A method for preparing and applying a 3D-printed strong and tough silk fibroin scaffold material

Abstract

This invention discloses a method for preparing and applying a 3D-printed strong and tough silk fibroin scaffold material. The method includes: incubating silk fibroin fibers in an aqueous lithium bromide solution; modifying with glycidyl methacrylate followed by freeze-drying; obtaining a bio-ink by dissolving in an aqueous photoinitiator solution; obtaining an intermediate solution by dissolving a calcium salt in a methacryloyl phosphate compound; obtaining a photocurable bio-ink by dissolving the intermediate solution in the bio-ink; and photocuring and printing in a 3D printer to obtain the 3D-printed strong and tough silk fibroin scaffold material. This method offers advantages such as good formability, flowability compatible with 3D printers, suitability for various 3D printing modes, and rapid photocuring performance. The prepared scaffold exhibits good biocompatibility, and the printed gel material shows better mechanical properties and stability compared to SilMA, with a slower degradation rate, enabling the scaffold to be widely used in multiple fields such as bone tissue engineering.

Description

Technical Field

[0001] This invention relates to a method for preparing a scaffold material, which relates to the field of biomaterials, and specifically to a method for preparing a 3D-printed strong and tough silk fibroin scaffold material. Background Technology

[0002] Silk fibroin biomaterials have attracted much attention due to their excellent mechanical properties, biocompatibility, lack of immune rejection, biodegradability, and natural protein structural characteristics. Among them, silkworm fibroin, tussah silk fibroin, and spider silk fibroin are representative silk fibroin biomaterials. These materials can be used to produce biomaterials with different shapes and functions, such as bone tissue repair, wound covering materials, and drug sustained-release materials, to meet the diverse needs of humankind for biomaterials.

[0003] 3D printing boasts advantages such as high customizability, high precision, high substitutability, high production efficiency, and high safety. 3D printing technology can produce biomaterials tailored to individual patient needs for implantation at damaged sites, enabling personalized medicine. By precisely controlling the shape and size of materials, 3D printing produces sophisticated biomaterials. This is crucial for complex tissue engineering and the manufacture of medical devices. Furthermore, 3D printing can produce biomaterials similar to human tissue, which can be used to repair or replace damaged tissues or organs. Therefore, it can be widely applied in the medical field, including tissue engineering, surgery, and dentistry. This technology can automate the production of biomaterials, significantly reducing production time and costs. Moreover, the higher precision of printed materials compared to handmade materials reduces errors from manual operations, thereby improving production safety and reliability. This is particularly important for high-risk medical applications.

[0004] SilMA, a bio-ink material used in photopolymer 3D printing, has been widely applied in the biomedical field since its development began in 2018. Its production process is simple, its composition is controllable, and it poses no toxicity or carcinogenic risk, making it suitable for internal use in the human body. Because it can be degraded by enzymes in the body and gradually metabolized and eliminated, it does not require secondary surgery for removal, thus reducing harm to the body. However, due to its high fluidity, SilMA can only be printed using photopolymer 3D printing technology. Furthermore, photopolymerized SilMA generally has poor formability and stability in aqueous solutions. In addition, different types and even different batches of bio-ink materials require different printing conditions, making accurate control and large-scale industrial production difficult. Moreover, extrusion 3D printing technology requires a moderate material viscosity—neither too thick to be difficult to extrude, nor too thin to maintain its shape. However, SilMA's high fluidity makes it unable to maintain its shape during extrusion, rendering it unsuitable for extrusion 3D printing. For these reasons, SilMA has limitations in various 3D printing technologies, further restricting its application in the biomedical field. Summary of the Invention

[0005] To address the problems existing in the background technology, the present invention provides a method for preparing and applying a 3D printed strong and tough silk fibroin scaffold material, which meets practical needs by improving the viscosity of SilMA before curing and the mechanical strength after curing.

[0006] The technical solution adopted in this invention is:

[0007] The preparation method of the 3D printed strong and tough silk fibroin scaffold material of the present invention includes the following steps:

[0008] 1) The silk fibroin fiber obtained after degumming silkworm cocoons is dissolved in lithium bromide aqueous solution and incubated to obtain silk fibroin solution.

[0009] 2) After modifying the silk fibroin solution with glycidyl methacrylate, the solution was freeze-dried to obtain methacrylated silk fibroin SilMA.

[0010] 3) Methacrylated silk fibroin SilMA was dissolved in an aqueous solution containing a photoinitiator to obtain bio-ink SilMA.

[0011] 4) Dissolve calcium salt in methacryloyl phosphate MAP compound to obtain intermediate solution CMAP; dissolve intermediate solution CMAP in bio-ink SilMA to obtain photocurable bio-ink BSC.

