CN110124113B - Oriented conductive collagen hydrogel, bionic conductive nerve scaffold material and preparation method thereof - Google Patents

Oriented conductive collagen hydrogel, bionic conductive nerve scaffold material and preparation method thereof Download PDF

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CN110124113B
CN110124113B CN201910459198.2A CN201910459198A CN110124113B CN 110124113 B CN110124113 B CN 110124113B CN 201910459198 A CN201910459198 A CN 201910459198A CN 110124113 B CN110124113 B CN 110124113B
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collagen
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CN110124113A (en
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范红松
吴承恒
陈素萍
陈露
孙静
罗红蓉
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Sichuan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/383—Nerve cells, e.g. dendritic cells, Schwann cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Abstract

The invention discloses an oriented conductive collagen hydrogel, a bionic conductive nerve scaffold material and a preparation method thereof. And adding cells in the hydrogel preparation process to obtain the bionic conductive nerve scaffold material with in-situ cell loading. The hydrogel fiber prepared by the invention has the conductivity matched with natural nervous tissue, similar mechanical property and good biocompatibility, can be directionally arranged along the direction of the hydrogel fiber on a micro-nano scale, and can simulate a directional structure in the natural nervous tissue; the prepared bionic conductive nerve scaffold material can simulate the process that neurons in nerve tissues are arranged along the direction of nerve fibers and electric signals are conducted along axons, and has good application prospect in the field of nerve tissue engineering.

Description

Oriented conductive collagen hydrogel, bionic conductive nerve scaffold material and preparation method thereof
Technical Field
The invention belongs to the technical field of biomedical materials and biomedical engineering, relates to hydrogel, and particularly relates to oriented conductive collagen hydrogel and a preparation method thereof.
Background
The nerve tissue is a special tissue with a multi-stage directional structure, and consists of nerve bundles with a directional structure, the nerve bundles are composed of micrometer-scale nerve fibers, the nerve fibers are directionally arranged by schwann cells to form myelin sheaths, and the nerve axons directionally grow along the myelin sheaths.
In neural tissue, neurons communicate with each other via electrical signals, which play a crucial role in the survival, differentiation and functional expression of neurons. In addition to supporting cell growth, the extracellular matrix of neurons, as a mediator of electrical signal transmission between neurons and between neuron-matrix, has electrical conductivity that directly affects the efficiency of electrical signal transmission. Therefore, the biocompatible conductive matrix has excellent application prospect in the field of nerve tissue repair. Among them, conductive hydrogels have attracted much attention of scientists in recent years and have made rapid progress due to their excellent biocompatibility, plasticity, and conductivity properties. The hydrogel material itself has the following advantages: (1) the hydrogel material can realize in-situ three-dimensional wrapping of cells, and as a high-water-content hydrophilic porous material, the hydrogel material can simulate a natural extracellular matrix microenvironment to transmit nutrient substances and metabolic waste between the matrix and the cells; (2) many hydrogel materials, such as natural proteins, polysaccharides, etc., have excellent biocompatibility, and can support survival, growth and functional expression of cells; (3) through special design, the hydrogel material can realize simulation of the morphological characteristics, mechanical properties and the like of natural nervous tissue, thereby realizing bionic construction of the nervous tissue repair material. Meanwhile, the hydrogel material is endowed with excellent conductivity by grafting conductive molecules on the molecular chain of the hydrogel or compounding conductive nano materials in the hydrogel, so that the hydrogel material is more suitable for repairing nervous tissues. In the prior literature reports, carbon-based nano materials (such as carbon nano tubes and graphene oxide), metal nano particles (such as gold nano particles) and conductive polymers (such as polypyrrole and polyaniline) are used to improve the conductivity of hydrogel materials and to be used for nerve repair, wherein polypyrrole is favored by many researchers due to its excellent biocompatibility and conductivity.
Sumi Yang et al reported that sodium alginate (Alg) and polypyrrole (PPy) are used for the research of nerve tissue engineering to prepare conductive hydrogel, and composite hydrogel with good conductivity is prepared by in-situ polymerization of PPy in the Alg hydrogel; human mesenchymal stem cells (hMSCs) cultured on the surface of the electrically conductive hydrogel exhibit a pronounced neuro-differentiating characteristic compared to pure Alg hydrogel (Yang S, Jang L K, Kim S, et al and soft biomaterials forhuman mesenchymal stem cell culture and potential neural tissue engineeringapplications[J]Macromolecular bioscience,2016,16(11): 1653) -1661.). However, this use of the oxidizing agent Fe in the hydrogel3+The in situ polymerization of pyrrole monomers is not conducive to three-dimensional encapsulation of cells, which are restricted to two-dimensional growth on the hydrogel surface, far from the natural extracellular three-dimensional matrix microenvironment. Jissoo Shin et al reported a study of improving conductivity of Hyaluronic Acid (HA) hydrogel by using PPy and modified carbon nanotubes and applying it to the effect of promoting neurogenesis of neural stem cells, improving dispersibility of carbon nanotubes in aqueous solution by catechol modification, compounding it into HA hydrogel together with PPy, improving conductivity of HA, realizing in-situ encapsulation of cells, and promoting differentiation of neural stem cells cultured therein toward neural direction (Shin J, choice E J, Cho J H, et al, three-dimensional electrically involved hydrophilic acids and neural stem cells cultured therein [ J, C]Biomacromolecules,2017,18(10): 3060-. Although the method for preparing the conductive hydrogel realizes three-dimensional encapsulation of cells, the HA HAs limited cell compatibility, limited cell adhesion sites and lack of oriented microstructures in the conductive hydrogel, and cannot well imitate oriented structures in natural nerve tissues, so the HA HAs limited application in nerve tissue engineering.
In summary, the conductive hydrogel reported at present is difficult to be applied to the neural tissue engineering research due to the difficulty in realizing three-dimensional encapsulation of cells, poor compatibility or lack of oriented microstructure. The hydrogel which can realize in-situ three-dimensional wrapping of cells and has good biocompatibility, orientation and electrical conductivity is researched, and the hydrogel has very important significance for the research of nervous tissue engineering.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the oriented conductive collagen hydrogel and the preparation method thereof, and the prepared conductive collagen hydrogel has an oriented microstructure, stable physical and chemical properties, excellent conductivity and good biocompatibility, and can realize in-situ three-dimensional wrapping of cells, so that a bionic construction of a nerve tissue repair material can be simulated.
The invention also aims to provide a simple and efficient bionic conductive nerve scaffold material loaded by three-dimensional cells and a preparation method thereof, the prepared bionic conductive nerve scaffold material can simulate a qualitative structure in a nerve tissue, and meanwhile, the bionic conductive nerve scaffold material has good conductivity and can support the growth of nerve cells and promote the expression of functions of the nerve cells.
