CN113842505A - Preparation method of porous double-network nano conductive hydrogel nerve scaffold for nerve repair - Google Patents
Preparation method of porous double-network nano conductive hydrogel nerve scaffold for nerve repair Download PDFInfo
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- 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
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
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- 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
- A61L27/02—Inorganic materials
- A61L27/08—Carbon ; Graphite
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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
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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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
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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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
- A61L27/50—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
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/108—Elemental carbon, e.g. charcoal
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Abstract
The invention discloses a preparation method of a porous double-network nano conductive hydrogel nerve scaffold for nerve repair, wherein the porous double-network nano conductive hydrogel nerve scaffold is mainly prepared from natural biological materials, graphene is selected for modifying and increasing the conductivity of materials, polyethylene glycol diacrylate is mixed for photo-crosslinking to increase the elasticity and crosslinking degree of the materials, and finally gelation is completed by utilizing self-assembly of molecules to generate the porous double-network nano conductive hydrogel nerve scaffold. The scaffold is composed of biological macromolecules, provides a guiding effect for the growth of tissues and cells, is favorable for accelerating the repair of nerve injury, and in addition, the used materials have certain mechanical properties, are favorable for the proliferation and growth of cells, have no toxicity and have good histocompatibility.
Description
Technical Field
The invention relates to a preparation method of a nerve scaffold.
Background
Because the nerve injuries caused by accident traffic accidents, natural disasters and war conflicts are rare, the current gold standard for treating various nerve injuries is still autologous transplantation, but the autologous nerve transplantation is always limited by various conditions such as insufficient source of donor grafts, insufficient length of injured nerves, postoperative dysfunction, poor regeneration capacity and the like, so that the artificial graft made of various biological materials is considered to be an effective alternative treatment method, and a better treatment effect is achieved. However, nerve regeneration is not only morphologically repaired but also requires functional recovery. One of the important functions of the nerve is to transmit the bioelectrical signal to the corresponding target cell, tissue or organ by indirect conduction. Therefore, the construction of the biological scaffold material with the electrical conductivity has important clinical significance and application value for better realizing nerve injury repair. To date, there have been few reports of constructing artificial nerve grafts having conductive properties for nerve damage repair.
Currently, conducting polymers with good biocompatibility, such as polyaniline, polypyrrole, polythiophene and their derivatives (mainly aniline oligomers and poly (3, 4-ethylenedioxythiophene)), are commonly used to prepare hydrogel nerve scaffolds. Subsequently, electrically conductive polymer composites based on electrically conductive polymers and biocompatible, biodegradable polymers (natural or synthetic) are also growing in force. However, these materials have poor electrical conductivity and mechanical tensile properties, and further, the long-term toxicity of their degradation products in vivo is unknown.
The construction of the artificial nerve graft with the electric conductivity is to select the substance with the electric conductivity and the biocompatibility as the raw material to prepare the corresponding stent or to modify the molecule with the excellent electric conductivity onto the known molecule of a certain biological material. In fact, blending molecules with conductive properties with known materials in solution state, or coating conductive molecules on known materials in a plating manner can also make the materials have certain conductivity. However, since a homogeneous system is not formed per se, its conductive properties may be lost with the degradation of the material.
Disclosure of Invention
The invention aims to provide a preparation method of a porous double-network nano conductive hydrogel nerve scaffold for nerve repair, which is favorable for accelerating nerve injury repair.
The technical solution of the invention is as follows:
a preparation method of a porous double-network nano conductive hydrogel nerve scaffold for nerve repair is characterized by comprising the following steps: the porous double-network nano conductive hydrogel nerve scaffold is mainly made of natural biological materials, graphene is selected to modify and increase the conductivity of the materials, polyethylene glycol diacrylate is mixed to increase the elasticity and the crosslinking degree of the materials through photo-crosslinking, and finally gelation is completed by utilizing self-assembly of molecules to generate the porous double-network nano conductive hydrogel nerve scaffold.
