and directly precipitates on the surface, resulting in continuous and uniform lateral growth of the carbonate coating. Under normal laboratory conditions (~25°C, 1 atm) the solution is allowed to spontaneously reach equilibrium without composition or pH control, and modification of the metal surface occurs simultaneously.

[0043] Figure 1 A shows the pure Mg intravascular absorbable stent before immersion in a carbonic acid solution. Figure 1The image in B shows the bubble columns generated from the scaffold surface and the bubbles attached to the surface after immersion. Continuous gas release of the Mg scaffold was observed for more than one hour, gradually losing strength and emitting fewer and fewer bubbles. After immersion for 1 hour, single bubbles were observed instead of columns of bursting bubbles. After immersion for more than 24 hours, no significant gas release was observed.

[0044] 2. Surface evolution of pure magnesium squares in carbonate solution

[0045] When pure Mg squares are immersed in carbonate solutions, the first effect after immersion is a loss of metallic luster, which is almost immediately observed as the surface darkens to various shades of gray as the carbonate solution degasses ( Figure 2 B). Before immersion, the square has a metallic luster ( Figure 2 A). Visual inspection of the square discs showed that the grey surface of the Mg discs became homogenized as the immersion time increased during the first hour: after 1 hour ( Figure 2 C), the original cut line on the metal surface is barely noticeable. After more than an hour of immersion, the surface modification slows down and the evolution of bubbles also slows down. Leaving the squares in the solution for longer periods of time, from 1 day to 1 week, results in lighter colored irregular deposits, which can be clearly observed on most of the square surface after 1 week of immersion ( Figure 2 D).

[0046] 3. XRD characterization shows the formation mechanism of the crystalline phase

[0047] XRD characterization of the square pieces immersed in carbonated water revealed the formation mechanism of the surface crystalline phase. Figure 3 a shows before immersion; Figure 3 b shows the evolution of the XRD pattern of the exposed Mg square after one hour of immersion. The XRD pattern after one hour shows a prominent Mg peak, marked with "m" in the figure, corresponding to the main peak of calcite calcium carbonate -(014)-, accompanied by secondary calcium carbonate features -(018) and (116)-. Increasing the immersion time to one day ( Figure 3 c), resulting in an increase in the intensity of the main carbonate peak relative to the metal peak, which is still clearly observable. After one week, no metal peak can be noticed in the XRD pattern, instead, additional small peaks, marked with "*", appear in the pattern ( Figure 3 d), which can be indexed as aragonite. Aragonite is another allotrope of calcium carbonate (CaCO3) with regular rhombohedral symmetry. The relative intensity of the aragonite peak is small, so it can be estimated that the amount of aragonite is about an order of magnitude lower than the amount of calcite, which is the main crystalline phase on the surface of the square plate. We also discuss below that the crystallization of the calcite coating along the surface by the addition of oriented particles explains the two-dimensional growth of the calcite coating, rather than the height, until it completely covers the Mg surface.

[0048] 4. Determination of the evolution of hydroxyl and carbonate groups in the formation of Mg surface coating using Raman spectroscopy

[0049] We used Raman spectroscopy to determine the presence of hydroxyl and carbonate groups in the coating film formed on Mg. Raman spectroscopy is a very powerful technique for phase identification of any calcium carbonate as it can clearly identify and differentiate between ACC and CaCO 3 It is a rapid, non-destructive technique suitable for studying thin films and for mapping the spatial distribution of each carbonate phase with a lateral resolution of a few micrometers. In addition, Raman spectroscopy is an excellent analytical method to identify the presence or absence of hydroxyl groups in the symmetric stretching vibrations of hydroxyl groups at 3600-3700 cm-1. Our study shows the Raman spectrum of an amorphous hydroxide layer formed on Mg immediately after immersion in a carbonic acid solution. The presence of a characteristic sharp band at 3650 cm-1 indicates the formation of a hydroxide layer on the Mg surface. The Raman spectrum of calcite has a main band at 1086 cm-1 and secondary bands at 711, 1433 and 1746 cm-1, respectively, in calcite CO 3 Based on these surface reaction scenarios, we used Raman bands showing the evolution of the intensity at 3650 cm-1 and 1086 cm-1, corresponding to the presence of hydroxyl and carbonate groups, respectively, to assess the extent and kinetics of carbonate substitution for hydroxyl groups. Figure 4 The evolution of the intensity of two Raman bands as a function of immersion time is shown, showing the coating mechanism of carbonate formation.

