Hydrogel stent with multiple physical gradients and preparation method thereof

Abstract

The invention discloses a hydrogel scaffold with multiple physical gradients. The hydrogel scaffold is prepared from the following synthetic raw materials: thermo-sensitive polyisocyanide polypeptide, silk nanofibers rich in beta lamellar structures and a conductive nanomaterial. The conductive nano material dispersion liquid and the BSNF solution are uniformly mixed according to a ratio of 1: 4; at 4 DEG C, adding a BSNF-loaded conductive nano material mixture into a PIC aqueous solution, uniformly mixing, adding into a square tank with an electrode, and applying a direct-current electric field to the hydrogel; after the electric field is removed, the temperature is raised to 25 DEG C, PIC is induced to form gel, and the conductive hydrogel stent with multiple physical gradients is obtained. The hydrogel contains multiple physical gradient structures such as rigidity, conductivity and morphology. The multi-gradient hydrogel with different patterns can be constructed by regulating and controlling the shape, the position and the electric field applying time of the electrode; the preparation method disclosed by the invention is simple, convenient to operate, simple in equipment and process requirements, low in cost and capable of realizing large-scale industrial production.

Description

Technical Field

[0001] The invention relates to a hydrogel scaffold material and a preparation method thereof, and belongs to the field of biomaterials. Background Art

[0002] In the process of nerve regeneration, the interaction between nerve cells and the extracellular matrix (ECM) plays a vital role. ECM is the microenvironment for cell growth, composed of various proteins and polysaccharides, which can regulate various cell behaviors such as cell growth, development and death. The simulation of the physical and biochemical properties in ECM through neural scaffold materials can well meet the needs of nerve cells for their growth microenvironment, and at the same time provide a wealth of regulatory factors for the behavior of nerve cells. Hydrogel is a three-dimensional network structure polymer material with water as the dispersion medium. Due to its good biocompatibility, unique porous structure, permeability and hydrophilicity, especially the water content and moisturizing ability similar to natural nerve tissue, it has always been a biomaterial simulating ECM with broad application prospects.

[0003] With the deepening of research on the nervous system, the function of ECM gradient cues has received extensive attention. Under the guidance of gradient cues, nerve cells can migrate, form cell-oriented arrangements and axon-oriented extensions, build new neural networks, and replace the functions of damaged neurons. Therefore, by introducing gradient signals into hydrogel neural scaffolds suitable for cell growth, there is great application potential in regulating nerve cell behavior and inducing nerve repair. Compared with biochemical gradient signals such as neurotrophic factors, physical gradient signals in hydrogels such as stiffness, conductivity, morphology and other gradient signals are relatively more stable and more designable, so they have received extensive attention in neural behavior regulation. However, most of the current hydrogel scaffolds only construct a single physical gradient signal to regulate the behavior of nerve cells. In vivo, it is the synergistic regulation of multiple physical gradient signals that achieves the regulation of cell behavior. Therefore, it is difficult for current hydrogel scaffolds to accurately simulate the in vivo microenvironment in which cells are located. It is of great significance to construct a conductive hydrogel scaffold with multiple physical gradients to simulate the complex in vivo and in vitro environment of nerve cells and achieve the regulation of the proliferation, migration, growth, differentiation and other behaviors of nerve cells. Summary of the invention

[0004] Technical problem: The first purpose of the present invention is to provide a hydrogel scaffold with multiple physical gradients, including stiffness gradient, conductivity gradient and morphology gradient, to simulate the complex in vivo and in vitro environment of nerve cells; the second purpose of the present invention is to provide a method for preparing the multiple physical gradient hydrogel scaffold.

[0005] Technical solution: A hydrogel scaffold with multiple physical gradients, wherein the hydrogel scaffold is a multiple physical gradient structure containing stiffness, conductivity and morphology; the preparation raw materials include thermosensitive polyisocyanate polypeptide, silk nanofibers rich in β-sheet structure and conductive nanomaterials; the conductive nanomaterial dispersion and the silk nanofiber solution rich in β-sheet structure are uniformly mixed; at 4°C, the conductive nanomaterial mixture loaded with silk nanofibers rich in β-sheet structure is added to the thermosensitive polyisocyanate polypeptide aqueous solution and mixed uniformly, a DC electric field is applied to the hydrogel, the temperature is raised to above 25°C, and the conductive material therein is induced by the electric field. The material is directional moved, and the temperature is controlled to induce the sol-gel transition of the thermosensitive polyisocyanate polypeptide hydrogel to fix the distribution of the conductive nanomaterials loaded on the silk nanofibers rich in β-sheet structure, and finally a multi-gradient distribution is formed. After the reaction is completed, the electric field is removed to obtain a conductive hydrogel scaffold with multiple physical gradients; the thermosensitive polyisocyanate polypeptide hydrogel is a highly bionic polypeptide hydrogel, which is liquid at 4°C and forms a gel above 25°C; the silk nanofibers rich in β-sheet structure carry a large amount of negative charge and can load conductive nanomaterials, thereby improving the dispersibility and stability of conductive nanomaterials in aqueous solution.

