A keratin-based hydrogel bio-ink and preparation method and application thereof

Abstract

The present application relates to a kind of keratin-based hydrogel biological ink and its preparation method and application, and the biological ink is constructed by keratin and metal ion coordination dynamic crosslinking.The preparation process of the present application is simple, does not need to modify substrate, has good biocompatibility and antibacterial performance, and can be used as 3D printing biological ink to construct keratin-based 3D support with clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of tissue engineering scaffolds, and particularly relates to a keratin-based hydrogel bio-ink as well as a preparation method and application thereof. BACKGROUND

[0002] 3D printing technology can precisely control the shape of the scaffold and construct complex porous structures, so it has attracted wide attention in the field of bone tissue engineering. At present, relevant researchers have developed some bio-inks for bone tissue engineering, but these bio-inks often limit the clinical development and application of this strategy due to poor biocompatibility or weak printing adaptability.

[0003] Keratin (KE) has abundant sulfhydryl groups and good biocompatibility, and can promote cell adhesion, proliferation and osteogenic differentiation. However, the abundant sulfhydryl groups of keratin can form strong disulfide covalent cross-linking, which reduces the shear thinning behavior, so that pure keratin bio-ink is difficult to be applied to extrusion 3D printing. Therefore, there are still many challenges in constructing ideal keratin-based bone repair materials through 3D printing technology. In view of all the above facts, a new design principle is needed, that is, to adjust the rheological properties of keratin hydrogel bio-ink to endow it with favorable properties. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a keratin-based hydrogel bio-ink as well as a preparation method and application thereof, which overcomes the inadaptability of pure keratin applied to 3D printing technology.

[0005] The present application provides a keratin-based hydrogel bio-ink, characterized in that the bio-ink is constructed by keratin and metal ion coordination dynamic cross-linking.

[0006] Preferably, the keratin is derived from chicken feathers, duck feathers, sheep wool and human hair or other keratin-containing materials.

[0007] Preferably, the metal ion is copper ion.

[0008] Preferably, the source of copper ion is one or more of copper sulfate pentahydrate, copper chloride and copper nitrate.

[0009] The present application also provides a preparation method of the keratin-based hydrogel bio-ink, comprising the following steps:

[0010] The keratin is dissolved in water, metal ions are added, and after uniform mixing, a photoinitiator is added under light shielding conditions and stirred to fully dissolve, to obtain the keratin-based hydrogel bio-ink; wherein the mass ratio of the keratin to the metal ion is 226-486:1.

[0011] Preferably, the water is one or more of deionized water, distilled water or ultrapure water.

[0012] Preferably, the photoinitiator comprises one or more of Irgacure 2959, lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP); the photoinitiator is added in an amount of 0.25%-3.00% wt / v.

[0013] The application also provides a use of the keratin-based hydrogel bio-ink in the preparation of a keratin-based 3D scaffold.

[0014] The specific preparation steps are as follows:

[0015] Under the control of an extrusion free-form fabrication system, the composite bio-ink is used for 3D printing: during the movement of the printing needle, the scaffold is assisted in curing using visible light, and after printing is completed, the keratin-based 3D scaffold is obtained.

[0016] The parameters of the 3D printing are as follows: extrusion air pressure 0.1-0.35 MPa, nozzle diameter 0.25-0.41 mm, filament spacing 1-1.5 mm, layer height 0.1-0.3 mm, printing speed 0.7-0.9 mm / s, and printing temperature 15-28℃.

[0017] The application adopts dynamic coordination cross-linking of keratin and metal ions to construct a keratin-based hydrogel bio-ink, and due to the existence of dynamic metal coordination cross-linking bonds, the keratin-based hydrogel bio-ink has shear thinning and self-healing properties to ensure the printability of the keratin-based hydrogel bio-ink at room temperature, and this property will also overcome the typical limitations of keratin not being easy to 3D print. Subsequently, visible light-induced disulfide covalent cross-linking improves the shape fidelity of the scaffold in the printing process.

[0018] The application adopts dynamic coordination cross-linking of keratin and metal ions to construct a keratin-based hydrogel bio-ink, and the system simplifies the complex into the simple, and realizes the maximization of functions by using two simple materials without modification. Keratin provides free thiol groups in the system, and keratin has cell adhesion peptides (RGD) that can promote cell adhesion and proliferation. The incorporated metal ions react with the keratin thiol groups to form dynamic metal coordination bonds, which endow the keratin hydrogel with shear thinning and self-healing properties. The metal ions have excellent antibacterial properties, and both materials provide structural and biological functions.