The preparation method of the oriented conductive collagen hydrogel provided by the invention comprises the following steps:
(1) preparation of Polymer nanoparticles
Uniformly dispersing a water-soluble high molecular polymer in deionized water at 50-80 ℃ to obtain a first dispersion liquid with the concentration of the water-soluble high molecular polymer being 0.075-0.15 g/mL, taking out the obtained first dispersion liquid, and cooling to room temperature; adding an oxidant into the first dispersion liquid, stirring until the oxidant is fully dispersed, and standing for at least 1h to obtain a second dispersion liquid with the oxidant concentration of 0.23-0.46 mol/L; then dropwise adding a conductive monomer into the second dispersion liquid under the stirring condition at the temperature of 0-5 ℃ until the concentration of the conductive monomer in the second dispersion liquid reaches 0.1-0.2 mol/L, then continuously stirring at the temperature of 0-5 ℃ for reaction for 3-5 hours, then centrifugally washing with deionized water to remove soluble impurities, freeze-drying the washed substance to determine the concentration of polymer nanoparticles in the washed substance, and then adding deionized water to dilute until the concentration of the polymer nanoparticles is 2-4 mg/mL to obtain a third dispersion liquid;
(2) preparation of hydrogel precursor mixture
Dissolving collagen in acetic acid to obtain an acetic acid solution with the collagen concentration of 8-12 mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a NaOH solution at 0-4 ℃, mixing the obtained solution with the third dispersion liquid obtained in the step (1), adding deionized water to adjust the collagen concentration to 5-8 mg/mL and the polymer nanoparticle concentration to 0.1-0.5mg/mL, and then oscillating and uniformly mixing the obtained mixed liquid to obtain a hydrogel precursor mixed liquid;
(3) preparation of electrically conductive collagen hydrogel
Injecting the hydrogel precursor mixed solution obtained in the step (2) and a PEG buffer solution from two inlets of a coaxial microfluid chip respectively according to a flow rate ratio of 1: 2-2: 1, and extruding the mixture into the PEG buffer solution through a coaxial pipeline to prepare the conductive collagen hydrogel microfiber.
According to the preparation method of the oriented conductive collagen hydrogel, the polymer nanoparticles with good water dispersibility and conductivity are prepared through a water-soluble high-molecular polymer/cationic oxidant complexing system, then the polymer nanoparticles and a collagen solution are uniformly mixed, and the assembly of collagen fibers is promoted through the dehydration effect of a PEG buffer solution, so that the polymer nanoparticle composite collagen hydrogel can be formed.
In the step (1), firstly, an oxidant is added into the first dispersion liquid dispersed with the water-soluble high molecular polymer, so that the oxidative metal cations and the high molecular polymer form complexation and are uniformly dispersed in water, and a second dispersion liquid is obtained. And dripping the conductive monomer into the second dispersion liquid, and carrying out oxidative polymerization reaction at 0-5 ℃ to obtain the conductive polymer nanoparticles coated by the molecular chain of the water-soluble high-molecular polymer. The water-soluble high molecular polymer molecular chains are attached to the surfaces of the conductive polymer particles, so that the dispersibility of the conductive polymer in water is enhanced. The water-soluble high molecular polymer adopted in the invention can improve the water dispersibility of the polymer nanoparticles, and the water-soluble high molecular polymer can be polyvinyl alcohol (PVA, Mw: 14500-45000), and is preferably polyvinyl alcohol with molecular weight of 31000. The oxidant adopted in the invention is FeCl3，Fe3+Has strong oxidizing property and can quickly form complexation with PVA molecules in water, so that oxidative polymerization can quickly occur on the reaction sites of the complex after the polymer conductive monomer is added. In the invention, the conductive polymer monomer is pyrrole, aniline or thiophene, and pyrrole (Py) is preferred, because polypyrrole (PPy) has excellent biocompatibility, and the conductive polymer monomer has wide application in the biomedical field.
In the step (2), firstly, the collagen is dissolved in acetic acid, and then the pH value of the acetic acid solution of the collagen is adjusted to 7 by using a NaOH solution at the temperature of 0-4 ℃ so as to obtain a neutral collagen solution. And (2) uniformly mixing the solution containing the collagen with the third dispersion liquid containing the polymer nanoparticles obtained in the step (1) to obtain a hydrogel precursor.
In the step (3), preparing hydrogel fibers by using a coaxial microfluidic chip and an injection pump, respectively connecting the hydrogel precursor solution and the PEG buffer solution prepared in the step with two inlets of the coaxial microfluidic chip through an injection pipeline provided with the injection pump, contacting the hydrogel precursor solution and the PEG in the coaxial pipeline of the microfluidic chip, promoting the assembly of the collagen fibers through the dehydration effect of the PEG-containing buffer solution to form the polymer nanoparticle-compounded collagen hydrogel fibers, extruding the hydrogel fibers into the PEG buffer solution, and further forming the fibers. The PEG buffer solution is prepared by dissolving polyethylene glycol-2000 in PBS buffer solution, and the concentration of polyethylene glycol in the buffer solution is 20%.
The oriented conductive collagen hydrogel prepared by the method has the conductivity matched with natural nervous tissues, similar mechanical properties and good biocompatibility. Due to the shearing force provided by PEG in the microfluidic chip, the collagen fibers are directionally arranged along the direction of the hydrogel fibers on the micro-nano scale, so that the directional structure in the natural nervous tissue can be simulated.
The invention further provides a preparation method of the bionic conductive nerve scaffold material, which comprises the following steps:
(1) preparation of Polymer nanoparticles
Uniformly dispersing a water-soluble high molecular polymer in deionized water at 50-80 ℃ to obtain a first dispersion, and taking out the first dispersion with the concentration of the water-soluble high molecular polymer of 0.075-0.15 g/mL and cooling to room temperature; adding an oxidant into the first dispersion liquid, stirring until the oxidant is fully dispersed, and standing for at least 1h to obtain a second dispersion liquid with the oxidant concentration of 0.23-0.46 mol/L; then dropwise adding a conductive monomer into the second dispersion liquid under the stirring condition at the temperature of 0-5 ℃ until the concentration of the conductive monomer in the second dispersion liquid reaches 0.1-0.2 mol/L, then continuously stirring at the temperature of 0-5 ℃ for reaction for 3-5 hours, then centrifugally washing with deionized water to remove soluble impurities, freeze-drying the washed substance to determine the concentration of polymer nanoparticles in the washed substance, and then adding deionized water to dilute until the concentration of the polymer nanoparticles is 2-4 mg/mL to obtain a third dispersion liquid;
(2) preparation of hydrogel precursor mixture
Dissolving collagen in acetic acid to obtain an acetic acid solution with the collagen concentration of 8-12 mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a NaOH solution at 0-4 ℃, mixing the obtained solution with the third dispersion liquid obtained in the step (1), adding deionized water to adjust the collagen concentration to 5-8 mg/mL and the polymer nanoparticle concentration to 0.1-0.5mg/mL, and then oscillating and uniformly mixing the obtained mixed liquid to obtain a hydrogel precursor mixed liquid;
(3) loading of cells
Uniformly mixing the hydrogel precursor mixed solution obtained in the step (2) with cells to obtain a cell-loaded hydrogel precursor mixed solution, wherein the cell density is 5 multiplied by 106～1×107Per mL;
(4) preparation of bionic conductive nerve scaffold material
Injecting the hydrogel precursor mixed solution loaded by the cells obtained in the step (3) and a buffer solution containing PEG from two inlets of a coaxial microfluid chip respectively according to a flow rate ratio of 1: 2-2: 1, and extruding the mixture into the PEG buffer solution through a coaxial pipeline to prepare the conductive collagen hydrogel microfiber, namely the bionic conductive nerve scaffold material.