[0006] The concentration of the conductive nanomaterial dispersion is 0.1 mg / mL to 5 mg / mL.

[0007] The conductive nanomaterial dispersion and the silk nanofiber solution rich in β-sheet structure are uniformly mixed in a ratio of 1:4.

[0008] The conductive nanomaterial is at least one of graphene, silver nanowires, carbon nanotubes or Mxene; the mass ratio of the conductive nanomaterial to the temperature-sensitive polyisocyanate polypeptide monomer is 0.01-0.25:1.

[0009] The electrodes of the direct current electric field include any of square, triangle, and point shapes. By regulating the shape, position, and time of applying the electric field, multi-gradient hydrogels with different patterns can be formed.

[0010] The method for preparing the hydrogel scaffold with multiple physical gradients comprises the following steps: uniformly mixing a conductive nanomaterial dispersion and a silk nanofiber solution rich in β-sheet structure, uniformly mixing the dispersion with a thermosensitive polyisocyanate polypeptide aqueous solution at 4°C, adding the dispersion into a square trough with electrodes, applying a direct current electric field to the hydrogel, raising the temperature to 25°C, inducing the movement of the charged conductive nanomaterial loaded by the silk nanofiber rich in β-sheet structure in the hydrogel by the direct current electric field, inducing the sol-gel transition of the thermosensitive polyisocyanate polypeptide hydrogel by controlling the temperature, fixing the distribution of the conductive nanomaterial loaded by the silk nanofiber rich in β-sheet structure, forming a multiple gradient distribution, and constructing a hydrogel scaffold with multiple physical gradients; after the reaction is completed, removing the electric field, and obtaining a conductive hydrogel scaffold with multiple physical gradients.

[0011] The method for preparing the hydrogel scaffold with multiple physical gradients comprises the following steps:

[0012] (1) dispersing a conductive nanomaterial in deionized water to obtain a conductive nanomaterial dispersion, adding the conductive nanomaterial dispersion to silk nanofibers rich in β-sheet structure, and rotating and stirring to obtain a conductive material dispersion loaded on silk nanofibers rich in β-sheet structure;

[0013] (2) adding a mixture of conductive materials loaded by silk nanofibers rich in β-sheet structure to a thermosensitive polyisocyanate polypeptide aqueous solution at 4°C and mixing the mixture evenly, adding the mixture to a square tank with electrodes, and applying a direct current electric field to the mixed hydrogel at room temperature to induce the directional movement of the conductive materials loaded by silk nanofibers rich in β-sheet structure;

[0014] (3) Raising the temperature to above 25°C induces the thermosensitive polyisocyanate to gel, forming a hydrogel with multiple gradients.

[0015] The mass concentration of the silk nanofibers rich in β-sheet structure in step (1) is 0.1wt% to 3wt%.

[0016] Beneficial effects:

[0017] (1) The conductive nanomaterials are loaded on the silk nanofibers SF with a large amount of negative charge, which improves the dispersibility of the conductive nanomaterials in the solution. At the same time, the directional movement of SF and the conductive nanomaterials in the electric field is achieved to form a gradient distribution.

[0018] (2) Using electric field to regulate the distribution of silk-loaded conductive nanomaterials in hydrogels, hydrogels with multiple physical gradients such as stiffness, conductivity, and morphology were prepared. Direct current electric field was used to induce the movement of charged β-sheet-rich silk nanofibers loaded with conductive nanomaterials in hydrogels to form a gradient distribution, thereby constructing hydrogel scaffolds with multiple physical gradients.

[0019] (3) By regulating the shape and position of the electrodes and the time of applying the electric field, multi-gradient hydrogels with different patterns can be constructed;

[0020] (4) By controlling the temperature to trigger the sol-gel transition of PIC hydrogel, the distribution of conductive nanomaterials loaded by BSNF can be fixed to achieve the construction of multi-gradient hydrogels; avoiding the addition of chemical reagents to trigger polymerization to fix the gradient distribution of nanomaterials;

[0021] (5) The preparation method is simple, easy to operate, has simple requirements on equipment and process, low cost, and can be realized in large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the preparation of conductive hydrogel scaffolds with multiple physical gradients.