[0019] Advantages

[0020] (1) In the application, only keratin and metal ions (especially copper ions) that do not need to be modified are used as substrates, which have good biocompatibility and are biodegradable, and the rich water environment can simulate the extracellular matrix to provide a good biological environment for cells.

[0021] (2) The metal ion coordination dynamic bond of the present application endows the keratin-based hydrogel with shear-thinning property, and the bio-ink exhibits excellent self-recovery property, which can quickly recover to the gel state after being extruded from the tip of the nozzle, which overcomes the limitation of keratin not being easy to 3D print. This property endows the keratin-based hydrogel with printability at room temperature.

[0022] (3) The composite bio-ink prepared in the present application can be used as printing ink for 3D printing technology to construct personalized complex scaffolds, avoiding the step of preparing a complex mold, so as to meet various needs.

[0023] (4) The present application improves the printability of keratin based on dynamic metal coordination bond, and at the same time, adopts the way of visible light induced rapid covalent cross-linking of disulfide bond to improve the shape fidelity of 3D printed keratin-based scaffold during printing.

[0024] (5) The 3D scaffold prepared in the present application has a pore diameter which can be controlled by computer, and has a certain pore in microstructure, and the preparation method is simple and has high automation level.

[0025] (6) The bio-ink prepared in the present application has good biocompatibility and does not produce cytotoxicity, has a certain promoting effect on cell late-stage proliferation, is biodegradable, and has a certain mechanical strength, which undoubtedly has broad prospects in actual biomedical applications. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the FTIR spectrum of 17KE and 17KE-0.065Cu hydrogel prepared in Example 1.

[0027] Figure 2 is the SEM image of 17KE-0.065Cu hydrogel prepared in Example 1 after freeze-drying.

[0028] Figure 3 is the curve graph of the viscosity of 17KE-0.065Cu bio-ink prepared in Example 1 with the change of shear rate.

[0029] Figure 4 is the macroscopic scaffold of 3D printed 17KE-0.065Cu hydrogel prepared in Example 1.

[0030] Figure 5 is the stress-strain curve of 17KE-0.065Cu hydrogel prepared in Example 1. DETAILED DESCRIPTION

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalents...

[0032] In this example, keratin was extracted as follows: 48g of urea, 5.76g of SDS, and 9.5g of sodium metabisulfite were weighed and dissolved in 100mL of deionized water, and heated and stirred at 70°C in a constant temperature water bath. 10g of duck feathers were added to the above solution, and the mixture was treated in a 70°C water bath for 1 hour until no obvious clumps of feathers remained. The supernatant was obtained by filtration. The supernatant was dialyzed (Mw = 8-14KD) for 3 days, and finally the dialysate was freeze-dried to obtain keratin powder.

[0033] Example 1

[0034] (1) Preparation of 17KE bio-ink: Accurately weigh 0.085g of keratin powder and dissolve it in 375μL of deionized water. Vortex and mix well. Add 125μL of 0.25% (wt / v) LAP at room temperature in the dark and mix well again by vortexing.

[0035] (2) Preparation of 17KE-0.065Cu hydrogel bio-ink: Accurately weigh 0.085g of keratin powder and 0.325mg of copper sulfate pentahydrate and dissolve them in 375μL of deionized water. Vortex the mixture until homogeneous. Add 125μL of 0.25% (wt / v) LAP at room temperature in the dark, and vortex the mixture again until homogeneous. Unless otherwise specified, all measurements in this invention are by mass-volume ratio.

[0036] (3) Using an external computer-controlled printing system, the air pressure is adjusted to be suitable for printing. The material is pre-extruded by air to make the prepared 17KE-0.065Cu bio-ink stack layer by layer on the worktable through the needle, forming a pre-modeled 3D model. The printing parameters are set as follows: extrusion air pressure 0.1-0.35MPa, nozzle diameter 0.3mm, filament spacing 1.2mm, layer height 0.15mm, printing speed 0.7-0.9mm / s, and visible light crosslinking is performed while printing at room temperature.