The preparation method of the bionic conductive nerve scaffold material is to add cells in the preparation process of the oriented conductive collagen hydrogel given above so as to realize in-situ loading of the cells. The loaded cells may be neural stem cells or neuronal cells. According to the bionic conductive nerve scaffold material prepared by the method, the micro-nano oriented hydrogel can promote the oriented growth of neurons along the fiber direction and simulate the arrangement of the neurons in a nerve tissue along the nerve fiber direction. In particular, the addition of PPy enhances the conductivity of the collagen hydrogel, promotes the extension of neurons and the expression of related functions of nerves, and shows good application prospects of nerve tissue engineering.
Compared with the prior art, the oriented conductive collagen hydrogel and the bionic conductive nerve scaffold material provided by the invention have the following beneficial technical effects:
1. the preparation method of the oriented conductive collagen hydrogel comprises the steps of firstly, taking a water-soluble high-molecular polymer, an oxidant and a conductive monomer as raw materials, preparing polymer nanoparticles with good water dispersibility and conductivity through an oxidative polymerization reaction, then taking a mixed solution of natural biological macromolecular collagen with excellent biocompatibility and the polymer nanoparticles as the raw material, and promoting the assembly of collagen fibers through the dehydration action of a PEG buffer solution to form polymer nanoparticle composite collagen hydrogel fibers, wherein the hydrogel fibers prepared by the method have the conductivity matched with natural nervous tissues, similar mechanical properties and good biocompatibility, can be directionally arranged along the direction of the hydrogel fibers on a micro-nano scale, and can simulate a directional structure in the natural nervous tissues;
2. according to the preparation method of the bionic conductive nerve scaffold material, cells are added in the hydrogel preparation process to obtain the bionic conductive nerve scaffold material with in-situ cell loading; because the collagen fibers can be directionally arranged along the direction of the hydrogel fibers on a micro-nano scale, the scaffold material can simulate the process that neurons in nerve tissues are arranged along the direction of the nerve fibers and an electric signal is conducted along an axon, and the addition of the polymer nanoparticles enhances the conductivity of the collagen hydrogel, can promote the extension of the neurons and the expression of related functions of nerves, and shows good application prospects in the field of nerve tissue engineering.
Drawings
Fig. 1 is a schematic diagram of a preparation process of an oriented conductive collagen hydrogel, wherein a is a schematic diagram of preparation of PPy nanoparticles, b is a schematic diagram of preparation of a cell-loaded oriented conductive collagen hydrogel, and c is a schematic diagram of an internal pipeline of a coaxial microfluidic chip.
FIG. 2 is a schematic diagram of structural property characterization of PPy nanoparticles prepared in example 1 of the present invention; wherein a is a scanning electron microscope atlas, b is a particle size distribution histogram, and c is a Fourier transform infrared spectrogram.
FIG. 3 is a bright field pattern of microfibers of the conductive collagen hydrogel prepared in comparative example 1, and the PPy-collagen composite hydrogel prepared in examples 1, 2 and 3 (i.e., the conductive collagen hydrogel), wherein Col0 indicates that the concentration of PPy in the hydrogel is 0mg/mL, Col0.1 indicates that the concentration of PPy in the hydrogel is 0.1mg/mL, Col0.2 indicates that the concentration of PPy in the hydrogel is 0.2mg/mL, and Col0.5 indicates that the concentration of PPy in the hydrogel is 0.5 mg/mL.
FIG. 4 is a scanning electron microscope chromatogram of the conductive collagen hydrogel prepared in comparative example 1 and the PPy-collagen composite hydrogel microfibers prepared in example 1, example 2 and example 3, in which Col0, Col0.1, Col0.2 and Col0.5 have the same meanings as defined above.
FIG. 5 is a graph showing the results of the electrical conductivity test of the electrically conductive collagen hydrogel prepared in comparative example 1 and the PPy-collagen composite hydrogels prepared in examples 1, 2 and 3 according to the present invention, in which Col0, Col0.1, Col0.2 and Col0.5 have the same meanings as above.
FIG. 6 is the FDA/PI staining laser confocal mapping of the bionic conductive nerve scaffold materials prepared in comparative example 2, example 6 and example 7 of the invention after 1, 4 and 7 days of culture, wherein Col0, Col0.2 and Col0.5 have the same meanings.
FIG. 7 shows the results of culturing the bionic conductive neural scaffold materials prepared in comparative example 2, example 6 and example 7; wherein a is F-Actin/DAPI staining laser confocal mapping of the bionic conductive nerve scaffold materials prepared in comparative example 2, example 6 and example 7 after culturing for 1, 4 and 7 days respectively, and b is cell length box line diagram counted according to staining results of 1 day and 4 days.
FIG. 8 is a Tubulin- β 3/DAPI staining laser confocal mapping of the biomimetic conductive neural scaffold material prepared in comparative example 2, example 6 and example 7 after 4 and 7 days of culture.
FIG. 9 is an L-VGCC/DAPI staining laser confocal map of the bionic conductive nerve scaffold material prepared in comparative example 2, example 6 and example 7 of the invention after 4 and 7 days of culture.
Detailed Description
The technical solutions of the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the specific method for determining the concentration of polymer nanoparticles in the washed material by lyophilization was as follows: firstly, taking 1mL of substance obtained by washing, then carrying out freeze-drying treatment on 1mL of substance obtained by washing by a conventional freeze-drying means to obtain dry polymer nanoparticles, weighing the polymer nanoparticles to obtain the weight of the polymer nanoparticles, and further obtaining the concentration of the polymer nanoparticles in the substance obtained by washing.
The schematic diagram of the internal channel of the coaxial microfluidic chip used in the following examples is shown in fig. 1c, and it is mainly composed of a conical glass capillary channel for injecting hydrogel and a cylindrical glass capillary channel for injecting PEG buffer, both inlets are connected to the injector with liquid through the needle and the plastic capillary, and the extrusion speed of the injector can be controlled by the injection pump to control the liquid flow rate of the coaxial channel in the chip. Two different liquids in the coaxial pipeline of the microfluid chip are contacted and then form a core-shell structure, wherein hydrogel precursor mixed liquid is used as a core, and PEG buffer liquid is used as a shell. When the two liquids are contacted, the two liquids cannot diffuse due to the laminar flow effect, so that a stable fluid shape is maintained, and the shape of the hydrogel fiber is maintained.