[0023] Figure 2 This is a fluorescence image of the multi-gradient hydrogel scaffold prepared by using square electrodes as cathode and anode in Example 1, in which BSNF and reduced graphene oxide (rGO) were stained with rhodamine.

[0024] Figure 3 This is a fluorescence image of the multi-gradient hydrogel scaffold prepared by using point electrodes and square electrodes as cathode and anode in Example 2, in which BSNF and rGO were stained with rhodamine.

[0025] Figure 4 The conductive gradient test diagrams of the multi-gradient hydrogel scaffold and the non-gradient hydrogel prepared in Example 1 are shown in FIG. The left side shows the result of the non-gradient hydrogel and the right side shows the result of the multi-gradient hydrogel.

[0026] Figure 5 This is a stiffness gradient test diagram of the hydrogel scaffold and the non-gradient hydrogel prepared in Example 1. DETAILED DESCRIPTION

[0027] The gradient hydrogel scaffold of the present invention comprises thermosensitive polyisocyanuric acid polypeptide (PIC) (reference: Nature, 2013, 493, 651), silk nanofiber (BSNF) rich in β-sheet structure (reference: Biomacromolecules, 2014, 15 (8): 3044), and conductive nanomaterials. The conductive nanomaterial dispersion and BSNF solution are uniformly mixed at a ratio of 1:4, the concentration of the conductive nanomaterial is 0.1 mg / mL to 5 mg / mL, and the concentration of BSNF is 0.1 wt% to 3 wt%. At 4°C, the BSNF-loaded conductive nanomaterial mixture is added to the PIC aqueous solution and mixed evenly, and then added to a square tank with electrodes, and a DC electric field is applied to the hydrogel. After removing the electric field, the temperature is raised to 25°C to obtain a conductive hydrogel scaffold with multiple physical gradients.

[0028] A method for preparing a hydrogel scaffold having multiple physical gradients comprises the following steps:

[0029] (1) Dispersing the conductive nanomaterial in deionized water to obtain a dispersion (0.1 mg / mL to 5 mg / mL). Adding the conductive nanomaterial dispersion to BSNF (0.1 wt% to 3 wt%), and rotating and stirring to obtain a BSNF-loaded conductive material dispersion;

[0030] (2) At 4°C, the BSNF-loaded conductive material mixture was added to the PIC aqueous solution and mixed evenly. The mixture was then added to a square tank with a pair of electrodes. A DC electric field was applied to the mixed hydrogel at room temperature to induce the directional movement of the BSNF-loaded conductive material.

[0031] (3) Heating to above 25°C induces PIC to gel, forming a hydrogel with multiple gradients.

[0032] The polyisocyanate polypeptide PIC is a thermosensitive hydrogel, which is a highly biomimetic polypeptide hydrogel. It is liquid at 4°C and forms a gel above 25°C. The silk nanofiber SF rich in β-sheet structure carries a large amount of negative charge and can load conductive nanomaterials to improve their dispersibility and stability in aqueous solution. Under a DC electric field, the conductive nanomaterials loaded with negatively charged SF will move toward the positive electrode to form a gradient distribution. The gradient distribution of SF-loaded conductive nanomaterials in the hydrogel will cause the hydrogel to present gradients such as stiffness, conductivity, and morphology.

[0033] Preferably, the conductive nanomaterial is at least one of graphene, silver nanowires, carbon nanotubes or Mxene; and the mass ratio of the conductive material to the monomer is 0.01 to 0.25:1.

[0034] Preferably, the multi-physical gradient hydrogel is prepared by inducing the movement of charged BSNF-loaded conductive nanomaterials in the hydrogel by a direct current electric field;

[0035] Preferably, the electrodes of the DC electric field may be in the shape of a square, a triangle, a dot, etc., and by regulating the shape, position, and time of applying the electric field of the electrodes, multi-gradient hydrogels with different patterns may be formed;

[0036] Preferably, the multi-physical gradient hydrogel scaffold refers to a hydrogel constructed with gradients of stiffness, conductivity, morphology, etc. at the same time;

[0037] Preferably, the hydrogel scaffold with multiple physical gradients is constructed by controlling the temperature to induce the sol-gel transition of the thermosensitive PIC hydrogel to fix the distribution of the conductive nanomaterials loaded by the BSNF, thereby realizing the construction of a hydrogel with multiple gradient distributions.

[0038] Embodiment 1:

[0039] The gradient hydrogel scaffold of the present invention comprises the following steps:

[0040] (1) 5 mg of rGO was dispersed in 1 mL of deionized water to obtain a rGO dispersion. The rGO dispersion was added to 2 wt% of BSNF and stirred at 800 rpm for 1 h to obtain a BSNF / rGO mixture.