The preparation method comprises the following steps:
step 1: modifying active protein groups on the side chains of natural high-molecular silk fibroin;
step 2: modifying the active functional groups on the surface of the nano conductive material graphene;
and step 3: graphene modification of silk fibroin: mixing the silk fibroin solution modified by the active protein group in the step 1 with the graphene solution modified by the active functional group in the step 2, and carrying out chemical reaction on the modifying groups of the silk fibroin solution and the graphene solution to generate covalent crosslinking, so that graphene is modified on silk fibroin molecules;
and 4, step 4: semi-crosslinking of the hydrogel: mixing the silk fibroin graphene mixed solution obtained in the step 3 with a polyethylene glycol diacrylate solution, and carrying out semi-crosslinking to generate a semi-gel product;
and 5: gelling: standing the semi-gel product obtained in the step 4 at room temperature, and completing gelation through molecular self-assembly.
The gelation is accomplished by self-assembly of molecules as follows: and standing the semi-crosslinked semi-gel product in a mold, accelerating the configuration transformation of natural biological macromolecular silk fibroin and the self-assembly of molecules by using the components of a photosensitive material solution, and generating the porous double-network nano conductive hydrogel nerve scaffold with a fixed form after about 2 hours.
Before the silk fibroin solution modified by the active protein groups and the graphene solution modified by the active functional groups are mixed, performing active protein reduction treatment on the silk fibroin solution modified by the active protein groups; and the active protein reduction treatment is to add TEPC-HCL into the silk fibroin solution modified by active protein groups to crack disulfide bonds so as to carry out oxidation reaction with active functional groups on graphene to generate covalent bonds.
And (3) when the silk fibroin is modified in the step (1), adjusting the pH value of the natural polymer silk fibroin solution to be 6.5.
The pH of the graphene solution in the step 2 is 7.5, and the substance with the corresponding functional group is dissolved in the graphene solution to perform the modification reaction while maintaining the shaking condition at 37 ℃.
In the step of semi-crosslinking of the hydrogel, when the silk fibroin graphene mixed solution and the polyethylene glycol diacrylate solution are mixed, the mixed solution is irradiated by ultraviolet light for 1-5 min.
When the mixed solution is irradiated by ultraviolet light, the distance between a light source and the solution needs to be kept at 1-2 cm so as to ensure that the solution can obtain enough light energy.
The invention is made of natural biological materials, silk fibroin is used as a substrate, graphene is used for modification, and the double-crosslinked nano conductive hydrogel nerve scaffold is formed by photo-crosslinking of polyethylene glycol diacrylate and self-assembly of silk fibroin molecules. The scaffold is composed of biological macromolecules, provides a guiding effect for the growth of tissues and cells, is favorable for accelerating the repair of nerve injury, and in addition, the used material has certain mechanical properties, is favorable for the proliferation and growth of cells, has no toxicity and has good histocompatibility. In addition, the nano conductive hydrogel nerve scaffold with different conductivities can be prepared by adjusting the modification amount of graphene, and different conductivity requirements can be met. The invention provides an idea for the conductive material in the aspect of nerve regeneration, and has important clinical test significance.
The present invention will be further described with reference to the following examples.
Detailed Description
Example 1:
(1) protein modification of silk fibroin:
the pH of a 20% solution of protofilament is adjusted to about 6.5, a certain amount of radical-activated molecules are added, and then the protein to be modified is added for chemical modification. Dialyzing and concentrating to the original concentration, and storing at 4 ℃ for later use.
(2) Active functional group modification of graphene:
adjusting the pH value of the graphene solution to about 7.5, dissolving a corresponding amount of molecules with active functional groups in the graphene solution, fully mixing, shaking at 37 ℃ for overnight reaction, and dialyzing and concentrating the product to 2 mg/ml.
