CN111359019A - Preparation method of graphene and chitosan composite conductive nerve scaffold with longitudinal pore channels - Google Patents
Preparation method of graphene and chitosan composite conductive nerve scaffold with longitudinal pore channels Download PDFInfo
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- CN111359019A CN111359019A CN202010180359.7A CN202010180359A CN111359019A CN 111359019 A CN111359019 A CN 111359019A CN 202010180359 A CN202010180359 A CN 202010180359A CN 111359019 A CN111359019 A CN 111359019A
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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
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/443—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with carbon fillers
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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
- A61L27/58—Materials at least partially resorbable by the body
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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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
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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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 graphene-chitosan composite conductive nerve repair scaffold with a longitudinal pore passage, which comprises the steps of uniformly mixing a chitosan solution and a graphene dispersion solution, adding 0.5-1.0% of genipin, mixing, and injecting the obtained mixed solution into a cylindrical mold with the inner diameter of 2-10 mm; soaking the mold into liquid nitrogen at a constant speed of 0.1-5 cm/min by adopting a one-way freezing method to fully freeze the mixed solution; and then freeze drying and fully crosslinking chitosan molecular chains to obtain the final product. The invention has simple process, mild reaction condition and easy operation, and the product has good mechanical toughness, conductivity, biocompatibility and in-vivo degradability and also has the functions of stimulating nerve fibers and regenerating blood vessels.
Description
Technical Field
The invention relates to the technical field of nano materials, in particular to a preparation method of a graphene-chitosan composite conductive nerve repair scaffold with a longitudinal ordered pore channel.
Background
Peripheral nerve injury is a common surgical disease in clinic, and accounts for 2.8% of surgical operations. The most common method for treating peripheral nerve injury at present is autologous nerve transplantation, but the autologous transplantation has many disadvantages, such as insufficient donors, difficult matching of sizes of donor nerves, injury of donor parts, immunological rejection of allogeneic nerves and the like. Therefore, a nerve repair scaffold with good biocompatibility is an important means for replacing auto/allogenic nerve transplantation in the future.
Excellent nerve repair scaffold requires materials with good biocompatibility and biodegradability and induces the growth of regenerative neurons to the far end of the injury. The chitosan is a natural biological material, has excellent biocompatibility and biodegradability, and can control the degradation rate of the material by controlling the molecular weight and the crosslinking degree of the chitosan. The chitosan has abundant amino groups, and is convenient for chemical modification. The chitosan oligosaccharide which is a degradation product of chitosan has the function of promoting Schwann cell proliferation and adhesion. Therefore, the chitosan is suitable to be used as a material of the nerve repair scaffold.
In the nerve repair process, proper electrical stimulation has a good promoting effect on the recovery of a patient, so that a certain electric conduction capability of a scaffold material is required to promote the directional growth of regenerative neurons. Graphene and oxidized derivatives thereof are nano materials with excellent conductivity, have good biocompatibility and in vivo degradability, and can be degraded in vivo by oxidase in neutrophils. In addition, graphene oxide can promote adhesion of neurons and angiogenesis, and is an excellent nerve regeneration material.
Good neural repair scaffolds are also required to be able to direct the growth of regenerative neuronal cells and their neurites to the distal end of the lesion. Therefore, compared with a disordered scaffold, the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal ordered pore channels has great advantages on nerve repair. The material is prepared by freezing pore-forming and freeze-drying methods, and the chemical components and physical properties of the material can be kept to the maximum extent.
Although a patent (CN 1385217A) for preparing a nerve repair scaffold by using chitosan and a patent (CN 109481736A) for preparing a cartilage-bone repair scaffold by using graphene in a bionic gradient exist in China, the search of the research shows that the patent or the research for preparing the ordered conductive nerve scaffold by using graphene composite chitosan is not reported yet.