Example 1
This example prepares an oriented conductive collagen hydrogel by the following steps:
(1) preparation of PPy nanoparticles
1.5g of PVA (molecular weight 31000) was dispersed in 20mL of deionized water, which was then placed in an oven at 60 ℃ for 30min to accelerate the dispersion to give a first dispersion, which was then taken out and cooled to room temperature. 1.2434g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.23 mol/L. Then, 140 mu L of conductive monomer Py is dropwise added into the second dispersion liquid (the concentration of Py is 0.1mol/L) at the temperature of 5 ℃ under the stirring condition, then, the mixture is continuously stirred and reacts for 4 hours at the temperature of 5 ℃, then, deionized water is used for centrifugally washing to remove soluble impurities, the concentration of PPy nano particles in the washed substance is determined through freeze-drying, and then, the washed substance is diluted by the deionized water until the concentration of the PPy nano particles is 3mg/mL, so that third dispersion liquid is obtained.
(2) Preparation of hydrogel precursor mixture
Dissolving 9mg of collagen in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a 5mol/L NaOH solution at 4 ℃, adding 0.05mL of the third dispersion liquid obtained in the step (1), adding 0.45mL of deionized water to adjust the collagen concentration to 6mg/mL and the PPy nanoparticle concentration to 0.1mg/mL, and then oscillating the obtained mixed liquid for 2min and uniformly mixing to obtain a hydrogel precursor mixed liquid.
(3) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer). And (3) respectively connecting the hydrogel precursor mixed solution obtained in the step (2) and an injector outlet of the PEG buffer solution with two inlets of a coaxial microfluid chip, respectively injecting the hydrogel precursor mixed solution and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:50) mu L/min, and extruding the mixture to a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PPy-collagen composite conductive collagen hydrogel microfiber.
Example 2
This example prepares an oriented conductive collagen hydrogel by the following steps:
(1) preparation of PPy nanoparticles
Dispersing 1.5g PVA (molecular weight of 31000) in 20mL deionized water, heating in an oven at 60 deg.C for 30min to accelerate its dispersion to obtain a first dispersion, taking out, and coolingAnd (4) cooling to room temperature. 1.2434g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.23 mol/L. Then, 140 mu L of conductive monomer Py is dropwise added into the second dispersion liquid (the concentration of Py is 0.1mol/L) at the temperature of 5 ℃ under the stirring condition, then, the mixture is continuously stirred and reacts for 4 hours at the temperature of 5 ℃, then, deionized water is used for centrifugally washing to remove soluble impurities, the concentration of PPy nano particles in the washed substance is determined through freeze-drying, and then, the washed substance is diluted by the deionized water until the concentration of the PPy nano particles is 3mg/mL, so that third dispersion liquid is obtained.
(2) Preparation of hydrogel precursor mixture
Dissolving 9mg of collagen in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a 5mol/L NaOH solution at 4 ℃, adding 0.1mL of the third dispersion liquid obtained in the step (1), adding 0.4mL of deionized water to adjust the collagen concentration to 6mg/mL and the PPy nanoparticle concentration to 0.2mg/mL, and then oscillating the obtained mixed liquid for 2min and uniformly mixing to obtain a hydrogel precursor mixed liquid.
(3) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer). And (3) respectively connecting the hydrogel precursor mixed solution obtained in the step (2) and an injector outlet of the PEG buffer solution with two inlets of a coaxial microfluid chip, respectively injecting the hydrogel precursor mixed solution and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:50) mu L/min, and extruding the mixture to a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PPy-collagen composite conductive collagen hydrogel microfiber.
Example 3
This example prepares an oriented conductive collagen hydrogel by the following steps:
(1) preparation of PPy nanoparticles
1.5g of PVA (molecular weight 31000) was dispersed in 20mL of deionized water, and then placed in an oven at 60 ℃ for 30minTo accelerate the dispersion to obtain a first dispersion, and then taking out and cooling to room temperature. 1.2434g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.23 mol/L. Then, 140 mu L of conductive monomer Py is dropwise added into the second dispersion liquid (the concentration of Py is 0.1mol/L) at the temperature of 5 ℃ under the stirring condition, then, the mixture is continuously stirred and reacts for 4 hours at the temperature of 5 ℃, then, deionized water is used for centrifugally washing to remove soluble impurities, the concentration of PPy nano particles in the washed substance is determined through freeze-drying, and then, the washed substance is diluted by the deionized water until the concentration of the PPy nano particles is 3mg/mL, so that third dispersion liquid is obtained.
(2) Preparation of hydrogel precursor mixture
Dissolving 9mg of collagen in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a 5mol/L NaOH solution at 4 ℃, adding 0.25mL of the third dispersion liquid obtained in the step (1), adding 0.25mL of deionized water to adjust the collagen concentration to 6mg/mL and the PPy nanoparticle concentration to 0.5mg/mL, and then oscillating the obtained mixed liquid for 2min and uniformly mixing to obtain a hydrogel precursor mixed liquid.
(3) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer). And (3) respectively connecting the hydrogel precursor mixed solution obtained in the step (2) and an injector outlet of the PEG buffer solution with two inlets of a coaxial microfluid chip, respectively injecting the hydrogel precursor mixed solution and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:50) mu L/min, and extruding the mixture to a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PPy-collagen composite conductive collagen hydrogel microfiber.
Example 4
This example prepares an oriented conductive collagen hydrogel by the following steps:
(1) preparation of PANI nanoparticles
Dispersing 1.5g of PVA (molecular weight 14500)In 20mL of deionized water, the mixture was then placed in an oven at 60 ℃ for 30min to accelerate the dispersion to give a first dispersion, which was then taken out and cooled to room temperature. 1.2434g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.23 mol/L. Then, 182 mu L of conductive monomer Aniline (ANI) is dripped into the second dispersion liquid (the concentration of the ANI is 0.1mol/L) under the stirring condition at the temperature of 5 ℃, the stirring reaction is continued for 3 hours at the temperature of 5 ℃, then deionized water is used for centrifugally washing to remove soluble impurities, the concentration of Polyaniline (PANI) nanoparticles in the washed substance is determined by freeze-drying, and then the washed substance is diluted to the concentration of the PANI nanoparticles of 2mg/mL by using the deionized water to obtain a third dispersion liquid.
(2) Preparation of hydrogel precursor mixture
Dissolving 8mg of collagen in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 8mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a 5mol/L NaOH solution at 0 ℃, adding 0.05mL of the third dispersion liquid obtained in the step (1), adding 0.45mL of deionized water to adjust the collagen concentration to 5.33mg/mL and the PANI nano-particle concentration to 0.1mg/mL, and then oscillating the obtained mixed liquid for 2min and uniformly mixing to obtain a hydrogel precursor mixed liquid.