[0037] Depend on Figure 1 It can be seen that the 17KE-0.065Cu hydrogel at 1630 cm⁻¹... -1 The absorption peak increased significantly at 1076 cm⁻¹. -1 The decrease in concentration indicates that copper ions formed Cu-S metal coordination bonds with the thiol groups in keratin. Figure 2It can be seen that the 17KE-0.065Cu hydrogel has a rich macroporous structure and good pore connectivity, so that the hydrogel has a high water content, provides sufficient space for cell adhesion and proliferation, and is also conducive to information transmission between cells and nutrient transport. Figure 3 It can be seen that the 17KE-0.065Cu hydrogel has a rich macroporous structure and good pore connectivity, so that the hydrogel has a high water content, provides sufficient space for cell adhesion and proliferation, and is also conducive to information transmission between cells and nutrient transport. Figure 4 It can be seen that the 17KE-0.065Cu hydrogel has a rich macroporous structure and good pore connectivity, so that the hydrogel has a high water content, provides sufficient space for cell adhesion and proliferation, and is also conducive to information transmission between cells and nutrient transport. Figure 5 It can be seen that the 17KE-0.065Cu hydrogel has a rich macroporous structure and good pore connectivity, so that the hydrogel has a high water content, provides sufficient space for cell adhesion and proliferation, and is also conducive to information transmission between cells and nutrient transport.

[0038] Example 2

[0039] (1) Preparation of 17KE bio-ink: accurately weigh 0.085 g of keratin powder and dissolve in 375 μL of deionized water, vortex and shake to mix, add 125 μL of 0.25% (wt / v) LAP under room temperature and light-proof conditions, and mix again.

[0040] (2) Preparation of 17KE-0.075Cu hydrogel bio-ink: accurately weigh 0.085 g of keratin powder and 0.375 mg of copper sulfate pentahydrate and dissolve in 375 μL of deionized water, vortex and shake to mix, add 125 μL of 0.25% (wt / v) LAP under room temperature and light-proof conditions, and mix again. In the present application, unless otherwise specified, the mass-volume ratio is used.

[0041] (3) Use an external computer-controlled printing system to adjust the air pressure suitable for printing, pre-extrude the prepared 17KE-0.075Cu bio-ink through the needle to stack layer by layer on the workbench to form a 3D model modeled in advance. The printing parameters are set as follows: extrusion air pressure 0.1-0.35 MPa, nozzle diameter 0.3 mm, filament spacing 1.2 mm, layer height 0.15 mm, printing speed 0.7-0.9 mm / s, and visible light crosslinking is performed at room temperature while printing.

[0042] The performance of the obtained 17KE-0.075Cu hydrogel is similar to that of the 17KE-0.065Cu hydrogel.

Claims

1. Use of a keratin-based hydrogel bio-ink for the preparation of a keratin-based 3D scaffold, characterized in that: The bio-ink is constructed by keratin and metal ion coordination dynamic cross-linking; the metal ion is copper ion; The preparation method of the bio-ink comprises the following steps: dissolving keratin in water, adding metal ions, stirring uniformly, adding a photoinitiator under light-proof conditions, stirring, and fully dissolving to obtain a keratin-based hydrogel bio-ink; wherein the mass ratio of the keratin to the metal ions is 226-486:

1. The specific steps for preparing the keratin-based 3D scaffold are as follows: under the control of an extrusion free-form fabrication system, using the composite bio-ink for 3D printing: using visible light to assist in curing the scaffold during the movement of the printing needle, and after the printing is completed, the keratin-based 3D scaffold is obtained.

2. Use according to claim 1, characterized in that: The keratin is derived from chicken feathers, duck feathers, sheep wool and human hair or other keratin-containing materials.

3. Use according to claim 1, characterized in that: The source of the copper ions is one or more of copper sulfate pentahydrate, copper chloride and copper nitrate.

4. Use according to claim 1, characterized in that: The water is one or more of deionized water, distilled water or ultrapure water.

5. The use according to claim 1, characterized in that: The photoinitiator comprises one or more of Irgacure 2959, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP); the addition amount of the photoinitiator is 0.25%-3.00%wt / v.

6. Use according to claim 1, characterized in that: The parameters of the 3D printing are as follows: extrusion air pressure 0.1-0.35 MPa, nozzle diameter 0.25-0.41 mm, filament spacing 1-1.5 mm, layer height 0.1-0.3 mm, printing speed 0.7-0.9 mm / s, and printing temperature 15-28 ℃.

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