[0012] 5) Place the photocurable bio-ink BSC into an extrusion 3D printer or a digital photocurable 3D printer, and perform photocuring and 3D printing under light to obtain a 3D printed tough silk fibroin scaffold material with excellent mechanical properties and biocompatibility.

[0013] In step 1), 20g of silk fibroin fiber obtained after degumming silkworm cocoons is dissolved in 100mL of 9.3M lithium bromide aqueous solution and incubated at 60℃ for 1h to obtain silk fibroin solution.

[0014] In step 2), 3-10 mL of glycidyl methacrylate is used to modify 100 mL of silk fibroin solution at 25-60 °C for 1-5 h, followed by freeze drying to obtain methacrylamide silk fibroin SilMA.

[0015] In step 3), the aqueous solution containing the photoinitiator is specifically a 0.25 wt% aqueous solution of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0016] Bio-ink SilMA was obtained by dissolving methacrylamide silk fibroin SilMA at a concentration of 10-20 wt% in an aqueous solution of 0.25 wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP.

[0017] In step 4), 0.04-0.05g of calcium salt is dissolved in 1mL of methacryloyl phosphate MAP compound to obtain intermediate solution CMAP; intermediate solution CMAP is dissolved in bio-ink SilMA at a concentration of 2-3wt% to obtain photocurable bio-ink BSC.

[0018] In step 5), the photocurable bio-ink BSC is placed in an extrusion 3D printer or a digital photocurable 3D printer and photocured under ultraviolet light with a wavelength of 450nm.

[0019] In step 4), the calcium salt is specifically calcium gluconate, calcium chloride, calcium hydroxide, or calcium lactate.

[0020] In step 4), the methacryloyl phosphate MAP compound is specifically 2-hydroxyethyl methacrylate phosphate HEMAP, di[2-(methacryloyloxy)ethyl] phosphate BMAP, 2-methacryloyloxyethyl phosphate, or polyethylene glycol methacrylate phosphate.

[0021] This invention employs a simple two-component mixing process. By adding a small amount of MAP to SilMA, a biocompatible bio-ink can be obtained. After photocuring, this ink exhibits an organic-inorganic composite structure without phase boundaries at the nanoscale, thereby improving its mechanical strength. Furthermore, its flowability is controllable by adjusting the concentration of MAP, allowing it to be printed using 3D printing technologies such as extrusion and photocuring.

[0022] The 3D-printed strong silk fibroin scaffold material is used for biomedical applications in the inoculation of adult cells and stem cells.

[0023] The 3D-printed strong silk fibroin scaffold can be used in experiments with human fibroblasts: human fibroblasts are seeded into the silk fibroin scaffold, and the cells can grow on the surface and inside the scaffold, exhibiting good cell proliferation ability.

[0024] The 3D-printed strong silk fibroin scaffold can be used in experiments with mesenchymal stem cells: human mesenchymal stem cells are seeded in the regular sheet structure of the silk fibroin scaffold. After one day of culture, they show a high adhesion effect. After two weeks of culture, they can promote the secretion of large amounts of collagen and alkaline phosphatase by cells, showing a good ability to promote the directed differentiation of stem cells.

[0025] The silk fibroin used in this invention is a natural active substance that is widely used in the biomedical industry and has no toxic reaction to cells or tissues. The addition of MAP transforms the liquid bio-ink into a viscous state while maintaining good molding properties, allowing SilMA, which was originally unsuitable for extrusion 3D printing, to be 3D printed using extrusion methods, greatly improving the printing efficiency of this three-dimensional biological scaffold.

[0026] This invention fully leverages the improved mechanical properties of its multi-component composition, enabling its application in extrusion 3D printing. Furthermore, the material combines the excellent biocompatibility of SilMA, making it an ideal three-dimensional scaffold material that can provide reference information for the design and fabrication processes of photopolymerizable 3D printing bio-inks.

[0027] The beneficial effects of this invention are:

[0028] 1) Excellent biocompatibility: The ingredients are safe and have been verified by CCK-8 to be non-toxic to biological substances. It is a material with no toxic side effects on body tissues.

[0029] 2) No environmental pollution: The preparation process does not use toxic reagents, the preparation conditions are mild, and no products that are toxic to humans and the environment are produced.

[0030] 3) Simple and fast process: Simply mix the prepared SilMA and CMAP and put them into an extrusion 3D printer or a photopolymer 3D printer to print the scaffold.

[0031] 4) Improved mechanical properties of the stent: Compared with SilMA, the BSC stent has significantly improved mechanical strength and better resistance to swelling and degradation, as well as better cyclic compression performance.

