Preparation method of articular cartilage three-dimensional nanofiber scaffold

Abstract

The invention discloses a preparation method of an articular cartilage three-dimensional nanofiber scaffold, and relates to the technical field of medical biological materials. The method comprises the following steps: dissolving a degradable polymer and a natural polymer material in a solvent to prepare a spinning solution; performing electrostatic spinning on the spinning solution to obtain a cartilage scaffold two-dimensional nanofiber membrane; performing foaming treatment on the cartilage scaffold two-dimensional nanofiber membrane, and converting the cartilage scaffold two-dimensional nanofiber membrane into a three-dimensional nanofiber cartilage scaffold; and soaking the freeze-dried three-dimensional nanofiber cartilage scaffold in a mixed solution of epigallocatechin gallate and SrCl2 for chemical modification, and freeze-drying the three-dimensional nanofiber cartilage scaffold to obtain a finished product of the three-dimensional nanofiber cartilage scaffold. The articular cartilage three-dimensional nanofiber cartilage scaffold prepared by the method can effectively simulate a three-dimensional ECM structure of natural cartilage, and cartilage regeneration is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical biomaterials, in particular to a preparation method of a three-dimensional nanofiber scaffold for articular cartilage. BACKGROUND

[0002] Articular cartilage defects caused by osteoarthritis, trauma or other diseases can greatly affect the quality of life of patients and can lead to serious disability. Due to the avascular and aneural structure of cartilage, its self-repairing ability is extremely limited, and new technologies are urgently needed to assist and promote the repair of damaged cartilage. Tissue engineering combines engineering technology with life science, aiming to restore or replace damaged tissues or organs. In this field, cartilage scaffolds play a crucial role. The raw materials of cartilage scaffolds can be derived from natural or synthetic substances, which simulate the extracellular matrix (ECM) of natural cartilage, provide binding sites for cells, support their proliferation, and ultimately form new tissues with similar structure and function to healthy cartilage.

[0003] In the preparation technology of cartilage scaffolds, electrospinning is widely used in the construction of cartilage tissue engineering scaffolds due to its wide material adaptability and high production efficiency. However, the nanofiber cartilage scaffolds produced by traditional electrospinning technology are usually dense two-dimensional nanofiber membranes, which are difficult for cells to penetrate, cannot effectively simulate the three-dimensional ECM structure of natural cartilage, and cannot achieve effective cartilage regeneration. SUMMARY

[0004] In view of the defects in the prior art, the technical problem to be solved by the present application is to provide a preparation method of a three-dimensional nanofiber cartilage scaffold for articular cartilage, which can effectively simulate the three-dimensional ECM structure of natural cartilage and is beneficial to cartilage regeneration.

[0005] To solve the above technical problems, the present application provides a preparation method of a three-dimensional nanofiber scaffold for articular cartilage, characterized in that it comprises the following steps:

[0006] S1: dissolving a degradable polymer and a natural high molecular material in a solvent to prepare a spinning solution;

[0007] The degradable polymer for preparing the spinning solution is selected from at least one of polylactic acid-caprolactone copolymer and polycaprolactone;

[0008] The natural high molecular material for preparing the spinning solution is selected from at least one of silk fibroin and collagen;

[0009] The solvent for preparing the spinning solution is selected from at least one of hexafluoroisopropanol and dichloromethane;

[0010] S2: electrospinning the prepared spinning solution in step S1 through an electrostatic spinning device to obtain a cartilage scaffold two-dimensional nanofiber membrane;

[0011] S3: foaming the cartilage scaffold two-dimensional nanofiber membrane obtained in step S2 by using a sodium borohydride aqueous solution to obtain a three-dimensional nanofiber cartilage scaffold after foaming;

[0012] S4: freeze-drying the three-dimensional nanofiber cartilage scaffold obtained in step S3;

[0013] S5: chemically modifying the three-dimensional nanofiber cartilage scaffold after freeze-drying by immersing it in a mixture solution of epigallocatechin gallate and SrCl2;

[0014] S6: freeze-drying the three-dimensional nanofiber cartilage scaffold after treatment in step S5 to obtain a three-dimensional nanofiber cartilage scaffold product.

[0015] Further, in the prepared spinning solution, the mass ratio of the degradable polymer to the natural high molecular material is 1:4.

