CN115838925A - A modified zinc alloy whose surface is coated with a zinc phosphate film layer and its preparation method and application - Google Patents

Abstract

The invention belongs to the field of surface modification of biomedical metal materials, and in particular relates to a modified zinc alloy whose surface is coated with a zinc phosphate film layer and a preparation method and application thereof. The invention provides a modified zinc alloy whose surface is coated with a zinc phosphate film layer, which comprises a zinc phosphate chemical conversion film that uses zinc alloy as a substrate and is attached to the surface of the zinc alloy substrate; the main phase composition of the zinc phosphate chemical conversion film is as follows: zinc phosphate. Utilizing the zinc phosphate chemical conversion film of the present invention can inhibit the excessive release of zinc ions in the process of zinc alloy degradation, realize effective protection of the zinc alloy substrate, improve the biocompatibility and antibacterial performance of the zinc alloy, and further improve the zinc alloy substrate implantation. The effect of in vivo clinical application has potential significance.

Description

A modified zinc alloy whose surface is coated with a zinc phosphate film layer and its preparation method and application

This application is a divisional application with an application date of July 13, 2021, an application number of 202110789515.4, and an invention title of "A Zinc Phosphate Chemical Conversion Coating on the Surface of a Zinc Alloy and Its Preparation and Application".

technical field

The invention relates to the field of surface modification of biomedical metal materials, in particular to a modified zinc alloy whose surface is coated with a zinc phosphate film layer and a preparation method and application thereof.

Background technique

The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

Zinc and zinc alloys have become a promising biodegradable metal because of their important physiological functions and good degradation properties. The strength and plasticity of zinc alloy are significantly improved, making up for the lack of pure zinc in mechanical properties. However, during the degradation process, due to the excessive release of zinc ions, the cytocompatibility and antibacterial properties of zinc alloys are affected. Therefore, surface modification of zinc alloys to endow them with biofunctionalized surfaces is an effective means to further improve their cytocompatibility and antibacterial properties. The phosphate chemical conversion method can form an insoluble film on the metal surface through chemical and electrochemical reactions at the interface between the base metal and the solution. The film prepared by this method is mainly composed of phosphate phase. By designing the conversion process parameters, it can be obtained Film layer system composed of different phases, components and structures.

Zinc is an essential element in the human body and is involved in many important physiological functions. Zinc ions can promote the differentiation of osteoblasts, inhibit the differentiation of osteoclasts, and play an important role in stimulating bone growth and mineralization. In addition, zinc can inhibit bacterial infection and maintain cardiac function through the redox signaling pathway. Therefore, the preparation of zinc-containing phosphate conversion coatings on the surface of degradable zinc alloys can endow zinc alloys with biofunctional surfaces and improve their biocompatibility and antibacterial properties. The surface morphology and structure of materials can significantly affect blood compatibility, cytocompatibility and antibacterial properties. It has been reported that micro/nano-scale coating structures can significantly improve the viability of preosteogenic cells and vascular endothelial cells, Adhesion and differentiation, significantly reducing the adhesion of platelets and Escherichia coli, and the biofunctionalized film layer can improve the biocompatibility and antibacterial performance of the material and control the degradation rate.

In the prior art, there are many methods for preparing chemical conversion coatings on the surface of titanium and titanium alloys. For example, the titanium substrate is pretreated with a strong alkali solution and then chemically converted, and finally a titanium-zinc phosphate chemical conversion coating is obtained by heat treatment; the chemical conversion of zinc phosphate is completed by applying an electric current field, and a zinc phosphate conversion coating is obtained on the titanium surface; The treated base titanium or titanium alloy is coupled with a metal with a relatively low corrosion potential, and after coupling, a chemical conversion film on the surface of titanium and titanium alloy is obtained through chemical conversion.

At present, the methods for preparing chemical conversion coatings on the surface of titanium and titanium alloys are relatively mature, but how to prepare zinc phosphate chemical conversion coatings on the surface of zinc alloys has not been reported yet.

