CN111001036B - Single-walled carbon nanotube composite material conductive nerve sleeve and preparation method and application thereof - Google Patents
Single-walled carbon nanotube composite material conductive nerve sleeve and preparation method and application thereof Download PDFInfo
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
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- C08J2479/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen, or carbon only, not provided for in groups C08J2461/00 - C08J2477/00
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- C08K3/041—Carbon nanotubes
Abstract
The invention discloses a single-walled carbon nanotube composite material conductive nerve sleeve and a preparation method and application thereof. The single-walled carbon nanotube composite material conductive nerve sleeve consists of a tube body and a coating covering the surface of the tube body; the tube body is a single-walled carbon nanotube containing carboxyl; the coating and the pipe body are combined together in a physical adsorption and chemical bond combination mode; the coating is composite hydrogel. The conductive nerve sleeve prepared by the invention has the excellent characteristics of good biocompatibility, conductivity and the like, and realizes the repair of large-section nerve defects and promotes the repair of peripheral nerves.
Description
Technical Field
The invention belongs to the field of biological materials, and relates to a single-walled carbon nanotube composite material conductive nerve sleeve and a preparation method and application thereof.
Background
The treatment of the quadriplegia and the dysfunction caused by the peripheral nerve injury is a worldwide problem of medical science, and with the rapid development of economy in China, the number of peripheral nerve injury patients caused by accidental injuries such as traffic and production accidents is increased year by year. In addition, diseases such as birth injury and tumor excision also increase the number of patients with peripheral nerve injury. After the peripheral nerve is damaged, the continuity of axons is blocked, nerve fibers from the far end to the damaged part are broken, and the axons are finally killed, so that the partial or complete loss of motor, sensory and autonomic nerve functions from the damaged nerve to the nerve loss innervated body is finally caused. Following peripheral nerve injury, the defect must be filled with a foreign graft, relying on restoration of nerve continuity to induce regeneration of broken axons, especially nerve defects of more than 4 cm. There are three main types of current grafts that fill a nerve defect: autologous nerve transplantation, allogeneic nerve transplantation and biological materials. The autograft adopts a method of 'dismantling east wall and supplementing west wall', although the autograft is in a gold standard, the nerve supply part loses nerve supply and then loses nerve dysfunction; allograft transplantation has many problems of immunological rejection, safety, ethics and the like; thus, biological materials are one of the current alternatives to solve the above problems.
Gelatin methacrylamide (GelMA) hydrogel is mainly produced by chemically crosslinking gelatin and methacrylamide, and because gelatin is derived from collagen hydrolysate which is an important component of natural extracellular matrix (ECM), and contains a large amount of RGD sequence, namely, arginine-glycine-aspartic acid (RGD) sequences capable of promoting cell adhesion, and FDA has approved gelatin-related products for clinical use, the GelMA hydrogel has excellent biocompatibility, low immunogenicity and good chemical modification potential.
Dopamine has a good adhesion effect on mussel foot protein extract, catechol residues in molecules of the dopamine are not only donors of hydrogen bonds but also receptors of the hydrogen bonds, and the hydrogen bonding effect is considered to be the reason of high adhesion of the dopamine. Current research also indicates that dopamine can promote cell adhesion, expansion and proliferation.
Carbon Nanotubes (CNTs) have excellent electrical conductivity and nanotopography, and are widely used in various fields. CNTs-based hydrogels can provide appropriate morphological, electrical and mechanical properties to tissues or cells, and can mimic the proliferation, elongation and differentiation facilitated by the extracellular matrix. When the CNTs hydrogel scaffold is used for filling peripheral nerve defects, the physical continuity and the electrical conduction of nerves are restored, and an excellent microenvironment is provided for the regeneration of axons.
Disclosure of Invention
The invention aims to provide a single-walled carbon nanotube composite material conductive nerve sleeve and a preparation method and application thereof. The single-walled carbon nanotube composite material conductive nerve sleeve can bridge the nerve broken ends at two ends, promotes the regeneration of peripheral nerves and has conductivity.
The invention provides a single-walled carbon nanotube composite material conductive nerve sleeve which comprises a tube body and a coating covering the surface of the tube body;
the tube body is a single-walled carbon nanotube containing carboxyl;
the coating and the pipe body are combined together in a physical adsorption and chemical bond combination mode;
the coating is composite hydrogel.
In the single-walled carbon nanotube composite material conductive nerve casing, the composite hydrogel is formed by compounding methacrylamide modified gelatin and dopamine.
The length of the single-walled carbon nanotube composite material conductive nerve sleeve is 15mm, the inner diameter is 1.2mm, and the outer diameter is 1.4 mm.
The invention provides a method for preparing a single-walled carbon nanotube composite material conductive nerve sleeve, which comprises the following steps:
1) uniformly mixing methacrylamide modified gelatin, the carboxyl-containing single-walled carbon nanotube and a photoinitiator, placing the mixture in a mold for shaping, and then carrying out a photo-crosslinking reaction to obtain a hydrogel support material;
2) mixing the hydrogel scaffold material obtained in the step 1) with dopamine to perform an oxidation grafting reaction to obtain the single-walled carbon nanotube composite material conductive nerve sleeve.