(3) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer). And (3) respectively connecting the hydrogel precursor mixed solution obtained in the step (2) and an injector outlet of the PEG buffer solution with two inlets of a coaxial microfluid chip, respectively injecting the hydrogel precursor mixed solution and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:100) mu L/min, and extruding the mixture into a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PANI-collagen composite conductive collagen hydrogel microfiber.
Example 5
This example prepares an oriented conductive collagen hydrogel by the following steps:
(1) preparation of PTH nanoparticles
3.0g of PVA (molecular weight: 45000) was dispersed in 20mL of deionized water, and then placed in an oven at 60 ℃ for 30min to accelerate its dispersion to give a first dispersion, which was then taken out and cooled to room temperature. 2.4868g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.46 mol/L. Then, 318 mu L of conductive monomer thiophene is dropwise added into the second dispersion liquid (the concentration of thiophene is 0.2mol/L) under the condition of stirring at 0 ℃, then, the stirring reaction is continued for 5 hours at 0 ℃, then, deionized water is used for centrifugally washing to remove soluble impurities, the concentration of polythiophene (PTh) nanoparticles in the substance obtained by washing is determined by freeze-drying, and then, the substance obtained by washing is diluted by the deionized water until the concentration of the PTh nanoparticles is 4mg/mL, so that the third dispersion liquid is obtained.
(2) Preparation of hydrogel precursor mixture
Dissolving 12mg of collagen in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 12mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a 5mol/L NaOH solution at 4 ℃, adding 0.25mL of the third dispersion liquid obtained in the step (1), adding 0.25mL of deionized water to adjust the collagen concentration to 8mg/mL and the PTh nanoparticle concentration to 0.5mg/mL, and then oscillating the obtained mixed liquid for 2min and uniformly mixing to obtain a hydrogel precursor mixed liquid.
(3) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer). And (3) respectively connecting the hydrogel precursor mixed solution obtained in the step (2) and an injector outlet of the PEG buffer solution with two inlets of a coaxial microfluid chip, respectively injecting the hydrogel precursor mixed solution and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (100:50) mu L/min, and extruding the mixture to a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PTh-collagen composite conductive collagen hydrogel microfiber.
Comparative example 1
The procedure for preparing the oriented collagen hydrogel of this comparative example was as follows:
(1) preparation of hydrogel precursor solution
Dissolving 9mg of collagen in 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a NaOH solution at 4 ℃, and then adding 0.5mL of deionized water to adjust the collagen concentration to 6mg/mL to obtain a hydrogel precursor solution.
(2) Preparation of collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer). And (2) respectively connecting the hydrogel precursor solution obtained in the step (1) and an injector outlet of the PEG buffer solution with two inlets of a coaxial microfluid chip, respectively injecting the hydrogel precursor solution and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:50) mu L/min, and extruding the hydrogel precursor solution and the PEG buffer solution into a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the conductive collagen hydrogel microfiber.
The hydrogel samples prepared in the above examples were characterized by the following structures and properties:
the conductive polymer nanoparticles are prepared as shown in FIG. 1a, first by oxidizing agent (FeCl)3) And a water-soluble high molecular Polymer (PVA) to form a complex and to be uniformly dispersed in water. And then adding a conductive monomer, and carrying out oxidative polymerization reaction at 0-5 ℃ to obtain the conductive polymer nanoparticles coated by the molecular chain of the water-soluble high-molecular polymer. In order to confirm that PPy was successfully synthesized, SEM, particle size and fourier transform infrared spectroscopy analysis of the PPy nanoparticles prepared in example 1 were performed, and the analysis results are shown in fig. 2, and it can be seen from fig. 2 that the polymer nanoparticles prepared have a particle size of about 70 nm. In the infrared spectrum, at 3419cm-1The peak value is caused by the N-H stretching vibration of the PPy ring, 1552cm-1The peak at (B) can be attributed to the five-membered ring stretch of PPy at 1317cm-1The peak observed therein is due to the stretching vibration of C-N adsorption at 1187cm-1And 1043cm-1The peak value at (C) corresponds to the in-plane deformation vibration of ═ C-N, and further, 917cm-1OfThe peak was due to the C-N out-of-plane deformation vibration, and successful synthesis of PPy was confirmed from the peaks appearing on the infrared spectrum.
The conductive collagen hydrogel prepared in comparative example 1 and the PPy-collagen composite conductive collagen hydrogel microfibers prepared in examples 1, 2 and 3 were analyzed by bright field microscopy, and the results are shown in fig. 3, in which Col0 represents the PPy concentration in the hydrogel to be 0mg/mL, Col0.1 represents the PPy concentration in the hydrogel to be 0.1mg/mL, Col0.2 represents the PPy concentration in the hydrogel to be 0.2mg/mL, and Col0.5 represents the PPy concentration in the hydrogel to be 0.5mg/mL (the same below). As can be seen from FIG. 3, with the increase of the concentration of the compounded PPy, the color of the hydrogel gradually turns black from transparent, the color is uniform, and the PPy in the hydrogel fiber prepared on the surface is uniformly distributed, because the surface of the PPy prepared by the method provided by the invention has a hydrophilic PVA molecular chain, the PPy has good hydrophilicity, and the diameters of the four groups of hydrogel fibers do not change obviously, which indicates that the addition of the PPy has no influence on the formation of the hydrogel microfiber.
SEM analysis of the conductive collagen hydrogel prepared in comparative example 1 and the PPy-collagen-composited conductive collagen hydrogel microfibers prepared in example 1, example 2, and example 3 was performed, and the results are shown in fig. 4. As can be seen from fig. 4, in the four groups of hydrogels, collagen fibers are directionally arranged in the micro-nano scale (as indicated by white arrows); the PPy nanoparticles were uniformly distributed in the hydrogels of groups col0.1, col0.2 and col0.5 without agglomeration, further indicating that PPy has good hydrophilicity, thus being able to be uniformly distributed in the collagen fibers.
The conductive collagen hydrogel prepared in comparative example 1 and the PPy-collagen composite conductive collagen hydrogel microfibers prepared in example 1, example 2 and example 3 were subjected to a conductivity test, and the results are shown in fig. 5. As can be seen from fig. 5, the conductivity of the hydrogel increased with the increase of the PPy concentration, but due to the lower PPy composite amount, the conductivity of group Col0.1 was not significantly different from that of Col0 prepared in comparative example 1, and when the PPy composite concentration was 0.5mg/mL, the conductivity was 8.8 times that of the pure collagen hydrogel group, indicating that the PPy-collagen composite conductive collagen hydrogel microfiber prepared by the method provided by the present invention has good conductivity.