Contents of the invention

In order to solve the problems in the prior art, the present invention provides a modified zinc alloy whose surface is coated with a zinc phosphate film layer and its preparation method and application. By adding film-forming substances to the chemical liquid, the phosphoric acid on the surface of the degradable zinc alloy can be realized. Preparation of Zinc Chemical Conversion Coatings. Through the coating of the zinc phosphate film layer, the biocompatibility and antibacterial performance of the zinc alloy matrix are further improved, and its clinical application effect is improved.

In order to solve the above technical problems, the technical solution of the present invention is:

The first aspect of the present invention provides a modified zinc alloy whose surface is coated with a zinc phosphate film layer, including a zinc phosphate chemical conversion coating that uses a zinc alloy as a substrate and is attached to the surface of the zinc alloy substrate; the zinc phosphate chemical conversion coating The main phase composition is zinc phosphate.

A second aspect of the present invention provides a method for preparing a modified zinc alloy whose surface is coated with a zinc phosphate film layer, comprising the following steps:

Prepare chemical conversion liquid, said chemical conversion liquid includes phosphoric acid solution, nitric acid solution and accelerator;

subjecting the chemical conversion liquid to slaking treatment of reduced iron powder to obtain a slaking liquid;

Coupling the cleaned and surface-adjusted zinc alloy substrate with iron clips, soaking it in the aging solution for chemical conversion treatment, coating the surface of the zinc alloy substrate with a zinc phosphate chemical conversion film, and obtaining the surface-coated phosphoric acid Modified zinc alloy of zinc film layer;

The surface adjustment includes the following steps: soaking in 2-5g/L colloidal titanium solution for 20-40s; the temperature of the zinc alloy substrate surface adjustment is 20-35°C.

The third aspect of the present invention provides the application of the above modified zinc alloy coated with zinc phosphate film layer or the modified zinc alloy coated with zinc phosphate film layer prepared by the above preparation method in the preparation of implant materials.

One or more embodiments of the present invention have the following beneficial effects:

(1) Starting from the unique biological function of zinc element, the present invention prepares a biologically functional film layer composed of zinc phosphate crystal phase on the surface of zinc alloy through a phosphate chemical conversion method. The chemical conversion coating endows the zinc alloy with a biofunctional surface, improving its biocompatibility and antibacterial properties.

(2) The present invention endows the zinc alloy substrate with a biologically functional surface, and the prepared zinc phosphate coating is not only compact and uniform, but also has fine zinc phosphate crystal particles, which can effectively protect the zinc alloy substrate and inhibit the excessive release of zinc ions , thereby improving the biocompatibility and antibacterial properties of zinc alloys, which has potential significance for further improving the clinical application effect of zinc alloy matrix implants.

(3) The preparation method of the present invention can be completed at normal temperature, the process is simple, the cost is low, the obtained film layer is uniform and complete, and the crystal grains are fine and dense.

(4) In the present invention, the zinc ions are provided by the zinc alloy matrix, and no external zinc source is required. By adjusting the conversion time, the preparation of the continuous zinc phosphate film can be realized without changing the film element and phase composition.

Description of drawings

The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention.

Fig. 1 is an X-ray diffraction pattern of a zinc phosphate chemical conversion coating on the surface of a zinc alloy prepared in Examples 1-4 of the present invention.

Fig. 2 is the field emission scanning electron microscope image of the zinc phosphate chemical conversion coating on the zinc alloy surface prepared by Examples 1-4 of the present invention;

A among Fig. 2 is the field emission scanning electron microscope image of the zinc phosphate chemical conversion coating on the surface of the zinc alloy prepared in Example 1,

Among Fig. 2, B is the field emission scanning electron microscope image of the zinc phosphate chemical conversion coating on the zinc alloy surface prepared in Example 2,

C among Fig. 2 is the field emission scanning electron microscope image of the zinc phosphate chemical conversion coating on the zinc alloy surface prepared by embodiment 3,

D in FIG. 2 is a field emission scanning electron microscope image of the zinc phosphate chemical conversion coating on the surface of the zinc alloy prepared in Example 4.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

In order to solve the problem that the zinc alloy's cell compatibility and antibacterial properties are affected due to the excessive release of zinc ions during the degradation process of the zinc alloy.