[0032] 5) Improved cell adhesion on the scaffold: Compared with SilMA, the BSC scaffold has significantly enhanced material rigidity, which is beneficial to cell adhesion and spread.

[0033] Therefore, the method of this invention has advantages such as good formability, fluidity compatible with 3D printers, suitability for various 3D printing modes, and rapid photopolymerization performance. The prepared printed scaffold has good biocompatibility. Compared with SilMA, the printed gel material has better mechanical properties and stability, and a slower degradation rate. It significantly improves the proliferation performance of human cells and the directed differentiation performance of stem cells, thereby enhancing the bone tissue repair function of the biological scaffold. This allows the printed scaffold to be widely used in multiple fields such as bone tissue engineering, and has broad application prospects in tissue engineering, drug sustained release, hemostatic materials, and filter membranes. Attached Figure Description

[0034] Figure 1 The images shown are scanning electron microscope (SEM) images of the BSC and SilMA scaffolds after photocuring in Example 1. Figure 1 Image A is a scanning electron microscope image of the BSC scaffold after photocuring in Example 1. Figure 1 B is a scanning electron microscope image of the SilMA scaffold after photocuring in Example 1;

[0035] Figure 2 The images shown are morphological images of BSC bio-ink and bio-ink SilMA from Example 1, as well as extrusion 3D printing images of BSC bio-ink and bio-ink SilMA. Figure 2 A is a morphology diagram of the BSC bio-ink in Example 1. Figure 2 Image B is a morphology diagram of the bio-ink SilMA from Example 1. Figure 2 C is the extrusion 3D printing diagram of BSC bio-ink in Example 1. Figure 2 D is the extrusion 3D printing diagram of the bio-ink SilMA in Example 1;

[0036] Figure 3 The figures show the compression and rheological curves of the BSC and SilMA scaffolds after photocuring in Example 3. Figure 3 A represents the compression curves of the BSC and SilMA scaffolds after photocuring in Example 3. Figure 3B is the rheological curve of BSC and SilMA scaffold after photocuring in Example 3;

[0037] Figure 4 The images show the optical microscopic morphology of human fibroblasts on BSC and SilMA scaffolds after photocuring in Example 4. Figure 4 Image A shows the optical microscopic morphology of human fibroblasts on a BSC scaffold after photocuring in Example 4. Figure 4 Image B is an optical microscope image of human fibroblasts on a SilMA scaffold. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Specific embodiments of the present invention are as follows:

[0040] Example 1:

[0041] 20g of silk fibroin fibers obtained after degumming silkworm cocoons were dissolved in 100mL of 9.3M lithium bromide aqueous solution and incubated at 60℃ for 1h to obtain a silk fibroin solution. 100mL of the silk fibroin solution was modified with 3mL of glycidyl methacrylate at 60℃ for 3h and then freeze-dried to obtain methacrylamide silk fibroin (SilMA). Methacrylamide silk fibroin (SilMA) was dissolved at a concentration of 15wt% in a 0.25wt% aqueous solution of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) to obtain bio-ink Sil. MA; 0.05g of calcium chloride (CaCl2) was dissolved in 1mL of bis[2-(methacryloyloxy)ethyl]phosphate (BMAP) to obtain intermediate solution CMAP; intermediate solution CMAP was dissolved in bio-ink SilMA at a concentration of 3wt% to obtain photocurable bio-ink BSC; the viscous photocurable bio-ink BSC was placed in an extrusion 3D printer or a digital photopolymerization 3D printer, and photocured under ultraviolet light at a wavelength of 450nm to obtain a 3D-printed strong and tough silk fibroin scaffold material with excellent mechanical properties and biocompatibility. The viscosity of photocurable bio-ink BSC increases with the increase of intermediate solution CMAP content.

[0042] The scanning electron microscope image of the scaffold cured with the photocurable bio-ink BSC in Example 1 is shown below. Figure 1 As shown in Figure A, the scanning electron microscope image of the scaffold cured with the bio-ink SilMA is as follows. Figure 1 As shown in Figure B; the optical morphology images of photocurable bio-ink BSC and bio-ink SilMA are shown in Figure B. Figure 2 A and Figure 2 As shown in Figure B, the printing processes of photopolymer bio-ink BSC and bio-ink SilMA in an extrusion 3D printer are as follows: Figure 2 C and Figure 2 As shown in Figure D, the roughness of the BSC in this invention is significantly improved compared to SilMA. BSC can be used for extrusion 3D printing, while SilMA cannot be formed by extrusion 3D printers due to its low viscosity and high flowability. That is, SilMA has poor printing accuracy in extrusion 3D printing.