[0016] Further, in the prepared spinning solution, the mass concentration of the degradable polymer and the natural high molecular material in the spinning solution is 10% to 12%.

[0017] Further, in the sodium borohydride aqueous solution used in step S3, the concentration of sodium borohydride is 0.5M to 1M.

[0018] Further, in the mixture solution of epigallocatechin gallate and SrCl2 used in step S5, the concentration of epigallocatechin gallate is 1mg / mL, and the concentration of SrCl2 is 0.1M.

[0019] The method for preparing the three-dimensional nanofiber cartilage scaffold provided by the application uses degradable polymer and natural high molecular material as raw materials, and sequentially performs electrospinning, gas foaming, freeze-drying and chemical modification to obtain a scaffold with stable mechanical structure and three-dimensional porous nanofiber morphology. The scaffold has rich porous structure on the surface and inside, can effectively simulate the three-dimensional ECM structure of natural cartilage, has good biocompatibility, and is beneficial to cartilage regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a scanning electron microscope image of the product obtained in each step of the method for preparing the three-dimensional nanofiber cartilage scaffold of the first embodiment of the application;

[0021] Figure 2 is an element distribution map of the three-dimensional nanofiber cartilage scaffold prepared by the method of the first embodiment of the application;

[0022] Figure 3 Figure is a curve diagram of the release of epigallocatechin gallate and strontium ions of the articular cartilage three-dimensional nanofiber cartilage scaffold prepared by the method of the first embodiment of the application over time;

[0023] Figure 4 Figure is a repair result diagram of the articular cartilage three-dimensional nanofiber cartilage scaffold prepared by the method of the first embodiment of the application on cartilage injury in rabbits;

[0024] Figure 5 Figure is a result diagram of the articular cartilage three-dimensional nanofiber cartilage scaffold prepared by the method of the first embodiment of the application in relieving inflammation in rabbits. DETAILED DESCRIPTION

[0025] The embodiments of the application will be further described in conjunction with the accompanying drawings, but the embodiments of the application are not used to limit the application, and any similar structure and similar changes thereof shall be included in the protection scope of the application, and the English letters in the application are case-sensitive.

[0026] The preparation method of the articular cartilage three-dimensional nanofiber scaffold provided by the application has the characteristics that it comprises the following steps:

[0027] S1: Dissolve the degradable polymer and the natural high molecular material in a solvent to prepare a spinning solution;

[0028] In the prepared spinning solution, the mass ratio of the degradable polymer to the natural high molecular material is 1:4, and the mass concentration of the degradable polymer and the natural high molecular material in the spinning solution is 10% to 12%, and the preferred scheme of the mass concentration of the degradable polymer and the natural high molecular material in the spinning solution is 10%;

[0029] The degradable polymer for preparing the spinning solution is selected from at least one of polylactic acid-caprolactone copolymer and polycaprolactone;

[0030] The natural high molecular material for preparing the spinning solution is selected from at least one of silk fibroin and collagen;

[0031] The solvent for preparing the spinning solution is selected from at least one of hexafluoroisopropanol and dichloromethane;

[0032] S2: The spinning solution prepared in step S1 is subjected to electrospinning through an electrospinning device to obtain a cartilage scaffold two-dimensional nanofiber membrane;

[0033] The process parameters for electrospinning by the electrospinning device are as follows: the voltage is 10kV to 12kV, the receiving distance is 8cm to 15cm, and the spinning solution flow rate is 1.0mL / h to 1.2mL / h;

[0034] S3: using sodium borohydride aqueous solution, the cartilage scaffold two-dimensional nanofiber membrane obtained in step S2 is foamed to obtain a foamed three-dimensional nanofiber cartilage scaffold;

[0035] The process parameters of foaming are: the concentration of sodium borohydride in the sodium borohydride aqueous solution is 0.5M-1M (M is mol per liter); the foaming time is 10 min or 20 min or 30 min;

[0036] The concentration of sodium borohydride in the sodium borohydride aqueous solution is preferably 0.5M, and the foaming time is preferably 10 min;

[0037] S4: freeze-drying the three-dimensional nanofiber cartilage scaffold obtained in step S3;

[0038] The environmental parameters for freeze-drying the three-dimensional nanofiber cartilage scaffold are: vacuum degree 0.5Pa-1Pa, drying temperature-80℃, and drying time 24h-48h;