The first aspect of the present invention provides a chemical conversion solution for forming a zinc phosphate chemical conversion film on a zinc alloy. The chemical conversion solution includes a phosphoric acid solution and a nitric acid solution. During the preparation process of the conversion coating on the surface of titanium and titanium alloy substrates, a dense oxide film is easily formed on the surface of the substrate. Due to the protective effect of the dense oxide film, the titanium and titanium alloy substrates only provide the potential difference during electrical coupling, but do not participate in the transformation reaction. The inventors found that the degradable zinc alloy can provide a certain amount of zinc ions through degradation during the conversion process, and this part of zinc ions can directly participate in chemical reactions to form a chemical conversion film. The chemical conversion solution of the present application does not provide zinc ions, and the zinc phosphate chemical conversion coating of the present application uses a zinc alloy matrix as the material providing zinc ions, and no external zinc source is required. The composition of the chemical conversion solution of the present application is simple, which can prevent the introduction of other cations to participate in the formation of the film layer, thereby interfering with the formation of the zinc phosphate crystal phase. The nitric acid solution not only participates in the film formation, but the introduced NO-3 can act as an accelerator without affecting the phase composition of the film layer.

In some embodiments, the chemical conversion liquid further includes an accelerator, preferably, the accelerator is Ca(NO ₃ ) ₂ ·4H ₂ O, NaClO ₃ , NaNO ₃ , C ₆ H ₈ O ₇ ·H ₂ One or more of O; preferably, the concentration of each substance in the chemical conversion liquid is: phosphoric acid 0.1-0.3 mol/L, nitric acid 0.3-0.6 mol/L, accelerator 0.01-0.1 mol/L. Adding an accelerator can speed up the reaction process, shorten the reaction time, and improve the density and uniformity of the film layer while promoting the film formation, so as to realize the optimization of the microstructure of the film layer. The substances that participate in the film formation are H ₃ PO ₄ and HNO ₃ . While providing phosphate, it is necessary to avoid introducing other cations to participate in the formation of the film layer and interfere with the formation of the zinc phosphate crystal phase. Zn ions can participate in the formation of the film, and Na ions do not participate in the formation of the film. So as not to interfere with the reaction process.

The second aspect of the present invention provides a method for preparing a zinc phosphate conversion film on the surface of a zinc alloy, using the above-mentioned chemical conversion liquid to prepare a zinc phosphate conversion film on the surface of a zinc alloy by a chemical conversion method;

Preferably, the substance providing phosphate ions is H ₃ PO ₄ ; the substance providing zinc ions is a zinc alloy matrix.

In some embodiments, the chemical conversion solution is treated with reduced iron powder to obtain a slaking solution, and the zinc alloy substrate is coupled with iron clips and soaked in the slaking solution for chemical conversion treatment to obtain a chemical conversion film;

Preferably, the method for slaking the reduced iron powder in the chemical conversion liquid is as follows: add 1-5 g of reduced iron powder to the chemical conversion liquid, and let it stand for slaking at 20-35°C for 12-24 hours to obtain the slaking liquid;

Preferably, the temperature of the chemical conversion treatment is 20-35° C., and the conversion time is 30 s-60 min. Further preferably, the conversion time is 30s-15min. After aging the chemical conversion liquid through the reduced iron powder, the obtained aging liquid contains a certain amount of ferrous ions. Ferrous ions can play a specific catalytic role, accelerate the chemical transformation process, and then promote the formation of phosphate crystal phase on the surface of the zinc alloy substrate.

In some embodiments, a zinc phosphate chemical conversion film on the surface of a degradable zinc alloy and a preparation method thereof include the following steps:

Preparation of chemical conversion liquid, mixing phosphorus-containing compounds and accelerators to obtain chemical conversion liquid;

Subjecting the above chemical conversion liquid to a reduced iron powder slaking treatment to obtain a slaking liquid;

Coupling the cleaned and polished zinc alloy substrate with iron clips for surface adjustment;

The treated zinc alloy substrate is soaked in a curing solution for chemical conversion treatment to obtain a chemical conversion film. The zinc phosphate chemical conversion coating on the surface of the degradable zinc alloy is realized by adding film-forming substances and accelerators to the conversion solution.