(3) Graphene modification of silk fibroin:
adjusting the PH of the fibroin solution modified with protein to 7, mixing different amounts of graphene (graphene: fibroin mass ratio = 0.5%, 1%, 1.5%, 2%) solution modified by active functional groups with the fibroin solution, and reacting for 2 h.
(4) Semi-crosslinking of the hydrogel:
mixing the silk fibroin graphene mixed solution with 10% polyethylene glycol diacrylate solution with the same volume as that of silk fibroin, and irradiating for 3min by using ultraviolet light of 365nm and 0.4W/cm2 to perform semi-crosslinking to generate a semi-gel product.
(5) Gelling:
standing the semi-gel product at room temperature for 2h, and completing gelation through molecular self-assembly.
Example 2:
before the silk fibroin solution modified by the active protein groups in the step (3) is mixed with the graphene solution modified by the active functional groups, the silk fibroin solution modified by the active protein groups is subjected to active protein reduction treatment; and the active protein reduction treatment is to add TEPC-HCL into the silk fibroin solution modified by active protein groups according to the concentration of 5mg/ml to crack disulfide bonds so as to carry out oxidation reaction with active functional groups on graphene to generate covalent bonds. The rest is the same as example 1.
Claims (6)
1. A preparation method of a porous double-network nano conductive hydrogel nerve scaffold for nerve repair is characterized by comprising the following steps: comprises the following steps:
step 1: modifying active protein groups on the side chains of natural high-molecular silk fibroin;
step 2: modifying the active functional groups on the surface of the nano conductive material graphene;
and step 3: graphene modification of silk fibroin: mixing the silk fibroin solution modified by the active protein group in the step 1 with the graphene solution modified by the active functional group in the step 2, and carrying out chemical reaction on the modifying groups of the silk fibroin solution and the graphene solution to generate covalent crosslinking, so that graphene is modified on silk fibroin molecules;
and 4, step 4: semi-crosslinking of the hydrogel: mixing the silk fibroin graphene mixed solution obtained in the step 3 with a polyethylene glycol diacrylate solution, and carrying out semi-crosslinking to generate a semi-gel product;
and 5: gelling: standing the semi-gel product obtained in the step 4 at room temperature, and completing gelation through molecular self-assembly.
2. The method for preparing the porous double-network nano conductive hydrogel nerve scaffold for nerve repair according to claim 1, which is characterized in that: before the silk fibroin solution modified by the active protein groups and the graphene solution modified by the active functional groups are mixed, performing active protein reduction treatment on the silk fibroin solution modified by the active protein groups; and the active protein reduction treatment is to add TEPC-HCL into the silk fibroin solution modified by active protein groups to crack disulfide bonds so as to carry out oxidation reaction with active functional groups on graphene to generate covalent bonds.
3. The method for preparing the porous double-network nano conductive hydrogel nerve scaffold for nerve repair according to claim 1, which is characterized in that: and (3) when the silk fibroin is modified in the step (1), adjusting the pH value of the natural polymer silk fibroin solution to be 6.5.
4. The method for preparing the porous double-network nano conductive hydrogel nerve scaffold for nerve repair according to claim 1, which is characterized in that: the pH of the graphene solution in the step 2 is 7.5, and the substance with the corresponding functional group is dissolved in the graphene solution to perform the modification reaction while maintaining the shaking condition at 37 ℃.
5. The method for preparing the porous double-network nano conductive hydrogel nerve scaffold for nerve repair according to claim 1, which is characterized in that: in the step of semi-crosslinking of the hydrogel, when the silk fibroin graphene mixed solution and the polyethylene glycol diacrylate solution are mixed, the mixed solution is irradiated by ultraviolet light for 1-5 min.
6. The method for preparing the porous double-network nano conductive hydrogel nerve scaffold for nerve repair according to claim 5, which is characterized in that: when the mixed solution is irradiated by ultraviolet light, the distance between a light source and the solution needs to be kept at 1-2 cm so as to ensure that the solution can obtain enough light energy.
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