In step 1) of the above method, the photoinitiator is Irgacure 2959;
the mass ratio of the methacrylamide modified gelatin to the carboxyl-containing single-walled carbon nanotube to the photoinitiator is 1 g: 5 mg: 5 mg; (ii) a
The methacrylamide modified gelatin takes part in the reaction in the form of aqueous solution; in the aqueous solution of the methacrylamide modified gelatin, the dosage ratio of the methacrylamide modified gelatin to water is 1 g: 10 ml;
in the shaping step, the temperature is minus 80 ℃ and is specifically 24 hours;
in the step of photo-crosslinking reaction, the temperature is room temperature; the time is 30 min; in the photo-crosslinking, the light used is ultraviolet light.
In this step, the method for preparing the methacrylamide modified gelatin (GelMA) comprises: 1g of gelatin was dissolved in 10ml of double distilled water (10% (w/v)), and the mixture was stirred in a water bath at 50 ℃ until completely dissolved. Subsequently, 0.6ml of methacrylic anhydride was added with constant stirring. After 3 hours, the reaction was quenched by addition of sufficient double distilled water to dilute. Putting the mixture solution into a dialysis bag with 12-14 kDa, putting into double distilled water with the temperature of 40 ℃, changing the double distilled water every 6 hours for dialysis for one week, finally freeze-drying, and storing at the temperature of 4 ℃.
In the step 2), the mass ratio of the hydrogel scaffold material to the dopamine is 1: 0.005;
in the step of the oxidation grafting reaction, the temperature is 50 ℃; the time is 3 hours;
the oxidation grafting reaction is carried out in a buffer solution; the buffer is specifically Tris-hydroxymethyl aminomethane-hydrochloric acid (Tris-HCI, pH 8.5);
the concentration of the dopamine in the buffer solution is 0.5 mg/ml.
The method further comprises the following steps: after the step 2) of oxidizing grafting reaction, soaking and cleaning the reaction product by using double distilled water, and freeze-drying.
In addition, the application of the single-walled carbon nanotube composite material conductive nerve casing pipe provided by the invention in the preparation of nerve repair and/or filling and a nerve repair and/or filling product containing the single-walled carbon nanotube composite material conductive nerve casing pipe also belong to the protection scope of the invention. Wherein the nerve is in particular a peripheral nerve.
Compared with the prior art, the invention has the beneficial effects that:
1. the composite hydrogel nerve cannula scaffold material is prepared by adopting GelMA and dopamine by using a photo-crosslinking method, so that the biocompatibility of the scaffold material is enhanced, and the scaffold material is changed into a nontoxic substance after reaction of a photo-crosslinking agent, so that the non-toxicity of hydrogel is ensured;
2. by adding the carbon nano tube, the nerve sleeve bracket material can have electrical properties, the electrical properties of nerve defects are repaired, and the mechanical properties of the nerve defects are enhanced;
3. the nerve sleeve material can restore the nerve defect structurally, is favorable for the cell to climb and form a microenvironment favorable for nerve repair.
Drawings
FIG. 1 is a schematic diagram of polydopamine production.
Fig. 2 is a schematic diagram of a single-walled carbon nanotube composite nerve cannula.
Fig. 3 is a mold.
Fig. 4 is the single-walled carbon nanotube composite nerve sleeve prepared in example 1.
Fig. 5 is an intraoperative view of a nerve defect repaired with the nerve cuff obtained in example 1.
Fig. 6 is a cannula with nerve tissue overgrowing.
Fig. 7 is a partially enlarged view of fig. 6.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Examples 1,
1) Preparation of methacrylamide modified gelatin (GelMA)
1g of gelatin was dissolved in 10ml of double distilled water (10% (w/v)), and the mixture was stirred in a water bath at 50 ℃ until completely dissolved. Subsequently, 0.6ml of methacrylic anhydride was added with constant stirring. After 3 hours, the reaction was quenched by addition of sufficient double distilled water to dilute. Putting the mixture solution into a dialysis bag with 12-14 kDa, putting into double distilled water with the temperature of 40 ℃, changing the double distilled water every 6 hours for dialysis for one week, and finally freeze-drying for later use.
2) GelMA and carboxyl-containing single-walled carbon nanotubes are placed in a mould for crosslinking reaction to prepare the hollow hydrogel support material
1g of the freeze-dried product of step 1) was dissolved in 10ml of double distilled water, and the solution was stirred in a water bath at 50 ℃ until it was completely dissolved. 5mg of carboxyl group-containing single-walled carbon nanotubes and 0.5mg of Irgacure 2959 were added, and stirred for 5 hours under a light-shielding condition, followed by pouring into a mold under a vacuum condition (as shown in FIG. 3) and then transferred to-80 ℃. And after 24 hours, removing the grinding tool, and irradiating for 30min under an ultraviolet lamp at room temperature.