[0039] S5: soaking the freeze-dried three-dimensional nanofiber cartilage scaffold in a mixture of epigallocatechin gallate and SrCl2 solution for chemical modification, and the soaking time is preferably 10 min;

[0040] In the mixture of epigallocatechin gallate and SrCl2 (strontium chloride), the concentration of epigallocatechin gallate is 1mg / mL, and the concentration of SrCl2 is 0.1M;

[0041] Epigallocatechin gallate can relieve inflammation, thereby slowing the degenerative changes of cartilage, and Sr 2+ Can promote the secretion of cartilage cell-specific matrix (such as type II collagen, glycosaminoglycan), so that the prepared three-dimensional nanofiber cartilage scaffold can be used to relieve inflammation at the joint injury site and promote joint repair;

[0042] S6: after taking out the three-dimensional nanofiber cartilage scaffold treated in step S5, washing with deionized water and freeze-drying, a three-dimensional nanofiber cartilage scaffold product is obtained;

[0043] The environmental parameters for freeze-drying the three-dimensional nanofiber cartilage scaffold are: vacuum degree 0.5Pa-1Pa, drying temperature-80℃, and drying time 24h-48h.

[0044] This invention utilizes biodegradable polymers and natural polymer materials to prepare a two-dimensional nanofiber membrane for cartilage scaffolds via electrospinning. After foaming, a three-dimensional nanofiber cartilage scaffold is obtained, increasing its porosity. The resulting three-dimensional nanofiber cartilage scaffold possesses a stable three-dimensional porous biomimetic structure, exhibits good biocompatibility, and is simple and easy to operate. Furthermore, the final three-dimensional nanofiber cartilage scaffold exhibits a sustained-release effect of epigallocatechin gallate and strontium ions, maintaining anti-inflammatory and cartilage repair functions for a prolonged period. In addition to effectively relieving joint inflammation, this scaffold can also promote the secretion of cartilage-specific matrix, showing broad application prospects in the treatment of cartilage injuries.

[0045] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0046] Example 1:

[0047] The first embodiment of the present invention provides a method for preparing a three-dimensional nanofiber scaffold for articular cartilage, comprising the following steps:

[0048] S1: Weigh 0.8g of polylactic acid-caprolactone copolymer (P(LLA-CL)) and 0.2g of silk fibroin (SF) and dissolve them in 10mL of hexafluoroisopropanol (the purity of hexafluoroisopropanol is greater than 99%) to prepare a P(LLA-CL) / SF spinning solution with a mass concentration of 10% of polylactic acid-caprolactone copolymer and silk fibroin;

[0049] S2: The P(LLA-CL) / SF spinning solution is electrospun using an electrospinning device. The electrospinning process parameters are: voltage 12kV, spinning solution flow rate 1.2mL / h, and the obtained cartilage scaffold two-dimensional nanofiber membrane is received by aluminum foil at a receiving distance of 8cm to 10cm.

[0050] S3: The two-dimensional nanofiber membrane of the cartilage scaffold was placed in a sodium borohydride aqueous solution with a concentration of 0.5M for foaming treatment for 10 minutes, and the thickness of the three-dimensional nanofiber cartilage scaffold obtained was 1 cm.

[0051] S4: Take out the three-dimensional nanofiber cartilage scaffold, rinse it three times in deionized water, and then freeze-dry it in a refrigerator for 24h to 48h. The vacuum degree in the refrigerator is 0.5Pa to 1Pa and the temperature in the refrigerator is -80℃. After taking it out, you will get a dried three-dimensional nanofiber cartilage scaffold.

[0052] S5: The three-dimensional nanofiber cartilage scaffold was chemically modified by immersing it in a mixed solution of epigallocatechin gallate and SrCl2, wherein the concentration of epigallocatechin gallate was 1 mg / mL and the concentration of SrCl2 was 0.1 M, the immersion time was 10 min, after which it was rinsed three times with deionized water and then freeze-dried in a refrigerator for 24 h to 48 h. The vacuum degree in the refrigerator was 0.5 Pa to 1 Pa and the temperature in the refrigerator was -80 °C. After removal, the functionalized three-dimensional nanofiber cartilage scaffold was obtained.