Example 6
The steps for preparing the bionic conductive nerve scaffold material in the embodiment are as follows:
(1) preparation of PPy nanoparticles
1.5g of PVA (molecular weight 31000) was dispersed in 20mL of deionized water, which was then placed in an oven at 60 ℃ for 30min to accelerate the dispersion to give a first dispersion, which was then taken out and cooled to room temperature. 1.2434g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.23 mol/L. Then, dropwise adding 140 mu L of conductive monomer Py into the second dispersion liquid (the concentration of Py is 0.1mol/L) at 5 ℃ under the stirring condition, continuously stirring at 5 ℃ for reaction for 4 hours, then centrifugally washing with deionized water to remove soluble impurities, determining the concentration of PPy nanoparticles in the substance obtained by washing through freeze-drying, and then diluting the substance obtained by washing with deionized water until the concentration of PPy nanoparticles is 3mg/mL to obtain a third dispersion liquid;
(2) preparation of hydrogel precursor mixture
Under the aseptic condition, 9mg of collagen is dissolved in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, then at 4 ℃, a 5mol/L NaOH solution is used for adjusting the pH value of the acetic acid solution of the collagen to 7, 0.1mL of the third dispersion liquid obtained in the step (1) is added, 0.4mL of deionized water is added for adjusting the collagen concentration to 6mg/mL and the PPy nano-particle concentration to 0.2mg/mL, and then the obtained mixed liquid is shaken for 2min and mixed evenly to obtain the hydrogel precursor mixed liquid.
(3) Loading of cells
Uniformly mixing the hydrogel precursor solution obtained in the step (2) with rat basophilic cytoma cells (PC12 cells widely used as neuron-like cells) induced and differentiated by NGF to obtain a hydrogel precursor mixed solution loaded by cells, wherein the cell density in the hydrogel precursor solution is 1 x 107one/mL.
(4) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer), and used as a buffer after sterilization. And (3) respectively connecting the outlet of the injector filled with the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution obtained in the step (3) with two inlets of a coaxial microfluid chip, then respectively injecting the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:50) mu L/min, and extruding the mixture into a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PPy-collagen composite conductive collagen hydrogel microfiber loaded by the cells, namely the bionic conductive nerve scaffold material.
Example 7
The steps for preparing the bionic conductive nerve scaffold material in the embodiment are as follows:
(1) preparation of PPy nanoparticles
1.5g of PVA (molecular weight 31000) was dispersed in 20mL of deionized water, which was then placed in an oven at 60 ℃ for 30min to accelerate the dispersion to give a first dispersion, which was then taken out and cooled to room temperature. 1.2434g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.23 mol/L. Then, 140 mu L of conductive monomer Py is dropwise added into the second dispersion liquid (the concentration of Py is 0.1mol/L) at the temperature of 5 ℃ under the stirring condition, then, the mixture is continuously stirred and reacts for 4 hours at the temperature of 5 ℃, then, deionized water is used for centrifugally washing to remove soluble impurities, the concentration of PPy nano particles in the washed substance is determined through freeze-drying, then, the washed substance is diluted by the deionized water until the concentration of the PPy nano particles is 3mg/mL, and the third dispersion liquid is obtained and sterilized for later use.
(2) Preparation of hydrogel precursor mixture
Under the aseptic condition, 9mg of collagen is dissolved in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, then at 4 ℃, a 5mol/L NaOH solution is used for adjusting the pH value of the acetic acid solution of the collagen to 7, 0.25mL of the third dispersion liquid obtained in the step (1) is added, 0.25mL of deionized water is added for adjusting the collagen concentration to 6mg/mL and the PPy nano-particle concentration to 0.5mg/mL, and then the obtained mixed liquid is shaken for 2min and mixed evenly to obtain the hydrogel precursor mixed liquid.
(3) Loading of cells
Uniformly mixing the hydrogel precursor solution obtained in the step (2) with rat basophilic cytoma cells (PC12 cells widely used as neuron-like cells) induced and differentiated by NGF to obtain a hydrogel precursor mixed solution loaded by cells, wherein the cell density in the hydrogel precursor solution is 1 x 107one/mL.
(4) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer), and used as a buffer after sterilization. And (3) respectively connecting the outlet of the injector filled with the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution obtained in the step (3) with two inlets of a coaxial microfluid chip, then respectively injecting the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:50) mu L/min, and extruding the mixture into a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PPy-collagen composite conductive collagen hydrogel microfiber loaded by the cells, namely the bionic conductive nerve scaffold material.
Example 8
The steps for preparing the bionic conductive nerve scaffold material in the embodiment are as follows:
(1) preparation of PPy nanoparticles
1.5g of PVA (molecular weight 31000) was dispersed in 20mL of deionized water, which was then placed in an oven at 60 ℃ for 30min to accelerate the dispersion to give a first dispersion, which was then taken out and cooled to room temperature. 1.2434g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.23 mol/L. Then, 140. mu.L of conductive monomer Py was added dropwise to the second dispersion solution (Py concentration 0.1mol/L) at 5 ℃ with stirring, and then the reaction was continued at 5 ℃ with stirring for 4 hours, after which the soluble impurities were removed by centrifugal washing with deionized water, and the PPy in the washed material was confirmed by lyophilizationAnd (4) diluting the substance obtained by washing with deionized water until the concentration of the PPy nanoparticles is 3mg/mL, and sterilizing for later use.
(2) Preparation of hydrogel precursor mixture
Under the aseptic condition, 9mg of collagen is dissolved in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, then at 4 ℃, a 5mol/L NaOH solution is used for adjusting the pH value of the acetic acid solution of the collagen to 7, 0.25mL of the third dispersion liquid obtained in the step (1) is added, 0.25mL of deionized water is added for adjusting the collagen concentration to 6mg/mL and the PPy nano-particle concentration to 0.5mg/mL, and then the obtained mixed liquid is shaken for 2min and mixed evenly to obtain the hydrogel precursor mixed liquid.
(3) Loading of cells
Uniformly mixing the hydrogel precursor solution obtained in the step (2) with rat basophilic cytoma cells (PC12 cells widely used as neuron-like cells) induced and differentiated by NGF to obtain a hydrogel precursor mixed solution loaded by cells, wherein the cell density in the hydrogel precursor solution is 1 x 107one/mL.
(4) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer), and used as a buffer after sterilization. And (3) respectively connecting the outlet of the injector filled with the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution obtained in the step (3) with two inlets of a coaxial microfluid chip, then respectively injecting the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:100) mu L/min, and extruding the mixture into a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PPy-collagen composite conductive collagen hydrogel microfiber loaded by the cells, namely the bionic conductive nerve scaffold material.