[0043] Example 2:

[0044] 20g of silk fibroin fibers obtained after degumming silkworm cocoons were dissolved in 100mL of 9.3M lithium bromide aqueous solution and incubated at 60℃ for 1h to obtain a silk fibroin solution. The 100mL silk fibroin solution was modified with 6mL of glycidyl methacrylate at 60℃ for 4h and then freeze-dried to obtain methacrylamide silk fibroin (SilMA). Methacrylamide silk fibroin (SilMA) was dissolved at a concentration of 10wt% in a 0.25wt% aqueous solution of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) to obtain bio-ink. SilMA; 0.04 g of calcium hydroxide Ca(OH)2 was dissolved in 1 mL of 2-hydroxyethyl methacrylate phosphate HEMAP to obtain intermediate solution CMAP; intermediate solution CMAP was dissolved in bio-ink SilMA at a concentration of 3 wt% to obtain photocurable bio-ink BSC; photocurable bio-ink BSC was placed in an extrusion 3D printer or a digital photopolymerization 3D printer, and photocured and 3D printed under ultraviolet light with a wavelength of 450 nm to obtain a 3D printed tough silk fibroin scaffold material with excellent mechanical properties and biocompatibility.

[0045] Example 3:

[0046] 20g of silk fibroin fibers obtained after degumming silkworm cocoons were dissolved in 100mL of 9.3M lithium bromide aqueous solution and incubated at 60℃ for 1h to obtain a silk fibroin solution. 100mL of the silk fibroin solution was modified with 10mL of glycidyl methacrylate at 30℃ for 1h and then freeze-dried to obtain methacrylamide silk fibroin (SilMA). Methacrylamide silk fibroin (SilMA) was dissolved at a concentration of 20wt% in 0.25wt% phenyl(2,4,6-trimethylbenzoyl)phosphate. Biological ink SilMA was obtained from an aqueous solution of lithium salt LAP; 0.05 g of calcium hydroxide Ca(OH)2 was dissolved in 1 mL of di[2-(methacryloyloxy)ethyl]phosphate BMAP to obtain intermediate solution CMAP; intermediate solution CMAP was dissolved in biological ink SilMA at a concentration of 2 wt% to obtain photocurable biological ink BSC; photocurable biological ink BSC was poured into a cylindrical mold and photocured under an external ultraviolet light source with a wavelength of 450 nm to obtain a strong silk fibroin scaffold material.

[0047] Comparative example of Example 3:

[0048] The bio-ink SilMA from Example 3 was poured into a cylindrical mold and photocured under an external ultraviolet light source with a wavelength of 450 nm to obtain a control material, which serves as a reference material for the strong silk fibroin scaffold material.

[0049] The photocured contrast-cured material and the strong silk fibroin scaffold material were subjected to compression tests using a universal testing machine. The resulting compressive stress-strain curves are shown below. Figure 3 As shown in Figure A; the changes in storage modulus and loss modulus (rheological properties) of bio-ink SilMA and photocurable bio-ink BSC under ultraviolet light irradiation are shown in Figure A. Figure 3 As shown in Figure B, the mechanical strength and photocuring rate of the BSC of this invention are significantly improved compared with SilMA.

[0050] Example 4:

[0051] 20g of silk fibroin fiber obtained after degumming silkworm cocoons was dissolved in 100mL of 9.3M lithium bromide aqueous solution and incubated at 60℃ for 1h to obtain silk fibroin solution; 100mL of silk fibroin solution was modified with 6mL of glycidyl methacrylate at 25℃ for 5h and then freeze-dried to obtain methacrylamide silk fibroin SilMA; 15wt% of methacrylamide silk fibroin SilMA was dissolved in 0.25wt% of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) aqueous solution to obtain bio-ink SilMA; 0.05g of calcium hydroxide Ca(OH)2 was dissolved in 1mL of polyethylene glycol methacrylate phosphate to obtain intermediate solution CMAP; intermediate solution CMAP was dissolved in bio-ink SilMA at a concentration of 3wt% to obtain photocurable bio-ink BSC; photocurable bio-ink BSC was coated in 24-well plates, photocured under ultraviolet light at a wavelength of 450nm, and then placed in human fibroblasts for culture.

[0052] Comparative example of Example 4:

[0053] The bio-ink SilMA from Example 4 was coated onto a 24-well plate, photocured under ultraviolet light at a wavelength of 450 nm, and then placed into human fibroblasts for culture.