In some embodiments, the cleaned and surface-adjusted zinc alloy substrate is soaked in a curing solution;

Preferably, the method for cleaning the zinc alloy substrate includes the following steps: ultrasonically clean the polished zinc alloy substrate with acetone, ethanol, and deionized water at room temperature for 10-15 minutes; then clean it with deionized water;

Preferably, the method for adjusting the surface of the zinc alloy substrate includes the following steps: soaking in 2-5g/L colloidal titanium solution for 20-40s;

Preferably, the adjusted temperature on the surface of the zinc alloy substrate is 20-35°C.

In some embodiments, before the chemical conversion treatment, the pH of the aging solution is adjusted to 2-3 with phosphoric acid or sodium hydroxide solution.

In some embodiments, the chemical conversion treatment is performed under ultrasonic field conditions;

Preferably, the ultrasonic power is 120-300W.

In some embodiments, the step of washing and drying the product is also included, the washing is rinsing with deionized water, and the drying temperature is 20-40°C. When the temperature is too low, the drying is slow, and when the temperature is too high, the film layer dries rapidly, which may cause some microcracks in the film layer.

The invention discloses a zinc phosphate chemical conversion film on the surface of a degradable zinc alloy and a preparation method thereof, which comprises the following steps: preparing a chemical conversion solution containing phosphate ions, nitrate ions and accelerators; powder aging treatment to obtain a curing solution; the cleaned and surface-activated zinc alloy substrate is coupled with iron clips and soaked in the curing solution for chemical conversion treatment to obtain a chemical conversion film. By designing the conversion time, the preparation of the zinc phosphate film on the surface of the degradable zinc alloy was realized. Zinc phosphate chemical conversion coating can inhibit the excessive release of zinc ions during the degradation process of zinc alloy, realize effective protection of zinc alloy matrix, improve the biocompatibility and antibacterial performance of zinc alloy, and further improve the clinical application of zinc alloy matrix implants. Applied effects are potentially meaningful.

The third aspect of the present invention provides a zinc phosphate chemical conversion coating on the surface of zinc alloy, the main phase composition is zinc phosphate (Hopeite, Zn ₃ (PO ₄ ) ₂ ·4H ₂ O); preferably, it is prepared by the above method .

The fourth aspect of the present invention provides a surface-modified zinc alloy, comprising a zinc alloy substrate and a zinc phosphate chemical conversion coating attached to the surface of the zinc alloy substrate.

The fifth aspect of the present invention provides the application of the above-mentioned surface-modified zinc alloy as an implant material.

Example 1

(1) Measure 30ml of HNO ₃ , 17ml of H ₃ PO ₄ , 2g of NaClO ₃ , 5g of Ca(NO ₃ ) ₂ 4H ₂ O, 5g of C ₆ H ₈ O ₇ .H ₂ O, dissolve in 1L of deionized water and mix well. Obtain chemical conversion base liquid;

(2) Weigh 1 g of reduced iron powder, pickle it with 7% v/v H ₃ PO ₄ for 5 minutes, add it to the chemical conversion base solution, let it stand at room temperature for aging for 24 hours, filter and take the supernatant to obtain the aging solution;

(3) Use 10wt% H ₃ PO ₄ or 5mol/L NaOH solution to adjust the pH value of the aging solution to 2.5;

(4) Clean the polished zinc alloy substrate sequentially with acetone, ethanol, and deionized water at room temperature for 10 minutes, and place it in a 3 g/L colloidal titanium solution for 30 seconds at room temperature after washing;

(5) Coupling the zinc alloy substrate treated in step (4) with iron clips, then soaking in the curing solution, and treating it in a 300W ultrasonic field environment for 1min at room temperature;

(6) Rinse the product obtained in step (5) with deionized water for several times, and dry at room temperature for 1 hour to obtain a zinc phosphate chemical conversion coating on the surface of the zinc alloy.