3) Step 2) grafting polydopamine on the scaffold material
An appropriate amount of dopamine was dissolved in 10mM Tris-hcl (Tris-HCI, pH 8.5) buffer to a final concentration of 0.5 mg/ml. And 2) putting 1g of the hydrogel stent material prepared in the step 2) into the solution, oxidizing 5mg of dopamine into polydopamine in the presence of oxygen, and grafting the polydopamine onto the material prepared in the step 2) to form a coating.
4) Freeze-drying the product of step 3):
soaking and cleaning the single-walled carbon nanotube composite material obtained in the step 3) with double distilled water, and freeze-drying to obtain the single-walled carbon nanotube composite material conductive nerve casing.
The single-walled carbon nanotube composite material conductive nerve casing prepared by the method has the length of 15mm, the inner diameter of 1.2mm and the outer diameter of 1.4mm as shown in figure 4.
The nerve defect is repaired by using the nerve sleeve of a 10mm model of the sciatic nerve defect of the SD rat, the intraoperative picture is shown in figure 5, the animal operation process is shown in the SD rat, 200 and 250g, and the male and female are unlimited. SD rats were isoflurane-induced anesthetized and maintained intraoperatively. The right hip was cut along the surface of the sciatic nerve to expose the sciatic nerve. Separating sciatic nerve peripheral tissue bluntly, cutting off 10mm nerve tissue to obtain nerve defect, suturing two ends of nerve defect with 10-0 suture sleeve, keeping the nerve defect position at 10mm, flushing wound, and suturing muscle and skin. Antibiotics were injected for three consecutive days.
After the nerve canula graft is transplanted into an SD rat body for 12 weeks, taking materials and carrying out HE staining, wherein the HE staining step comprises the following steps: under the anesthesia state of SD rat, paraformaldehyde is perfused through heart, right side nerve tissue is taken out together with nerve sleeve, and after fixation and dehydration of 30% sucrose, OCT embedding medium is used for embedding. The sections were subsequently frozen to a thickness of 8 um. Finally, the resin is sealed and photographed under an optical microscope through the conventional procedure of HE dyeing (machine integrated dyeing).
The overgrowth of nerve tissue within the cannula can be observed from fig. 6 and 7.
Claims (10)
1. A single-walled carbon nanotube composite material conductive nerve sleeve comprises a tube body and a coating covering the surface of the tube body;
the tube body is a single-walled carbon nanotube containing carboxyl;
the coating and the pipe body are combined together in a physical adsorption and chemical bond combination mode;
the coating is composite hydrogel;
the composite hydrogel is compounded by methacrylamide modified gelatin and dopamine.
2. The single wall carbon nanotube composite conductive nerve cannula of claim 1, wherein: the length of the single-walled carbon nanotube composite material conductive nerve sleeve is 15mm, the inner diameter is 1.2mm, and the outer diameter is 1.4 mm.
3. A method of making the single-walled carbon nanotube composite conductive nerve sleeve of claim 1 or 2, comprising:
1) uniformly mixing methacrylamide modified gelatin, the carboxyl-containing single-walled carbon nanotube and a photoinitiator, placing the mixture in a mold for shaping, and then carrying out a photo-crosslinking reaction to obtain a hydrogel support material;
2) mixing the hydrogel scaffold material obtained in the step 1) with dopamine to perform an oxidation grafting reaction to obtain the single-walled carbon nanotube composite material conductive nerve sleeve.
4. The method of claim 3, wherein: in the step 1), the photoinitiator is Irgacure 2959;
the mass ratio of the methacrylamide modified gelatin to the carboxyl-containing single-walled carbon nanotube to the photoinitiator is 0.1 g: 5 mg: 5 mg;
the methacrylamide modified gelatin takes part in the reaction in the form of aqueous solution; in the aqueous solution of the methacrylamide modified gelatin, the dosage ratio of the methacrylamide modified gelatin to water is 1 g: 10 mL;
in the shaping step, the temperature is-80 ℃ and the time is 24 hours;
in the step of photo-crosslinking reaction, the temperature is room temperature; the time is 30 min; in the photo-crosslinking, the light used is ultraviolet light.
5. The method according to claim 3 or 4, characterized in that: in the step 2), the dosage ratio of the hydrogel scaffold material to the dopamine is 1: 0.5;
in the step of the oxidation grafting reaction, the temperature is 50 ℃; the time is 3 hours;
the oxidation grafting reaction is carried out in a buffer solution; the buffer is Tris-hydroxymethyl aminomethane-hydrochloric acid (Tris-HCI, pH 8.5);
the concentration of the dopamine in the buffer is 0.5 mg/mL.
6. The method according to claim 3 or 4, characterized in that: the method further comprises the following steps: after the step 2) of oxidizing grafting reaction, soaking and cleaning the reaction product by using double distilled water, and freeze-drying.
7. Use of the single-walled carbon nanotube composite electrically conductive nerve sleeve of claim 1 or 2 for the preparation of a nerve repair and/or filling material.
8. Use according to claim 7, characterized in that: the nerve is a peripheral nerve.
9. A nerve repair and/or filling product comprising the single wall carbon nanotube composite conductive nerve sleeve of claim 1 or 2.
10. The product of claim 9, wherein: the nerve is a peripheral nerve.
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