[0053] Testing revealed that the interlayer spacing of the three-dimensional nanofiber cartilage scaffold prepared in the first embodiment of this invention was 30 μm to 65 μm, and the pore size was 35 μm. 2 ~65μm 2 .

[0054] Example 2:

[0055] The second embodiment of the present invention provides a method for preparing a three-dimensional nanofiber scaffold for articular cartilage, comprising the following steps:

[0056] S1: Weigh 0.8g of polycaprolactone (PCL) and 0.2g of collagen (COL) and dissolve them in 10mL of hexafluoroisopropanol (the purity of hexafluoroisopropanol is greater than 99%) to prepare a PCL / COL spinning solution with a mass concentration of 10% of polycaprolactone and collagen.

[0057] S2: The PCL / COL spinning solution is electrospun using an electrospinning device. The electrospinning process parameters are: voltage 12kV, spinning solution flow rate 1.2mL / h, and the obtained cartilage scaffold two-dimensional nanofiber membrane is received by aluminum foil at a receiving distance of 8cm to 10cm.

[0058] S3: The two-dimensional nanofiber membrane of the cartilage scaffold was placed in a sodium borohydride aqueous solution with a concentration of 0.5M for foaming treatment for 10 minutes, and the thickness of the three-dimensional nanofiber cartilage scaffold obtained was 1 cm.

[0059] S4: Take out the three-dimensional nanofiber cartilage scaffold, rinse it three times in deionized water, and then freeze-dry it in a refrigerator for 24h to 48h. The vacuum degree in the refrigerator is 0.5Pa to 1Pa and the temperature in the refrigerator is -80℃. After taking it out, you will get a dried three-dimensional nanofiber cartilage scaffold.

[0060] S5: The three-dimensional nanofiber cartilage scaffold was chemically modified by immersing it in a mixed solution of epigallocatechin gallate and SrCl2, wherein the concentration of epigallocatechin gallate was 1 mg / mL and the concentration of SrCl2 was 0.1 M, the immersion time was 10 min, after which it was rinsed three times with deionized water and then freeze-dried in a refrigerator for 24 h to 48 h. The vacuum degree in the refrigerator was 0.5 Pa to 1 Pa and the temperature in the refrigerator was -80 °C. After removal, the functionalized three-dimensional nanofiber cartilage scaffold was obtained.

[0061] Testing revealed that the interlayer spacing of the three-dimensional nanofiber cartilage scaffold prepared according to the second embodiment of the present invention was 35 μm to 60 μm, and the pore size was 40 μm. 2 ~65μm 2 .

[0062] Example 3:

[0063] The third embodiment of the present invention provides a method for preparing a three-dimensional nanofiber scaffold for articular cartilage, comprising the following steps:

[0064] S1: Weigh 0.8g of polylactic acid-caprolactone copolymer (P(LLA-CL)) and 0.2g of silk fibroin (SF) and dissolve them in 10mL of hexafluoroisopropanol (the purity of hexafluoroisopropanol is greater than 99%) to prepare a P(LLA-CL) / SF spinning solution with a mass concentration of 10% of polylactic acid-caprolactone copolymer and silk fibroin;

[0065] S2: The P(LLA-CL) / SF spinning solution is electrospun using an electrospinning device. The electrospinning process parameters are: voltage 12kV, spinning solution flow rate 1.2mL / h, and the obtained cartilage scaffold two-dimensional nanofiber membrane is received by aluminum foil at a receiving distance of 8cm to 10cm.

[0066] S3: The two-dimensional nanofiber membrane of the cartilage scaffold was placed in a sodium borohydride aqueous solution with a concentration of 0.5M for foaming treatment for 30 minutes, and the thickness of the three-dimensional nanofiber cartilage scaffold obtained was 3cm.

[0067] S4: Take out the three-dimensional nanofiber cartilage scaffold, rinse it three times in deionized water, and then freeze-dry it in a refrigerator for 24h to 48h. The vacuum degree in the refrigerator is 0.5Pa to 1Pa and the temperature in the refrigerator is -80℃. After taking it out, you will get a dried three-dimensional nanofiber cartilage scaffold.