Example 2

(1) Measure 30ml of HNO ₃ , 17ml of H ₃ PO ₄ , 2g of NaClO ₃ , 5g of Ca(NO ₃ ) ₂ 4H ₂ O, 5g of C ₆ H ₈ O ₇ .H ₂ O, dissolve in 1L of deionized water and mix well. Obtain chemical conversion base fluid;

(2) Weigh 1 g of reduced iron powder, pickle it with 7% v/v H ₃ PO ₄ for 5 minutes, add it to the chemical conversion base solution, let it stand at room temperature for aging for 24 hours, filter and take the supernatant to obtain the aging solution;

(3) Use 10wt% H ₃ PO ₄ or 5mol/L NaOH solution to adjust the pH value of the aging solution to 2.5;

(4) Clean the polished zinc alloy substrate sequentially with acetone, ethanol, and deionized water at room temperature for 10 minutes, and place it in a 3 g/L colloidal titanium solution for 30 seconds at room temperature after washing;

(5) Coupling the zinc alloy substrate treated in step (4) with iron clips, then soaking in the curing solution, and treating it in a 300W ultrasonic field environment for 5 minutes at room temperature;

(6) Rinse the product obtained in step (5) with deionized water for several times, and dry at room temperature for 1 hour to obtain a zinc phosphate chemical conversion coating on the surface of the zinc alloy.

Example 3

(1) Measure 30ml of HNO ₃ , 17ml of H ₃ PO ₄ , 2g of NaClO ₃ , 5g of Ca(NO ₃ ) ₂ 4H ₂ O, 5g of C ₆ H ₈ O ₇ .H ₂ O, dissolve in 1L of deionized water and mix well. Obtain chemical conversion base fluid;

(2) Weigh 1 g of reduced iron powder, pickle it with 7% v/v H ₃ PO ₄ for 5 minutes, add it to the chemical conversion base solution, let it stand at room temperature for aging for 24 hours, filter and take the supernatant to obtain the aging solution;

(3) Use 10wt% H ₃ PO ₄ or 5mol/L NaOH solution to adjust the pH value of the aging solution to 2.5;

(4) Clean the polished zinc alloy substrate sequentially with acetone, ethanol, and deionized water at room temperature for 10 minutes, and place it in a 3 g/L colloidal titanium solution for 30 seconds at room temperature after washing;

(5) Connect the zinc alloy substrate treated in step (4) with iron clips, then soak in the curing solution, and treat it in a 300W ultrasonic field environment for 10 minutes at room temperature;

(6) Rinse the product obtained in step (5) with deionized water for several times, and dry at room temperature for 1 hour to obtain a zinc phosphate chemical conversion coating on the surface of the zinc alloy.

Example 4

(1) Measure 30ml of HNO ₃ , 17ml of H ₃ PO ₄ , 2g of NaClO ₃ , 5g of Ca(NO ₃ ) ₂ 4H ₂ O, 5g of C ₆ H ₈ O ₇ .H ₂ O, dissolve in 1L of deionized water and mix well. Obtain chemical conversion base liquid;

(2) Weigh 1 g of reduced iron powder, pickle it with 7% v/v H ₃ PO ₄ for 5 minutes, add it to the chemical conversion base solution, let it stand at room temperature for aging for 24 hours, filter and take the supernatant to obtain the aging solution;

(3) Use 10wt% H ₃ PO ₄ or 5mol/L NaOH solution to adjust the pH value of the aging solution to 2.5;

(4) Clean the polished zinc alloy substrate sequentially with acetone, ethanol, and deionized water at room temperature for 10 minutes, and place it in a 3 g/L colloidal titanium solution for 30 seconds at room temperature after washing;

(5) Coupling the zinc alloy substrate treated in step (4) with iron clips, then soaking in the curing solution, and treating it in a 300W ultrasonic field environment for 15 minutes at room temperature;

(6) Rinse the product obtained in step (5) with deionized water for several times, and dry at room temperature for 1 hour to obtain a zinc phosphate chemical conversion coating on the surface of the zinc alloy.

Figure 1 describes the X-ray diffraction pattern of the zinc phosphate chemical conversion coating on the surface of the zinc alloy. It can be seen from the spectrum analysis that the phase composition of the conversion film obtained in Examples 1-4 (that is, under the conditions of different conversion times in the conversion liquid) is zinc phosphate crystal phase (Hopeite, Zn ₃ (PO ₄ ) ₂ ·4H ₂ O) .