Disclosure of Invention
The invention aims to provide a preparation method of a graphene-chitosan composite conductive nerve repair scaffold with a longitudinal ordered pore canal, which has the advantages of simple process, mild reaction conditions and easy operation, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
the preparation method of the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal pore channel is characterized by comprising the following steps of:
a) dissolving chitosan powder in 1-10% acetic acid solution (or phosphoric acid, citric acid and lactic acid), and stirring for 2-10 h to form 5-50 mg/mL chitosan solution;
b) adding graphene powder into deionized water, stirring vigorously for 1h, and then dispersing strongly for 4-10 h by using ultrasonic to form graphene dispersion liquid with the concentration of 2-10 mg/mL;
c) slowly adding the graphene dispersion liquid into a chitosan solution according to the mass ratio of graphene powder to chitosan of 1: 20-100, continuously stirring for 1h, and then strongly dispersing the mixed solution for 2-10 h by an ultrasonic oscillation method to obtain a mixed solution A with uniformly dispersed graphene;
d) slowly dripping 10mg/ml of genipin solution into the mixed solution A according to the mass ratio of 0.1-5% of the chitosan, and stirring for 1-4 h at 20-30 ℃ to obtain a mixed solution B containing a genipin cross-linking agent;
e) injecting the B into a cylindrical mold with the inner diameter of 2-10 mm and the height of 1-20 cm, and vertically immersing the mold into liquid nitrogen at a constant speed of 0.1-5 cm/min; until the liquid nitrogen completely submerges the mould, the mixed liquid B is completely frozen; and taking out the graphene-chitosan composite conductive nerve repair scaffold, removing the mold, and freeze-drying to obtain the final product, namely the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal ordered pore channel.
As a further scheme of the invention: in the step a), the cohesive relative molecular weight of the chitosan is 10,000-1,000,000.
As a further scheme of the invention: in the step b), the used graphene further comprises graphene oxide, carboxylated graphene oxide, aminated graphene oxide and reduced graphene oxide.
As a further scheme of the invention: in the step c), the mass ratio of the added graphene to the chitosan is 1: 20-100.
As a further scheme of the invention: in the step d), slowly dripping 10mg/ml genipin solution into the mixed solution A according to the mass ratio of 0.5-4% of the chitosan.
As a further scheme of the invention: in the step e), the mold is vertically immersed into liquid nitrogen at the speed of 0.1-5 cm/min until the liquid nitrogen completely immerses the mold, and the mixed liquid B is sequentially frozen repeatedly to form a pore channel.
Compared with the prior art, the invention has the beneficial effects that:
the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal pore passage is prepared. The invention has simple process, mild reaction condition and easy operation, and the prepared nerve scaffold not only has good biocompatibility, but also has longitudinally and orderly arranged pore canals, thereby providing good physical guide function for the directional growth of the nerve axon. The scaffold also has good mechanical toughness and electrical conductivity, and has potential application value in promoting repair of peripheral nerves.
Drawings
Fig. 1 is a longitudinal scanning electron microscope photograph of a graphene-chitosan composite conductive nerve repair scaffold with longitudinal channels.
Fig. 2 is a cross-sectional scanning electron microscope photograph of the graphene-chitosan composite conductive nerve repair scaffold with longitudinal pores.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
Example 1
Dissolving 2g of chitosan with the viscosity-average relative molecular weight of 100 ten thousand in 100mL of weak acid solution, wherein the weak acid solution is acetic acid (or lactic acid), the concentration of the acid is 1-10% (for example: 4%, 6% and 8%), and stirring strongly for 6 hours until the chitosan is fully dissolved; dissolving 20mg of graphene oxide in 10mL of deionized water, stirring for turbidity, and then oscillating for 6 hours by using ultrasonic waves to uniformly disperse the graphene oxide; slowly adding the fully dispersed graphene oxide dispersion liquid into the stirring chitosan solution, and continuously stirring for 1h until the mixture is turbid; and then, using ultrasonic oscillation for 4h to uniformly disperse the graphene oxide in the chitosan solution. 2mL of genipin with the concentration of 10mg/mL is dissolved and slowly dripped into the stirring mixed solution, and the mixed solution is kept at 30 ℃ and stirred for 2 hours to ensure that the chitosan is fully crosslinked. Injecting the crosslinked mixed solution into a cylindrical mold with the inner diameter of 2mm and the length of 10 cm; then, the mould is immersed into liquid nitrogen at the speed of 1cm/min until the mould is completely immersed below the liquid level of the liquid nitrogen; subsequently, the mould is taken out of the liquid nitrogen and removed; and finally, freeze-drying the material to obtain the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal pore channel.