[0050] 5. SEM and AFM show the morphological evolution of hydrochloride coating

[0051] Microscale morphological assessments revealed how calcite crystal layers grew by attaching carbonate (ACC) nanoparticles. Figure 5 The surface morphology of the carbonate coating formed after immersion in a carbonic acid solution for 20 minutes is shown. Scanning electron microscope (SEM) image ( Figure 5 a) shows the formation of submicron-sized aggregates formed from primary nanoparticles with a size of approximately 30 nm. Atomic force microscopy (AFM) image analysis was performed to determine the nanoscale topography of the coated samples immediately after immersion ( Figure 5 b), highlighting the nanoscale roughness / waviness of amorphous calcium carbonate (ACC) films. Figure 5 Two-dimensional (b, c) AFM images of the Mg square disk after immersion in a carbonate solution for 15 min are shown, with a scale of (1 μm × 1 μm), and Figure 5An example of a characteristic line scan (surface profile) in c. The nanoscale roughness of the ACC coating is reflected in the roughness parameters as follows: the peak-to-valley parameters of Rq = 36.9 nm and Rpv = 182.3 nm reflect the presence of steps between the nanoscale roughness and the aggregates of hundreds of nanoparticles. On the other hand, the AFM image shows the comprehensive coverage of the Mg surface by the ACC layer.

[0052] 6. AFM shows the morphological growth of Mg surface before and after carbonate treatment

[0053] Figure 6 A shows a 2D AFM image of a bare Mg square, scanned over an area of ​​5 μm × 5 μm, with “enhanced contrast”, and a typical profile, which is a line scan collected along the scan direction, using the red line markers in the figure to guide the scan head. The untreated Mg square exhibits a series of wrinkles and valleys, preferentially aligned along one direction, probably formed during the cutting process. However, it can be observed from the line scan in that the surface is flat, indicating that the surface features are located in the vertical range of -80 nm (see the vertical scale of the surface profile, from -40 to +40 nm). The RMS roughness of the image is 18.5 nm, while the average roughness value is equal to 14.1 nm.

[0054] Figure 6 B shows an AFM image of a Mg square after immersion in a carbonate solution for 15 minutes, with a scale of (8 μm × 8 μm). It shows a compact ACC layer produced by carbonation, forming a large amount of material aggregation covering the Mg surface, and a top layer of round protruding aggregates with a diameter of hundreds of nanometers, with increased roughness values, such as Rq = 328.7 nm, and a peak-to-valley parameter of Rpv ~ 1.93 μm for the entire area, estimated from the 2D AFM image in the figure, and therefore about 25 times higher than the bare Mg surface.

[0055] Figure 6 C shows the AFM image of the Mg square after immersion for 1 h, showing a completely different morphology (scanning an area of ​​8 μm × 8 μm), consisting of stacked polyhedral crystals of hundreds of nanometers, overlapping in the form of steps and terraces (similar to stairs). The spatial arrangement of the polyhedral crystals leads to the formation of a stepped surface, which is evidenced by the increased values ​​of the roughness parameters: Rq = 509.6 nm, Rpv = 3.69 μm.

[0056] Figure 6 D shows a 2D AFM image of Mg after 1 day of immersion: 8 μm × 8 μm; showing larger faceted crystals, the coating retains a compact morphology, but the appearance of some exposed crystal faces suggests a finer surface texture. However, at the (8 μm × 8 μm) scale, the roughness parameters are similar to the values ​​obtained for the sample immersed for 1 hour: Rq = 478.1 nm, Rpv = 3.21 μm.

[0057] 7. Study on Cell Viability of Carbonate Coatings

[0058] Cell biocompatibility and toxic effects were evaluated in two cell lines (NCTC and HUVEC) by MTT test to assess mitochondrial dehydrogenase activity and LDH test to investigate cell membrane integrity by quantifying the amount of LDH enzyme released into the culture medium after cell lysis. The results of MTT test on NCTC mouse fibroblasts showed that Mg and the material coated with calcite-type calcium carbonate after immersion for 1 hour, named Mg-CC1h, were found not to induce cytotoxic effects after two exposures for 72 hours. The cell viability percentages of the tests were higher than 99% ( Figure 7 ).