[0041] (2) At 4°C, BSNF / rGO was added to the PIC aqueous solution and mixed evenly. 25 μL of PIC / BSNF / rGO hydrogel was added to a 5 mm × 5 mm × 1 mm square groove. Square copper electrodes were placed at both ends of the square groove as anode and cathode. A 1.5 V / mm -1 Direct current electric field was applied and the electric field was removed after 3 min.

[0042] (3) Raising the temperature to 25°C to initiate PIC gelation, thereby obtaining a conductive hydrogel scaffold with multiple physical gradients.

[0043] Embodiment 2:

[0044] The gradient hydrogel scaffold of the present invention comprises the following steps:

[0045] (1) 3 mg of rGO was dispersed in 1 mL of deionized water to obtain rGO dispersion. The rGO dispersion was added to 2 wt% BSNF and stirred at 800 rpm for 1 h to obtain a BSNF / rGO mixture.

[0046] (2) At 4°C, BSNF / rGO was added to the PIC aqueous solution and mixed evenly. 25 μL of PIC / BSNF / RGO hydrogel was added to a 5 mm × 5 mm × 1 mm square groove. A dot-shaped copper electrode and a square copper electrode were placed at both ends of the square groove as the anode and cathode, respectively. A 1.5 V.mm -1 Direct current electric field was applied and the electric field was removed after 5 min.

[0047] (3) raising the temperature to 30°C to induce PIC gelation, thereby obtaining a conductive hydrogel scaffold with multiple physical gradients;

[0048] Embodiment 3:

[0049] The preparation method of the gradient hydrogel of the present invention comprises the following steps:

[0050] (1) 3 mg of carboxylated carbon nanotubes were dispersed in 1 mL of deionized water to obtain a CNT dispersion. The CNT dispersion was added to 2 wt % of BSNF and stirred at 800 rpm for 1 h to obtain a BSNF / CNT mixture.

[0051] (2) At 4°C, BSNF / CNT was added to the PIC aqueous solution and mixed evenly. 25 μL of PIC / BSNF / CNT hydrogel was added to a 5 mm × 5 mm × 1 mm square groove. A square copper electrode and a triangular copper electrode were placed at both ends of the square groove as the anode and cathode, respectively. A 1.5 V.mm -1 Direct current electric field was applied and the electric field was removed after 5 min.

[0052] (3) Raising the temperature to 37°C to induce PIC gelation and obtain a conductive hydrogel scaffold with multiple physical gradients

[0053] Example 4

[0054] The gradient hydrogel scaffold of the present invention comprises the following steps:

[0055] (1) Disperse 5 mg of carboxylated carbon nanotubes in 1 mL of deionized water to obtain a CNT dispersion. Add the CNT dispersion to 2 wt % BSNF and stir at 800 rpm for 1 h to obtain a BSNF / CNT mixture.

[0056] (2) At 4°C, BSNF / CNT was added to the PIC aqueous solution and mixed evenly. 25 μL of PIC / BSNF / CNT hydrogel was added to a 5 mm × 5 mm × 1 mm square groove. Dot-shaped copper electrodes were placed at both ends of the square groove as anode and cathode. A 1.5 V.mm -1 Direct current electric field was applied and the electric field was removed after 5 min.

[0057] (3) Raising the temperature to 25°C to induce PIC gelation, obtaining a conductive hydrogel scaffold with multiple physical gradients; Performance testing

[0058] like Figure 2 As shown, the fluorescence intensity of the gradient hydrogel prepared in Example 1 changes along the direction of the electric field and gradually increases from the cathode to the anode, indicating that the SF-loaded rGO presents a gradient distribution in the hydrogel.

[0059] like Figure 3 As shown, the gradient hydrogel prepared in Example 2 uses a dot electrode and a square electrode as the cathode and anode, and the fluorescence intensity shows that the rGO loaded by SF can form a gradient distribution with an arc in the hydrogel;

[0060] like Figure 4 As shown, the multi-gradient hydrogel prepared in Example 1 has a gradient distribution of SF and conductive nanomaterials in the hydrogel, and its conductivity also shows an obvious gradient distribution along the electric field direction; while the conductivity of each part of the non-gradient hydrogel is basically the same.

[0061] like Figure 5 As shown, the multi-gradient hydrogel prepared in Example 1 has a gradient distribution of SF and conductive materials in the hydrogel, and its stiffness also shows an obvious gradient distribution along the electric field direction; while the stiffness of each part of the non-gradient hydrogel is basically the same.

