A hydrogel scaffold with multiple physical gradients and its preparation method

Abstract

The present invention discloses a hydrogel scaffold with multiple physical gradients. The synthetic raw materials include thermosensitive polyisocyanurate, silk nanofibers rich in β-sheet structure, and conductive nanomaterials. The conductive nanomaterial dispersion and BSNF solution are uniformly mixed in a ratio of 1:4. At 4°C, the BSNF-loaded conductive nanomaterial mixture is added to a PIC aqueous solution and mixed evenly. The mixture is then added to a square trough 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 induce PIC to gel, resulting in a conductive hydrogel scaffold with multiple physical gradients. The hydrogel contains multiple physical gradient structures, including stiffness, conductivity, and morphology. By regulating the shape and position of the electrodes and the duration of the electric field application, multi-gradient hydrogels with different patterns can be constructed. The preparation method of the present invention is simple, easy to operate, requires simple equipment and processes, is low-cost, and can be implemented in large-scale industrial production.

Description

Technical Field

[0001] The present 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 of 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 that simulates ECM with broad application prospects.

[0003] With the deepening of research on the nervous system, the function of gradient cues in the electrochemical matrix (ECM) has garnered widespread attention. Guided by gradient cues, neural cells can migrate, form directional cell alignment and axon extension, construct new neural networks, and replace the functions of damaged neurons. Therefore, introducing gradient signals into hydrogel neural scaffolds suitable for cell growth has great potential for regulating neural cell behavior and inducing neural repair. Compared to biochemical gradient signals such as neurotrophic factors, physical gradient signals in hydrogels, such as those of stiffness, conductivity, and topography, are relatively more stable and designable, and therefore have garnered widespread attention in regulating neural behavior. However, most current hydrogel scaffolds only incorporate a single physical gradient signal to regulate neural cell behavior. In vivo, however, it is the synergistic regulation of multiple physical gradient signals that achieves cell behavior regulation. Therefore, current hydrogel scaffolds struggle to accurately mimic the in vivo microenvironment in which cells reside. Constructing conductive hydrogel scaffolds with multiple physical gradients to mimic the complex in vivo and in vitro environments of neural cells and to regulate their proliferation, migration, growth, and differentiation is of great significance. 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 vitro and in vivo environment of nerve cells; the second purpose of the present invention is to provide a preparation method of the multiple physical gradient hydrogel scaffold.

[0005] Technical solution: A hydrogel scaffold with multiple physical gradients, wherein the hydrogel scaffold is a multi-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 evenly, and a DC electric field is applied to the hydrogel, and the temperature is raised to above 25°C, and the conductive material is induced by the electric field. The material moves in a directional manner, and the temperature is controlled to trigger the sol-gel transition of the thermosensitive polyisocyanate polypeptide hydrogel to fix the distribution of the conductive nanomaterial loaded on the silk nanofibers rich in β-sheet structure, ultimately forming a multi-gradient distribution. After the reaction is completed and the electric field is removed, a conductive hydrogel scaffold with multiple physical gradients is obtained; the thermosensitive polyisocyanate polypeptide hydrogel is a highly biomimetic 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 the 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 to 0.25:1.

[0009] The electrodes of the DC electric field include any of square, triangular, 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 temperature-sensitive polyisocyanate polypeptide aqueous solution at 4°C, adding the mixture to a square trough with electrodes, applying a DC electric field to the hydrogel, raising the temperature to 25°C, inducing the movement of the charged conductive nanomaterial loaded on the silk nanofiber rich in β-sheet structure in the hydrogel by the DC electric field, inducing the sol-gel transition of the temperature-sensitive polyisocyanate polypeptide hydrogel by controlling the temperature, fixing the distribution of the conductive nanomaterial loaded on 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, the electric field is removed to obtain 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) At 4°C, a mixture of conductive materials loaded on silk nanofibers rich in β-sheet structure was added to a thermosensitive polyisocyanate polypeptide aqueous solution and mixed evenly. The mixture was then placed in a square trough with electrodes. A DC electric field was applied to the mixed hydrogel at room temperature to induce the directional movement of the conductive materials loaded on silk nanofibers rich in β-sheet structure.

[0014] (3) Heating 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 0.1 wt% to 3 wt%.

[0016] Beneficial effects:

[0017] (1) The conductive nanomaterials are loaded on 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, forming a gradient distribution.