[0068] S5: The three-dimensional nanofiber cartilage scaffold was chemically modified by immersing it in a mixed solution of epigallocatechin gallate and SrCl2, wherein the concentration of epigallocatechin gallate was 1 mg / mL and the concentration of SrCl2 was 0.1 M, the immersion time was 10 min, then rinsed three times with deionized water, and then freeze-dried in a refrigerator for 24 h to 48 h, the vacuum degree in the refrigerator was 0.5 Pa to 1 Pa, and the temperature in the refrigerator was -80 °C. After removal, the functionalized three-dimensional nanofiber cartilage scaffold was obtained.

[0069] Testing revealed that the interlayer spacing of the three-dimensional nanofiber cartilage scaffold prepared according to the first embodiment of the present invention was 50 μm to 75 μm, and the pore size was 45 μm. 2 ~75μm 2 .

[0070] In the first embodiment of this invention, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), inductively coupled plasma optical emission spectrometry (ICP-OES), and ultraviolet spectrophotometry were used to test the performance of the prepared three-dimensional nanofiber cartilage scaffold. The tests included the morphology, chemical composition, sustained release of epigallocatechin gallate and strontium ions from the articular cartilage nanofiber scaffold. Furthermore, the three-dimensional nanofiber cartilage scaffold was implanted into the joint cavity of rabbits to evaluate its application potential in alleviating inflammation and promoting joint regeneration. Specific test results are as follows:

[0071] (1) SEM testing

[0072] Figure 1 These are scanning electron microscope images of the products obtained in each step of the preparation of a three-dimensional nanofiber cartilage scaffold for articular cartilage according to the method of the first embodiment of the present invention. Figure 1 The 2DS image is a scanning electron microscope (SEM) image of a two-dimensional nanofiber membrane for cartilage scaffolds obtained by electrospinning (hereinafter referred to as the 2DS scaffold). The 3DS image is a scanning electron microscope (SEM) image of a three-dimensional nanofiber cartilage scaffold obtained by foaming treatment (hereinafter referred to as the 3DS scaffold). The 3DS-E image is a scanning electron microscope (SEM) image of a three-dimensional nanofiber cartilage scaffold obtained by chemical modification with a mixed solution of epigallocatechin gallate and SrCl2 (hereinafter referred to as the 3DS-E scaffold). All scaffolds show the surface morphology of nanofibers. The three-dimensional nanofiber cartilage scaffold obtained by foaming treatment has a porous structure with increased pore size and interlayer spacing. In the three-dimensional nanofiber cartilage scaffold obtained by chemical modification with a mixed solution of epigallocatechin gallate and SrCl2, some small particles can be observed distributed in the scaffold.

[0073] (2) Element detection

[0074] Figure 2The three-dimensional nanofiber cartilage scaffold for articular cartilage prepared by the method of the first embodiment of the present invention was analyzed by energy dispersive spectroscopy (EDS) to determine the elemental distribution of C, O, and Sr on the surface of the 3DS-E scaffold. The EDS spectra further illustrate the relationship between the three-dimensional nanofiber scaffold and epigallocatechin gallate / Sr. 2+ The interaction between them reveals the presence of strontium in the particles distributed in the 3DS-E scaffold.

[0075] (3) Release test of epigallocatechin gallate and strontium

[0076] Figure 3 This is a graph showing the release of epigallocatechin gallate and strontium ions over time of the three-dimensional nanofiber cartilage scaffold prepared by the method of the first embodiment of the present invention. Figure 3 The left figure shows the release behavior of EGCG in the 3DS-E stent, with a release rate of 33.61% on day 1, reaching 72.31% by day 9, and a total release rate of 77.71% by day 28. Figure 3 The right figure in the image shows Sr 2+ The release behavior was initially rapid, with 36.78% released on the first day, reaching 80.46% after 9 days, and 90.81% after 28 days. These findings indicate that the 3DS-E stent can sustainably release EGCG and Sr. 2+ .

[0077] (4) Testing the in vivo cartilage repair capacity of the scaffold

[0078] Figure 4 This is a diagram showing the repair results of cartilage damage in rabbits using the three-dimensional nanofiber cartilage scaffold prepared by the method of the first embodiment of the present invention. Figure 4 The Gross view in the image is a panoramic view of the rabbit full-thickness cartilage defect model. HE is a hematoxylin-eosin staining image of rabbit articular cartilage. Saf-O / FG is a Safranbon-Fix Green staining image of rabbit articular cartilage. COL II is a type II collagen staining image of rabbit articular cartilage. COL I is a type I collagen staining image of rabbit articular cartilage.