Fig. 2 is a field emission scanning electron microscope image of a zinc phosphate chemical conversion film on the surface of a zinc alloy. Figure 2.A, Figure 2.B, Figure 2.C, and Figure 2.D correspond to the morphology characteristics of the zinc phosphate conversion coating obtained in Example 1, Example 2, Example 3, and Example 4, respectively. It can be seen from the figure that when the conversion time is short, the zinc phosphate conversion film can evenly cover the surface of the zinc alloy substrate, the grains are fine and uniform, and the film layer is dense and complete. As the reaction time was extended to 10min, the film layer became thicker, and some areas of the film layer fell off. But during this period, the change of conversion time has little effect on the morphology of zinc phosphate conversion coating. When the reaction time was 15 minutes, the morphology of the zinc phosphate film changed. At this time, the film could tightly cover the zinc alloy substrate, but the film still fell off in some areas. The above phenomena indicate that the preparation of the zinc phosphate chemical conversion coating on the surface of the zinc alloy substrate can be realized in a relatively short period of time.

What is disclosed above is only a preferred embodiment of the present invention, and of course it cannot limit the scope of rights of the present invention. Therefore, equivalent changes made according to the patent scope of the present invention still fall within the scope of the present invention.

Claims (9)

1. A modified zinc alloy whose surface is coated with a zinc phosphate film layer, comprising a zinc alloy as a substrate and a zinc phosphate chemical conversion coating coated on the surface of the zinc alloy substrate; The main phase composition of the zinc phosphate chemical conversion coating is zinc phosphate. 2. a preparation method of a modified zinc alloy whose surface is coated with a zinc phosphate film layer, comprising the following steps: Prepare chemical conversion liquid, said chemical conversion liquid includes phosphoric acid solution, nitric acid solution and accelerator; subjecting the chemical conversion liquid to slaking treatment of reduced iron powder to obtain a slaking liquid; Coupling the cleaned and surface-adjusted zinc alloy substrate with iron clips, soaking it in the aging solution for chemical conversion treatment, coating the surface of the zinc alloy substrate with a zinc phosphate chemical conversion film, and obtaining the surface-coated phosphoric acid Modified zinc alloy of zinc film layer; The surface adjustment includes the following steps: soaking in 2-5g/L colloidal titanium solution for 20-40s; the temperature of the zinc alloy substrate surface adjustment is 20-35°C. 3. The preparation method according to claim 2, characterized in that, the accelerator is Ca(NO ₃ ) ₂ ·4H ₂ O, NaClO ₃ , NaNO ₃ and C ₆ H ₈ O ₇ ·H ₂ O one or more; The concentration of each substance in the chemical conversion liquid is: phosphoric acid 0.1-0.3 mol/L, nitric acid 0.3-0.6 mol/L, accelerator 0.01-0.1 mol/L. 4. The preparation method according to claim 2, characterized in that, the aging treatment is as follows: adding 1-5g of reduced iron powder to the chemical conversion liquid, standing and aging at 20-35°C for 12-24h to obtain aging liquid; The temperature of the chemical conversion treatment is 20-35°C, and the conversion time is 30s-60min. 5. The preparation method according to claim 2, wherein said cleaning comprises the steps of: ultrasonically cleaning the polished zinc alloy substrate with acetone, ethanol, and deionized water at room temperature for 10-15min successively; Rinse with deionized water. 6. The preparation method according to claim 2, characterized in that, before the chemical conversion treatment, the pH value of the aging solution is adjusted to 2-3 with phosphoric acid or sodium hydroxide solution. 7. The preparation method according to claim 2, characterized in that, after the chemical conversion treatment, it also includes the step of cleaning and drying the obtained product, the cleaning is rinsing with deionized water, and the drying temperature is 20-40°C . 8. The preparation method according to claim 2, characterized in that, the chemical conversion treatment is carried out under the condition of an ultrasonic field; the power of the ultrasonic field is 120-300W. 9. the modified zinc alloy of the surface coating zinc phosphate film layer described in claim 1 or the modified zinc alloy of the surface coating zinc phosphate film layer that the preparation method described in any one of claim 2～8 makes Applications in body materials.

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