[0059] Cells cultured in the presence of Mg and Mg-CC1h were studied from the perspective of cell membrane integrity by measuring the amount of LDH released in the culture medium. The results obtained showed that the levels of LDH released in the culture medium were low and similar to those in the control samples, indicating that the integrity of the cell membrane and, therefore, the viability of the cells was not affected.

[0060] 8. Cell morphology study

[0061] like Figure 8 Aβγ showed no significant changes in morphology and cell density in the case of NCTC mouse fibroblasts cultured with Mg and Mg-CC1h extracts, and the appearance and density of the cells were normal and similar to those of the control samples ( Figure 8 Aα). The culture almost reached a cell monolayer, and the cells showed a normal, slightly polygonal appearance with 2-3 cytoplasmic extensions and fine cytoplasm. The results obtained with SaOS-2 human osteoblasts were similar to those observed with NCTC fibroblasts ( Figure 8 Bβγ). The cells showed a normal osteoblast-like appearance with only one nucleus and many nucleosomes. In addition, the cell density was similar to that of the control samples ( Figure 8 Bα), the culture covered about 70-75% of the surface of the square.

[0062] Cell morphology and viability were also assessed by fluorescence microscopy after staining live NCTC and SaOS-2 cells with calcein (green) and ethylenediaminetetraacetic acid (red) after 24 h of treatment with Mg and Mg-CC1h samples. NCTC mouse fibroblasts maintained their viability after 24 h of treatment, and few dead cells were observed ( Figure 8 Cβγ). In addition, the treated cells showed no morphological changes, maintained a normal morphology, and exhibited a density similar to that of cells in the control group ( Figure 8Cα). In SaOS-2 cells treated with Mg and carbonate-coated samples ( Figure 8 Dβγ), comparable results were observed, retaining a phenotype and cell density similar to that of the control ( Figure 8 Dα). These results are consistent with those obtained by quantitative MTT and LDH assays, indicating that the coatings treated with pure magnesium and carbonate have good biocompatibility and no toxic effects on NCTC, HUVEC and SaOS-2 cells.

[0063] The above technical solutions only reflect the preferred technical solutions of the technical solutions of the present invention. Some changes that may be made to certain parts thereof by technicians in this technical field all reflect the principles of the present invention and fall within the protection scope of the present invention.

Claims

1. A green chemical method for growing carbonate film on magnesium alloy surface, characterized in that: The following steps are involved: Step 1: Clean the surface of the magnesium material; Step 2: The magnesium material is immersed in a carbonate buffer solution for surface treatment; Step 3: Adjust the pH; Step 4: Rinse with deionized water and dry.

2. A green chemical method for growing carbonate film on the surface of magnesium alloy according to claim 1, characterized in that: The magnesium material is pure magnesium or a magnesium alloy material.

3. A green chemical method for growing carbonate film on magnesium alloy surface according to claim 2, characterized in that: The working environment of step 2 is: immersing the magnesium material in the following carbonate buffer solution under standard laboratory conditions of 25° C. and 1 atmosphere pressure.

4. A green chemical method for growing a carbonate film on a magnesium alloy surface according to claim 3, characterized in that: The carbonate buffer contains Ca 2+ The concentration is 5.7-7.7mmol / L, HCO3 － 23.1-31.0mmol / L, Mg 2+ 2.7-4.7mmol / L, Na + 1.6-3.6mmol / L, K + 0.21-0.41mmol / L,Cl － 2.5-3.5mmol / L; HPO4 － 0.3-0.4mmol / L and CO236.8-56.8mmol / L.

5. The green chemical method for growing carbonate film on magnesium alloy surface according to claim 1, characterized in that: In the step 1, surface cleaning, the magnesium material is cleaned with acetone and anhydrous ethanol for 10 minutes each under ultrasound, then cleaned with double distilled deionized water for 5 times, and dried at 60° C. for 1 hour.

6. The green chemical method for growing carbonate film on the surface of magnesium alloy according to claim 1, characterized in that: The pH in step three is adjusted to be between 7.5 and 11.

9.

7. A green chemical method for growing a carbonate film on a magnesium alloy surface according to claim 6, characterized in that: The pH value was adjusted to 8.0.

Images