Example 2
Dissolving 3g of chitosan with the viscosity-average relative molecular weight of 80 ten thousand in 100mL of weak acid-removed solution, wherein the weak acid solution is acetic acid (or citric acid), the concentration of the acid is 2-6%, and stirring for 6h until the chitosan is fully dissolved; dissolving 150mg of carboxylated graphene oxide in 30mL of deionized water, stirring for turbidity, and dispersing for 6 hours by using ultrasonic waves until the graphene oxide is uniformly dispersed; and slowly adding the fully dispersed graphene oxide dispersion liquid into the stirring chitosan solution, continuously stirring for 1h until the solution is turbid, and then uniformly dispersing the graphene oxide in the chitosan solution through ultrasonic dispersion for 6 h. 3mL of genipin with the concentration of 10mg/mL is dissolved and slowly dripped into the stirring mixed solution, and the mixed solution is kept at 30 ℃ and stirred for 2 hours to ensure that the chitosan is fully crosslinked. Injecting the crosslinked mixed solution into a cylindrical mold with the inner diameter of 2mm and the length of 10 cm; then, the mould is immersed into liquid nitrogen at the speed of 1cm/min until the mould is completely immersed below the liquid level of the liquid nitrogen; subsequently, the mould is taken out of the liquid nitrogen and removed; and finally, freeze-drying the material to obtain the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal pore channel.
Example 3
Dissolving 2g of chitosan with the viscosity-average relative molecular weight of 100 ten thousand in 100mL of weak acid solution, wherein the weak acid solution is acetic acid (or lactic acid), the concentration of the acid is 1-10% (for example: 4%, 6% and 8%), and stirring strongly for 6 hours until the chitosan is fully dissolved; dissolving 20mg of carboxylated graphene oxide into 10mL of deionized water, stirring for turbidity, and then oscillating for 6 hours by using ultrasonic waves to uniformly disperse the graphene oxide; slowly adding the fully dispersed graphene oxide dispersion liquid into the stirring chitosan solution, and continuously stirring for 1h until the mixture is turbid; and then, using ultrasonic oscillation for 4h to uniformly disperse the graphene oxide in the chitosan solution. 2mL of genipin with the concentration of 10mg/mL is dissolved and slowly dripped into the stirring mixed solution, and the mixed solution is kept at 30 ℃ and stirred for 2 hours to ensure that the chitosan is fully crosslinked. Injecting the crosslinked mixed solution into a cylindrical mold with the inner diameter of 2mm and the length of 10 cm; then, the mould is immersed into liquid nitrogen at the speed of 1cm/min until the mould is completely immersed below the liquid level of the liquid nitrogen; subsequently, the mould is taken out of the liquid nitrogen and removed; and finally, freeze-drying the material to obtain the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal pore channel.
Example 4
Dissolving 2g of chitosan with the viscosity-average relative molecular weight of 80 ten thousand in 100mL of weak acid solution, wherein the weak acid solution is acetic acid (or lactic acid), the concentration of the acid is 1-10% (for example: 4%, 6% and 8%), and stirring strongly for 6 hours until the chitosan is fully dissolved; dissolving 20mg of reduced graphene oxide in 10mL of deionized water, stirring for turbidity, and then oscillating for 6 hours by using ultrasonic waves to uniformly disperse the graphene oxide; slowly adding the fully dispersed graphene oxide dispersion liquid into the stirring chitosan solution, and continuously stirring for 1h until the mixture is turbid; and then, using ultrasonic oscillation for 4h to uniformly disperse the graphene oxide in the chitosan solution. 2mL of genipin with the concentration of 10mg/mL is dissolved and slowly dripped into the stirring mixed solution, and the mixed solution is kept at 30 ℃ and stirred for 2 hours to ensure that the chitosan is fully crosslinked. Injecting the crosslinked mixed solution into a cylindrical mold with the inner diameter of 2mm and the length of 10 cm; then, the mould is immersed into liquid nitrogen at the speed of 1cm/min until the mould is completely immersed below the liquid level of the liquid nitrogen; subsequently, the mould is taken out of the liquid nitrogen and removed; and finally, freeze-drying the material to obtain the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal pore channel.