[0079] In a rabbit full-thickness cartilage defect model, normal cartilage tissue was used as the normal group, and no treatment was given after defect formation as the control group. 2DS, 3DS, and 3DS-E scaffolds were transplanted into the joint as experimental groups. Twelve weeks after transplantation, section scanning of the target area revealed significant cartilage defects in the control group, with almost no new tissue formation in the defect area. In the 2DS group, the defect was filled with white new cartilage tissue, accompanied by fine fissures. In the 3DS group, the newly formed cartilage-like tissue partially fused with adjacent tissue, but some fine fissures remained. In contrast, the 3DS-E group showed that the new cartilage tissue filled the defect and fused with the surrounding cartilage tissue, with a smooth and even surface. Subsequent histological and immunohistochemical evaluations of the collected samples showed that the defect area in the control and 2DS groups contained only sparse fibrous tissue, with poor integration of new tissue with the original cartilage, weak staining for safranin and type II collagen, and strong staining for type I collagen. In contrast, the regenerated tissue of 3DS-E showed a large amount of ECM deposition and was completely integrated with the native cartilage, reflecting the best cartilage regeneration effect.

[0080] (5) In vivo inflammation relief test of the stent

[0081] Figure 5 This image shows the effect of the three-dimensional nanofiber cartilage scaffold prepared by the method of the first embodiment of the present invention on the relief of inflammation in rabbits. Figure 5 In the image, DAPI is a nuclear staining image of rabbit articular cartilage, TNF-α is a tumor necrosis factor staining image of rabbit articular cartilage, IL-1β is an interleukin-1β staining image of rabbit articular cartilage, and Merge is a combined image of the three staining methods mentioned above.

[0082] The inflammatory response of damaged cartilage was detected by immunofluorescence staining of TNF-α and IL-1β. No obvious fluorescence signal was observed in the normal group. The fluorescence expression of the 3DS-E group was weaker than that of the control group, 2DS group and 3DS group, indicating that 3DS-E can promote cartilage tissue regeneration by alleviating inflammation.

Claims

1. A method for preparing a three-dimensional nanofibrous scaffold for articular cartilage, characterized by, The method comprises the following steps: S1: dissolving degradable polymer and natural polymer material in a solvent to prepare a spinning solution; The degradable polymer for preparing the spinning solution is at least one selected from polylactic acid-caprolactone copolymer and polycaprolactone; The natural polymer material for preparing the spinning solution is at least one selected from silk fibroin and collagen; The solvent for preparing the spinning solution is at least one selected from hexafluoroisopropanol and dichloromethane; S2: electrospinning the spinning solution prepared in step S1 through an electrospinning device to obtain a two-dimensional nanofiber membrane of a cartilage scaffold; S3: foaming the two-dimensional nanofiber membrane of the cartilage scaffold obtained in step S2 by using a sodium borohydride aqueous solution to obtain a three-dimensional nanofiber cartilage scaffold after foaming; S4: freeze-drying the three-dimensional nanofiber cartilage scaffold obtained in step S3; S5: chemically modifying the three-dimensional nanofiber cartilage scaffold after freeze-drying by immersing it in a mixed solution of epigallocatechin gallate and SrCl2; S6: freeze-drying the three-dimensional nanofiber cartilage scaffold after the treatment in step S5 to obtain a finished product of the three-dimensional nanofiber cartilage scaffold.

2. The method of claim 1, wherein the method further comprises the step of: In the prepared spinning solution, the mass ratio of the degradable polymer to the natural polymer material is 1:

4.

3. The method of claim 1, wherein the method further comprises the step of: In the prepared spinning solution, the mass concentration of the degradable polymer and the natural polymer material in the spinning solution is 10% to 12%. ​ 4. The method of claim 1, wherein the method further comprises the step of: In the sodium borohydride aqueous solution used in step S3, the concentration of sodium borohydride is 0.5M to 1M. ​ 5. The method of claim 1, wherein the method further comprises the step of: In the mixed solution of epigallocatechin gallate and SrCl2 used in step S5, the concentration of epigallocatechin gallate is 1mg / mL, and the concentration of SrCl2 is 0.1M. ​

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