Example 9
The steps for preparing the bionic conductive nerve scaffold material in the embodiment are as follows:
(1) preparation of PPy nanoparticles
1.5g of PVA (molecular weight 31000) was dispersed in 20mL of deionized waterThen, the mixture was placed in an oven at 60 ℃ and heated for 30min to accelerate the dispersion thereof to obtain a first dispersion, and then taken out and cooled to room temperature. 1.2434g of oxidizing agent FeCl3·6H2Adding O into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for 1h to obtain FeCl3A second dispersion having a concentration of 0.23 mol/L. Then, 140 mu L of conductive monomer Py is dropwise added into the second dispersion liquid (the concentration of Py is 0.1mol/L) at the temperature of 5 ℃ under the stirring condition, then, the mixture is continuously stirred and reacts for 4 hours at the temperature of 5 ℃, then, deionized water is used for centrifugally washing to remove soluble impurities, the concentration of PPy nano particles in the washed substance is determined through freeze-drying, then, the washed substance is diluted by the deionized water until the concentration of the PPy nano particles is 3mg/mL, and the third dispersion liquid is obtained and sterilized for later use.
(2) Preparation of hydrogel precursor mixture
Under the aseptic condition, 9mg of collagen is dissolved in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, then at 4 ℃, a 5mol/L NaOH solution is used for adjusting the pH value of the acetic acid solution of the collagen to 7, 0.25mL of the third dispersion liquid obtained in the step (1) is added, 0.25mL of deionized water is added for adjusting the collagen concentration to 6mg/mL and the PPy nano-particle concentration to 0.5mg/mL, and then the obtained mixed liquid is shaken for 2min and mixed evenly to obtain the hydrogel precursor mixed liquid.
(3) Loading of cells
Uniformly mixing the hydrogel precursor solution obtained in the step (2) with rat basophilic cytoma cells (PC12 cells widely used as neuron-like cells) induced and differentiated by NGF to obtain a hydrogel precursor mixed solution loaded by cells, wherein the cell density in the hydrogel precursor solution is 5 x 106one/mL.
(4) Preparation of electrically conductive collagen hydrogel
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer), and used as a buffer after sterilization. And (3) respectively connecting the outlet of the injector filled with the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution obtained in the step (3) with two inlets of a coaxial microfluid chip, then respectively injecting the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (100:100) mu L/min, and extruding the mixture into a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PPy-collagen composite conductive collagen hydrogel microfiber loaded by the cells, namely the bionic conductive nerve scaffold material.
Comparative example 2
The steps for preparing the bionic nerve scaffold material in the comparative example are as follows:
(1) preparation of hydrogel precursor solution
Under the aseptic condition, 9mg of collagen is dissolved in 1mL of 0.5mol/L acetic acid to obtain an acetic acid solution with the collagen concentration of 9mg/mL, then at the temperature of 4 ℃, 5mol/L NaOH solution is used for adjusting the pH value of the acetic acid solution of the collagen to 7, and then 0.5mL of deionized water is added for adjusting the collagen concentration to 6mg/mL, so that the hydrogel precursor solution is obtained.
(2) Loading of cells
Uniformly mixing the hydrogel precursor solution obtained in the step (1) with rat basophilic cytoma cells (PC12 cells widely used as neuron-like cells) induced and differentiated by NGF to obtain a hydrogel precursor mixed solution loaded by cells, wherein the cell density in the hydrogel precursor solution is 1 x 107one/mL.
(3) Preparation of bionic nerve scaffold material
Polyethylene glycol-2000 (PEG-2000) was dissolved in PBS buffer to obtain 20% PEG-2000 concentration in PBS buffer (i.e., PEG buffer), and used as a buffer after sterilization. And (3) respectively connecting the outlet of the injector filled with the hydrogel precursor mixed solution loaded by the cells obtained in the step (2) and the PEG buffer solution with two inlets of a coaxial microfluid chip, then respectively injecting the hydrogel precursor mixed solution loaded by the cells and the PEG buffer solution into the coaxial microfluid chip from the two inlets according to the flow rate ratio (50:50) mu L/min, and extruding the mixture into a culture dish filled with the PEG buffer solution through a coaxial pipeline of the coaxial microfluid chip to obtain the PPy-collagen composite conductive collagen hydrogel microfiber loaded by the cells, namely the bionic conductive nerve scaffold material.
The preparation process of the cell-loaded oriented conductive collagen hydrogel is shown in fig. 1b, firstly, cells are loaded into a mixed solution of collagen and polymer nanoparticles to obtain a cell-loaded hydrogel precursor mixed solution, then the cell-loaded hydrogel precursor mixed solution and a PEG PBS buffer solution are injected into a coaxial microfluidic chip together, and the assembly of collagen fibers is promoted through the dehydration effect of the PEG buffer solution to form the cell in-situ loaded bionic conductive nerve scaffold material. In order to illustrate the biocompatibility and the effect of the bionic nerve scaffold sample prepared by the method of the present invention in promoting the elongation of neurons and the expression of nerve-related functions, the following bionic conductive nerve scaffold materials prepared in comparative example 2, example 6 and example 7 were subjected to related performance tests.
The bionic conductive nerve scaffold materials prepared in comparative example 2, example 6 and example 7 were respectively subjected to FDA/PI staining on PC12 cells loaded in the materials after 1, 4 and 7 days of loading, and then laser confocal microscope analysis was performed on the stained scaffold materials, and the results are shown in fig. 6. As can be seen from the figure, the PC12 cells have good activity in each group of scaffolds, which indicates that the bionic conductive nerve scaffold material prepared by the invention has excellent biocompatibility.
The bionic conductive nerve scaffold materials prepared in comparative example 2, example 6 and example 7 were subjected to F-actin/DAPI staining on PC12 cells loaded in the materials after 1, 4 and 7 days of cell loading, and then the stained scaffold materials were subjected to laser confocal microscope analysis, and the results are shown in FIG. 7 (a). As can be seen in fig. 7(a), the PC12 cells elongated in the hydrogel along the hydrogel axis. The length of the cells in the micrograph of the stained corresponding cells loaded for 1 and 4 days was counted, and the result is shown in FIG. 7 (b). As can be seen from fig. 7(b), the spreading of cells was faster in the col0.5 group with higher conductivity.
The bionic conductive nerve scaffold materials prepared in comparative example 2, example 6 and example 7 were respectively subjected to the immunofluorescence staining of the neurogenesis-related protein Tubulin- β 3 on the PC12 cells loaded in the scaffold material after 4 and 7 days of cell loading, and then the stained scaffold material was subjected to laser confocal microscopy analysis, and the result is shown in FIG. 8. from FIG. 8, it can be seen that at two time points, the expression level of Tubulin- β 3 of the PC12 cells in the hydrogel group with stronger conductivity is higher, which indicates that the cells are better connected with each other in the conductive matrix through an electric signal, so that the expression of Tubulin- β 3 is promoted.