The figure is a picture of a scanning electron microscope of an ordered scaffold prepared from 800000 molecular weight chitosan composite graphene oxide. FIG. 1 is a scanning electron micrograph of a cross section of a stent, from which it can be seen that channels are arranged in parallel along the longitudinal direction of the stent, the height is 20 to 50 μm, and a small number of fibers are connected between layers. FIG. 2 is a scanning electron micrograph of a cross section of the stent, with a pore size of 20-50 μm, suitable for neuron adhesion and growth along the longitudinal channels.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (6)
1. The preparation method of the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal pore channel is characterized by comprising the following steps of:
a) adding chitosan powder into 1-10% acetic acid solution (or phosphoric acid, citric acid and lactic acid), and stirring for 2-10 h to fully dissolve the chitosan powder to form 5-50 mg/mL chitosan solution;
b) adding graphene powder into deionized water, stirring vigorously for 1h, and then carrying out ultrasonic treatment for 4-10 h to fully disperse the graphene powder to form graphene dispersion turbid liquid with the concentration of 2-10 mg/mL;
c) slowly adding the graphene dispersion liquid into a chitosan solution according to the mass ratio of graphene powder to chitosan of 1: 20-100, continuously stirring for 1h, and continuously dispersing the mixed solution for 2-10 h by an ultrasonic method to obtain a mixed solution A with uniformly dispersed graphene;
d) slowly dripping 10mg/ml of genipin solution into the mixed solution A according to the mass ratio of 0.1-5% of the genipin solution to the chitosan, and stirring for 1-4 h at 20-30 ℃ to obtain a mixed solution B containing a genipin cross-linking agent;
e) injecting the B into a cylindrical mold with the inner diameter of 2-10 mm and the height of 1-20 cm, and vertically immersing the mold into liquid nitrogen at a constant speed of 0.1-5 cm/min; and after the liquid nitrogen completely immerses the mold, taking out the mold, and then carrying out freeze drying and genipin full cross-linking reaction to obtain the graphene-chitosan composite conductive nerve repair scaffold with the longitudinal ordered pore channel as a final product.
2. The preparation method of the graphene-chitosan composite conductive nerve repair scaffold with longitudinally ordered pore canals as claimed in claim 1, wherein in the step a), the cohesive relative molecular weight of the chitosan used is 10,000-1,000,000.
3. The method for preparing the graphene-chitosan composite conductive nerve repair scaffold with the longitudinally ordered pore channels according to claim 1, wherein in the step b), the used graphene further comprises graphene oxide, carboxylated graphene oxide, aminated graphene oxide and reduced graphene oxide.
4. The preparation method of the graphene-chitosan composite conductive nerve repair scaffold with the longitudinally ordered pore channels as claimed in claim 1, wherein in the step c), the mass ratio of the added graphene to the chitosan is 1: 20-100.
5. The preparation method of the graphene-chitosan composite conductive nerve repair scaffold with the longitudinally ordered pore channels according to claim 1, wherein in the step d), a 10mg/ml genipin solution is slowly added dropwise into the mixed solution A according to a mass ratio of 0.1-5% of chitosan.
6. The preparation method of the graphene-chitosan composite conductive nerve repair scaffold with the longitudinally ordered pore canals as claimed in claim 1, wherein in the step e), the mold is vertically immersed into liquid nitrogen at a speed of 0.1-5 cm/min until the liquid nitrogen completely submerges the mold.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115463251A (en) * | 2022-09-09 | 2022-12-13 | 四川大学 | Optogenetic nerve repair scaffold compounded with up-conversion nanoparticles and preparation method thereof |
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