Claims

1. A hydrogel scaffold with multiple physical gradients, characterized in that: The hydrogel scaffold is a multi-physical gradient structure containing stiffness, conductivity and morphology; the preparation raw materials include thermosensitive polyisocyanuric acid polypeptide, silk nanofibers rich in β-sheet structure and conductive nanomaterials; the conductive nanomaterial dispersion and the silk nanofiber solution rich in β-sheet structure are evenly mixed; at 4°C, the conductive nanomaterial mixture loaded by silk nanofibers rich in β-sheet structure is added to the thermosensitive polyisocyanuric acid polypeptide aqueous solution and mixed evenly, a direct current electric field is applied to the hydrogel, the temperature is raised to above 25°C, the conductive material therein is induced to move directional by the electric field, and the temperature is controlled to induce the directional movement of the conductive material. The sol-gel transition of the thermosensitive polyisocyanate polypeptide hydrogel is used to fix the distribution of the conductive nanomaterials loaded on the silk nanofibers rich in β-sheet structure, and finally a multi-gradient distribution is formed. After the reaction is completed, the electric field is removed to obtain a conductive hydrogel scaffold with multiple physical gradients. The thermosensitive polyisocyanate polypeptide hydrogel is a highly bionic polypeptide hydrogel, which is liquid at 4°C and forms a gel above 25°C. The silk nanofibers rich in β-sheet structure carry a large amount of negative charge and can load conductive nanomaterials, thereby improving the dispersibility and stability of conductive nanomaterials in aqueous solution.

2. The hydrogel scaffold with multiple physical gradients according to claim 1, characterized in that: The mass concentration of the conductive nano material dispersion is 0.1 mg / mL to 5 mg / mL.

3. The hydrogel scaffold with multiple physical gradients according to claim 1, characterized in that: The conductive nanomaterial dispersion and the silk nanofiber solution rich in β-sheet structure are uniformly mixed in a ratio of 1:

4.

4. The hydrogel scaffold with multiple physical gradients according to claim 1, characterized in that: The conductive nanomaterial is at least one of graphene, silver nanowires, carbon nanotubes or Mxene; the mass ratio of the conductive nanomaterial to the temperature-sensitive polyisocyanate polypeptide monomer is 0.01-0.25:

1.

5. The hydrogel scaffold with multiple physical gradients according to claim 1, characterized in that: The electrodes of the direct current electric field include any of square, triangle, and point shapes. By regulating the shape, position, and time of applying the electric field, multi-gradient hydrogels with different patterns can be formed.

6. A method for preparing a hydrogel scaffold having multiple physical gradients according to any one of claims 1 to 6, characterized in that: The conductive nanomaterial dispersion and the silk nanofiber solution rich in β-sheet structure are evenly mixed, and then evenly mixed with the thermosensitive polyisocyanate polypeptide aqueous solution at 4°C, and then added to a square trough with electrodes, and a DC electric field is applied to the hydrogel, and the temperature is raised to 25°C. The DC electric field is used to induce the movement of the conductive nanomaterials loaded on the charged silk nanofibers rich in β-sheet structure in the hydrogel, and the sol-gel transition of the thermosensitive polyisocyanate polypeptide hydrogel is induced by controlling the temperature to fix the distribution of the conductive nanomaterials loaded on the silk nanofibers rich in β-sheet structure to form a multiple gradient distribution, so as to construct a hydrogel scaffold with multiple physical gradients. After the reaction is completed, the electric field is removed to obtain a conductive hydrogel scaffold with multiple physical gradients.

7. The method for preparing a hydrogel scaffold with multiple physical gradients according to claim 7, characterized in that: The following steps are involved: (1) dispersing a conductive nanomaterial in deionized water to obtain a conductive nanomaterial dispersion, adding the conductive nanomaterial dispersion to silk nanofibers rich in β-sheet structure, and rotating and stirring to obtain a conductive material dispersion loaded on silk nanofibers rich in β-sheet structure; (2) adding a mixture of conductive materials loaded by silk nanofibers rich in β-sheet structure to a thermosensitive polyisocyanate polypeptide aqueous solution at 4°C and mixing the mixture evenly, adding the mixture to a square tank with electrodes, and applying a direct current electric field to the mixed hydrogel at room temperature to induce the directional movement of the conductive materials loaded by silk nanofibers rich in β-sheet structure; (3) Raising the temperature to above 25°C induces the thermosensitive polyisocyanate to gel, forming a hydrogel with multiple gradients.

8. The method for preparing a hydrogel scaffold with multiple physical gradients according to claim 7, characterized in that: The mass concentration of the silk nanofibers rich in β-sheet structure in step (1) is 0.1wt% to 3wt%.

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