Calcium ions play an important role in the excitation activity of nerve cells, and researches show that the high expression of calcium ion channel proteins can promote the functional expression and neurogenesis of neurons. In view of this, the biomimetic conductive nerve scaffold materials prepared in comparative example 2, example 6 and example 7 were subjected to calcium ion channel protein L-VGCC immunofluorescence staining on PC12 cells loaded in the scaffold material 4, 7 days after cell loading, and then the stained scaffold material was subjected to laser confocal microscopy analysis, with the results shown in fig. 9. As can be seen from fig. 9, the expression of PC12 cells L-VGCC was higher in the conductive hydrogel group (especially the col0.5 group) than in the pure collagen hydrogel group at both time points.
From the analysis, the bionic conductive nerve scaffold material obtained by the preparation method provided by the invention has the advantages that the PPy nanoparticles enhance the conductivity of the hydrogel, so that the extension of neurons and the expression of related functions of nerves can be promoted, and the bionic conductive nerve scaffold material has a good application prospect in the field of cell tissue engineering.
Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.

Claims (9)

1. A preparation method of oriented conductive collagen hydrogel is characterized by comprising the following steps:
(1) preparation of Polymer nanoparticles
Uniformly dispersing a water-soluble high molecular polymer in deionized water at 50-80 ℃ to obtain a first dispersion liquid with the concentration of the water-soluble high molecular polymer being 0.075-0.15 g/mL, taking out the obtained first dispersion liquid, and cooling to room temperature; adding an oxidant into the first dispersion liquid, stirring until the oxidant is fully dispersed, standing for at least 1h to obtain a second component with the oxidant concentration of 0.23-0.46 mol/LDispersing; then dropwise adding a conductive monomer into the second dispersion liquid under the stirring condition at the temperature of 0-5 ℃ until the concentration of the conductive monomer in the second dispersion liquid reaches 0.1-0.2 mol/L, then continuously stirring at the temperature of 0-5 ℃ for reaction for 3-5 hours, then centrifugally washing with deionized water to remove soluble impurities, freeze-drying the washed substance to determine the concentration of polymer nanoparticles in the washed substance, and then adding deionized water to dilute until the concentration of the polymer nanoparticles is 2-4 mg/mL to obtain a third dispersion liquid; the oxidant is FeCl3；
(2) Preparation of hydrogel precursor mixture
Dissolving collagen in acetic acid to obtain an acetic acid solution with the collagen concentration of 8-12 mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a NaOH solution at 0-4 ℃, mixing the obtained solution with the third dispersion liquid obtained in the step (1), adding deionized water to adjust the collagen concentration to 5-8 mg/mL and the polymer nanoparticle concentration to 0.1-0.5mg/mL, and then oscillating and uniformly mixing the obtained mixed liquid to obtain a hydrogel precursor mixed liquid;
(3) preparation of electrically conductive collagen hydrogel
Injecting the hydrogel precursor mixed solution obtained in the step (2) and a PEG buffer solution from two inlets of a coaxial microfluid chip respectively according to a flow rate ratio of 1: 2-2: 1, and extruding the mixture into the PEG buffer solution through a coaxial pipeline to prepare the conductive collagen hydrogel microfiber.
2. The method for preparing oriented conductive collagen hydrogel according to claim 1, wherein in step (1), the water-soluble high molecular polymer is polyvinyl alcohol with a molecular weight of 14500-45000, and the oxidant is FeCl3The conductive monomer is pyrrole, aniline or thiophene.
3. The method for preparing an oriented electrically conductive collagen hydrogel according to claim 1, wherein in step (3), the PEG buffer is prepared by dissolving polyethylene glycol in PBS buffer, and the concentration of polyethylene glycol in the buffer is 20%.
4. An oriented electrically conductive collagen hydrogel prepared by the method of any one of claims 1 to 3.
5. A preparation method of a bionic conductive nerve scaffold material is characterized by comprising the following steps:
(1) preparation of Polymer nanoparticles
Uniformly dispersing a water-soluble high molecular polymer in deionized water at 50-80 ℃ to obtain a first dispersion liquid with the concentration of the water-soluble high molecular polymer being 0.075-0.15 g/mL, taking out the obtained first dispersion liquid, and cooling to room temperature; adding an oxidant into the first dispersion liquid, stirring until the oxidant is fully dispersed, and standing for at least 1h to obtain a second dispersion liquid with the oxidant concentration of 0.23-0.46 mol/L; then dropwise adding a conductive monomer into the second dispersion liquid under the stirring condition at the temperature of 0-5 ℃ until the concentration of the conductive monomer in the second dispersion liquid reaches 0.1-0.2 mol/L, then continuously stirring at the temperature of 0-5 ℃ for reaction for 3-5 hours, then centrifugally washing with deionized water to remove soluble impurities, freeze-drying the washed substance to determine the concentration of polymer nanoparticles in the washed substance, and then adding deionized water to dilute until the concentration of the polymer nanoparticles is 2-4 mg/mL to obtain a third dispersion liquid; the oxidant is FeCl3；
(2) Preparation of hydrogel precursor mixture
Dissolving collagen in acetic acid to obtain an acetic acid solution with the collagen concentration of 8-12 mg/mL, adjusting the pH value of the acetic acid solution of the collagen to 7 by using a NaOH solution at 0-4 ℃, mixing the obtained solution with the third dispersion liquid obtained in the step (1), adding deionized water to adjust the collagen concentration to 5-8 mg/mL and the polymer nanoparticle concentration to 0.1-0.5mg/mL, and then oscillating and uniformly mixing the obtained mixed liquid to obtain a hydrogel precursor mixed liquid;
(3) loading of cells
Uniformly mixing the hydrogel precursor mixed solution obtained in the step (2) with cells to obtain a cell-loaded hydrogel precursor mixed solution, wherein the cell density is 5 multiplied by 106～1×107Per mL;
(4) preparation of bionic conductive nerve scaffold material
Injecting the hydrogel precursor mixed solution loaded by the cells obtained in the step (3) and a buffer solution containing PEG from two inlets of a coaxial microfluid chip respectively according to a flow rate ratio of 1: 2-2: 1, and extruding the mixture into the PEG buffer solution through a coaxial pipeline to prepare the conductive collagen hydrogel microfiber, namely the bionic conductive nerve scaffold material.
6. The preparation method of the bionic conductive nerve scaffold material according to claim 5, wherein in the step (1), the water-soluble high polymer is polyvinyl alcohol with a molecular weight of 14500-45000, and the oxidant is FeCl3The conductive monomer is pyrrole, aniline or thiophene.
7. The method for preparing the bionic conductive nerve scaffold material according to claim 5, wherein in the step (3), the cells are neural stem cells or neuron cells.
8. The method for preparing the bionic conductive nerve scaffold material according to claim 5, wherein in the step (4), the buffer solution containing PEG is obtained by dissolving polyethylene glycol in PBS buffer solution, and the concentration of the polyethylene glycol in the buffer solution is 20%.
9. The bionic conductive nerve scaffold material prepared by the method of any one of claims 5 to 8.
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