[0054] After 7 days of culture, the cell morphology of human fibroblasts on the solidified BSC scaffold obtained in Example 4 is as follows: Figure 4 As shown in Figure A. The cell morphology of human fibroblasts on the cured SilMA scaffold is as follows. Figure 4As shown in Figure B. Cell culture results indicate that human fibroblasts can adhere and proliferate on the BSC scaffold in the early stages, exhibiting good biocompatibility; while the SilMA control group scaffold has fewer cells and a smaller cell spreading area.

[0055] In summary, this invention provides a novel silk fibroin bio-ink that significantly improves the viscosity, formability, and mechanical properties of SilMA, while shortening its curing time. This allows SilMA, which was previously unsuitable for extrusion 3D printing, to be formed through extrusion. This greatly enhances the efficiency of large-scale production of silk fibroin 3D printing materials. Cells can adhere, proliferate, and differentiate well on this BSC scaffold. The improved mechanical strength also accelerates osteogenic differentiation efficiency. Therefore, the method of this invention can prepare a bioscaffold with good biocompatibility, significant cell culture effects, and the ability to induce mesenchymal stem cell differentiation.

Claims

1. A method for preparing a 3D-printed strong and tough silk fibroin scaffold material, characterized in that: The method includes the following steps: 1) Dissolve silk fibroin fibers in lithium bromide aqueous solution and incubate to obtain silk fibroin solution; 2) After modifying the silk fibroin solution with glycidyl methacrylate, the solution was freeze-dried to obtain methacrylamide silk fibroin SilMA; 3) Methacrylamide silk fibroin SilMA was dissolved in an aqueous solution containing a photoinitiator to obtain bio-ink SilMA; 4) Dissolve calcium salts in methacryloyl phosphate (MAP) compounds to obtain intermediate solution CMAP; dissolve intermediate solution CMAP in bio-ink SilMA to obtain photocurable bio-ink BSC; 5) Place the photocurable bio-ink BSC into an extrusion 3D printer or a digital photocurable 3D printer, and perform photocuring and 3D printing under light to obtain a 3D printed tough silk fibroin scaffold material with an organic-inorganic composite structure without phase boundaries.

2. The method for preparing a 3D-printed strong and tough silk fibroin scaffold material according to claim 1, characterized in that: In step 1), 20 g of silk fibroin fiber is dissolved in 100 mL of 9.3 M lithium bromide aqueous solution and incubated at 60 °C for 1 h to obtain silk fibroin solution.

3. The method for preparing a 3D-printed strong and tough silk fibroin scaffold material according to claim 1, characterized in that: In step 2), 3-10 mL of glycidyl methacrylate is used to modify 100 mL of silk fibroin solution at 25-60℃ for 1-5 h, followed by freeze drying to obtain methacrylamide silk fibroin SilMA.

4. The method for preparing a 3D-printed strong and tough silk fibroin scaffold material according to claim 1, characterized in that: In step 3), the aqueous solution containing the photoinitiator is specifically a 0.25 wt% aqueous solution of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP). Bio-ink SilMA was obtained by dissolving methacrylamide silk fibroin SilMA at a concentration of 10-20 wt% in an aqueous solution of 0.25 wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP.

5. The method for preparing a 3D-printed strong and tough silk fibroin scaffold material according to claim 1, characterized in that: In step 4), 0.04-0.05 g of calcium salt is dissolved in 1 mL of a methacryloyl phosphate MAP compound to obtain an intermediate solution CMAP; the intermediate solution CMAP is dissolved in bio-ink SilMA at a concentration of 2-3 wt% to obtain a photocurable bio-ink BSC.

6. The method for preparing a 3D-printed strong and tough silk fibroin scaffold material according to claim 1, characterized in that: In step 5), the photocurable bio-ink BSC is placed in an extrusion 3D printer or a digital photocurable 3D printer and photocured under ultraviolet light with a wavelength of 450 nm.

7. The method for preparing a 3D-printed strong and tough silk fibroin scaffold material according to claim 1, characterized in that: In step 4), the calcium salt is specifically calcium gluconate, calcium chloride, calcium hydroxide, or calcium lactate.

8. The method for preparing a 3D-printed strong and tough silk fibroin scaffold material according to claim 1, characterized in that: In step 4), the methacryloyl phosphate MAP compound is specifically 2-hydroxyethyl methacrylate phosphate HEMAP, di[2-(methacryloyloxy)ethyl] phosphate BMAP, 2-methacryloyloxyethyl phosphate, or polyethylene glycol methacrylate phosphate.

9. The 3D printed strong and tough silk fibroin scaffold material prepared by the preparation method of any one of claims 1-8.

10. The 3D-printed strong and tough silk fibroin scaffold material prepared by the method described in any one of claims 1-8 is used for seeding adult cells and stem cells.

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