[0018] (2) Using electric fields to regulate the distribution of silk-loaded conductive nanomaterials in hydrogels, the preparation of hydrogels with multiple physical gradients such as stiffness, conductivity, and morphology was achieved. A DC electric field was used to induce the movement of charged β-sheet-rich silk nanofiber-loaded conductive nanomaterials in the hydrogel, forming a gradient distribution to construct a hydrogel scaffold 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 BSNF-loaded conductive nanomaterials is fixed, realizing the construction of multi-gradient hydrogels; avoiding the addition of external 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 for 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 with 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. 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 is synthesized from raw materials including thermosensitive polyisocyanate (PIC) (reference: Nature, 2013, 493, 651), β-sheet-rich silk nanofiber (BSNF) (reference: Biomacromolecules, 2014, 15(8):3044), and a conductive nanomaterial. The conductive nanomaterial dispersion and BSNF solution are uniformly mixed at a ratio of 1:4, with the conductive nanomaterial concentration being 0.1 mg / mL to 5 mg / mL and the BSNF concentration being 0.1 wt% to 3 wt%. At 4°C, the BSNF-loaded conductive nanomaterial mixture is added to the PIC aqueous solution and mixed uniformly. The mixture is 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 with 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 trough 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 (PIC) is a thermosensitive hydrogel, a highly biomimetic polypeptide hydrogel that is liquid at 4°C and forms a gel above 25°C. Silk nanofibers (SF), rich in β-sheet structures, carry a large amount of negative charge and can load conductive nanomaterials, improving their dispersibility and stability in aqueous solutions. Under a DC electric field, the negatively charged SF-loaded conductive nanomaterials migrate toward the positive electrode, forming a gradient distribution. This gradient distribution of SF-loaded conductive nanomaterials in the hydrogel results in the hydrogel exhibiting gradients in 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 DC electric field;

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

[0036] Preferably, the multi-physical gradient hydrogel scaffold refers to a hydrogel having gradients in stiffness, conductivity, morphology, etc.;

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

[0038] Example 1:

[0039] The preparation method of 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 an 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 trough. Square copper electrodes were placed at both ends of the square trough 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] Example 2:

[0044] The preparation method of 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 an 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.

[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 trough. A dot-shaped copper electrode and a square copper electrode were placed at both ends of the square trough 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] Example 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) Add BSNF / CNT to PIC aqueous solution at 4°C and mix well. Add 25 μL of PIC / BSNF / CNT hydrogel to a 5 mm × 5 mm × 1 mm square trough. Place a square copper electrode and a triangular copper electrode at both ends of the square trough as the anode and cathode, respectively. Apply 1.5 V.mm to the hydrogel. -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, obtaining a conductive hydrogel scaffold with multiple physical gradients

[0053] Example 4

[0054] The preparation method of 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) Add BSNF / CNT to PIC aqueous solution at 4°C and mix well. Add 25 μL of PIC / BSNF / CNT hydrogel to a 5 mm × 5 mm × 1 mm square trough. Place dot-shaped copper electrodes at both ends of the square trough as anode and cathode. Apply 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. The fluorescence intensity shows that the SF-loaded rGO 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 direction of the electric field; while the conductivity of each part of the non-gradient hydrogel is basically not much.

[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 raw materials for preparation 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 evenly mixed; at 4°C, the conductive nanomaterial mixture loaded by silk nanofibers rich in β-sheet structure is added to the thermosensitive polyisocyanate polypeptide aqueous solution and mixed evenly, and then a DC electric field is applied to the hydrogel, and the temperature is raised to above 25°C. The electric field is used to induce the directional movement of the conductive nanomaterials therein, and the temperature is controlled to induce the sol-gel transition of the thermosensitive polyisocyanate polypeptide hydrogel, thereby fixing the distribution of the conductive nanomaterial loaded by silk nanofibers rich in β-sheet structure, and finally forming a multi-gradient distribution. 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 biomimetic polypeptide hydrogel. It is liquid when heated to 25°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 conductive nanomaterial dispersion has a mass concentration of 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 DC electric field include any of square, triangular, 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 5, 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. The mixture is added to a square trough with electrodes, and a DC electric field is applied to the hydrogel. The temperature is raised to 25°C. The DC electric field is used to induce the movement of the charged conductive nanomaterial loaded on the silk nanofiber rich in β-sheet structure in the hydrogel. The sol-gel transition of the thermosensitive polyisocyanate polypeptide hydrogel is induced by controlling the temperature to fix the distribution of the conductive nanomaterial loaded on the silk nanofiber rich in β-sheet structure, forming a multiple gradient distribution 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 6, characterized in that: The following steps are involved: (1) dispersing the conductive nanomaterial in deionized water to obtain a conductive nanomaterial dispersion, adding the conductive nanomaterial dispersion to a silk nanofiber solution rich in β-sheet structure, and rotating and stirring to obtain a conductive nanomaterial mixture loaded with silk nanofibers rich in β-sheet structure; (2) At 4°C, a mixture of conductive nanomaterials loaded on silk nanofibers rich in β-sheet structure was added to a thermosensitive polyisocyanate polypeptide aqueous solution and mixed evenly. The mixture was then added to a square trough with electrodes. A DC electric field was applied to the mixed hydrogel at room temperature to induce the directional movement of the conductive material loaded on silk nanofibers rich in β-sheet structure. (3) Heating 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 β-sheet-rich silk nanofiber solution in step (1) is 0.1 wt% to 3 wt%.

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