CN110368532B

CN110368532B - Preparation method of conductive polymer-based graphene composite porous scaffold

Info

Publication number: CN110368532B
Application number: CN201910543478.1A
Authority: CN
Inventors: 张超; 王心如; 臧鑫; 黄爱成; 陈颖; 金汉邦; 李国龙
Original assignee: Huaiyin Institute of Technology
Current assignee: Suzhou 30 Billion Technology Co ltd
Priority date: 2019-06-21
Filing date: 2019-06-21
Publication date: 2021-08-17
Anticipated expiration: 2039-06-21
Also published as: CN110368532A

Abstract

The invention relates to the fields of tissue engineering and biomedical materials, and discloses a preparation method of a conductive polymer-based graphene composite porous scaffold, comprising step 1) degumming silk to obtain an aqueous solution of regenerated silk fibroin (RSF) and concentrating to a certain concentration ; Step 2) Electrospin the RSF aqueous solution with graphene oxide (GO)/ethanol aqueous solution as the receiving electrode to prepare the RSF/GO composite fiber scaffold; Step 3) Treat the RSF/GO scaffold with hydrothermal method to prepare the RSF /Reduced graphene oxide (RGO) composite fiber scaffold. Compared with the prior art, the present invention introduces GO into the RSF fiber scaffold during the fiber forming process, prepares the composite fiber with the shell (GO)-core (RSF) structure, and then reduces the GO in situ by the hydrothermal method, Make the bracket conductive. The method has few preparation steps, simple method, common preparation raw materials, low price, no chemical pollution, and forms a tissue engineering scaffold with both electrical conductivity and biocompatibility.

Description

Preparation method of conductive polymer-based graphene composite porous scaffold

Technical Field

The invention relates to the field of tissue engineering and biomedical materials, in particular to a preparation method of a conductive polymer-based graphene composite porous scaffold.

Background

In recent years, the number of cases of nerve damage caused by accidents has increased year by year. How to repair the nerve defects and injuries is a great problem in clinical treatment. The biological conduit or the bracket is transplanted into a human body, so that the aims of inducing the growth, proliferation and differentiation of nerve cells can be fulfilled.

The Regenerated Silk Fibroin (RSF) has good biocompatibility and degradability, and is widely applied to the fields of biological medicine and tissue engineering. The electrical signals have important functions on the transmission of intercellular information and the regulation of cell physiological activities (such as proliferation, differentiation and spreading). Cells are inoculated on the conductive biological material, and certain electric stimulation is applied, so that differentiation and growth of cells such as nerves, bones, cardiac muscles, myoblasts and the like are facilitated, and more researchers are dedicated to development and research of conductive tissue engineering scaffolds. However, the conventional regenerated silk fibroin RSF fiber has no conductivity and has no promotion effect on the differentiation of nerve cells, so that the conductive regenerated silk fibroin RSF fiber scaffold is prepared, has good conductivity and biocompatibility, and has very important significance in nerve tissues.

In recent years, graphene (or Reduced Graphene Oxide (RGO)), silver nanoparticles, and carbon nanotubes have been reported to improve electrical properties and antibacterial properties of RSF fibers. The graphene serving as a two-dimensional carbon nano material has a unique layered structure, good mechanical property and electrical property, and is an excellent nano filler. At present, the following two strategies are mainly adopted for modifying the RSF fiber scaffold by adopting graphene:

adding graphene into RSF solution in a solution blending mode, and preparing the scaffold by an electrostatic spinning method. According to the scaffold prepared by the method, the graphene materials are compounded in the fibers in a discrete state, and gaps exist among the fibers, so that graphite particles in the fibers cannot be effectively connected, and further, a complete conductive network structure cannot be formed in the material. Therefore, the prepared scaffold shows a certain conductivity, but the conductivity of the two blended fiber scaffolds is still poor compared with other conductive materials.

And secondly, dipping the RSF fiber support in a Graphene Oxide (GO) solution by adopting a solution dipping method, taking out and drying the RSF fiber support, and reducing GO into RGO by using ascorbic acid as a reducing agent to prepare the RSF/RGO composite fiber support. The composite scaffold not only maintains the surface appearance of the fiber, but also has good conductivity. However, the disadvantages are that the preparation process is complicated and time-consuming, and that chemical contamination is easily caused by the use of reducing agents.

In the two methods, from the angles of before and after fiber forming, graphene or Graphene Oxide (GO) is introduced into the RSF fiber or the bracket. However, in the fiber forming process, graphene or Graphene Oxide (GO) can be introduced into the RSF scaffold, so that the preparation process is reduced, no chemical pollution is generated, and the tissue engineering scaffold with biocompatibility and conductivity is constructed, which has led to the thinking of researchers.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a preparation method of a conductive polymer-based graphene composite porous scaffold. The method is simple, common in preparation raw materials and low in price, and does not produce chemical pollution, so that the tissue engineering scaffold with biocompatibility and conductivity is formed.

The technical scheme is as follows: the invention provides a preparation method of a conductive polymer-based graphene composite porous scaffold, which comprises the following steps:

step 1) dissolving degummed silk in a lithium bromide (LiBr) aqueous solution, removing impurities and LiBr through centrifugation, filtration and dialysis to obtain a regenerated silk fibroin aqueous solution, and further concentrating until the mass percentage of the regenerated silk fibroin reaches a certain percentage to form a solution S;

step 2) dispersing GO in an ethanol aqueous solution by ultrasonic, recording as a mixed solution A, carrying out electrostatic spinning on the solution S in the step 1) by taking the mixed solution A as a receiving electrode, taking out and drying to obtain a composite fiber scaffold taking GO as a skin layer and RSF as a core layer;

and 3) treating the RSF/GO composite fiber scaffold in the step 2) by adopting a hydrothermal method, reducing GO on the surface of the composite fiber scaffold into RGO, taking out the RGO and drying to finally obtain the composite porous fiber scaffold taking the RGO as a skin layer and the RSF as a core layer.

Preferably, the RSF mass percentage of the solution S in the step 1) is 27-35%.

Preferably, the molar concentration of the LiBr aqueous solution in the step 1) is 9.0 +/-0.5 mol/L.

Preferably, the size of GO in the step 2) is 1-5 μm, the concentration of GO in the mixed solution A is 0.5-5 mg/mL, and the volume fraction of the ethanol water solution is 70-80%.

Preferably, the ultrasonic dispersion treatment of the step 2) uses water bath ultrasound, and the ultrasound time is 5-10 min.

Preferably, the conditions of the electrospinning method of the step 2) are as follows: the environment temperature is 15-25 ℃, the humidity is 40-60 RH%, the voltage is 16-20 kV, the receiving distance from the needle head to the receiver is 10-15 cm, and the extrusion speed of the micro-injection pump is 0.6-1.2 mL/h.

Preferably, the conditions of the hydrothermal method of step 3) are: the hydrothermal temperature is 120-180 ℃, and the hydrothermal time is 3-8 h.

Preferably, the medium for processing the RSF/GO composite fiber scaffold by the hydrothermal method is an ethanol water solution with the volume fraction of 80-100%.

Has the advantages that:

1. the invention creatively provides a preparation method of a conductive polymer-based graphene composite porous scaffold, and obtains a composite fiber scaffold taking RGO as a skin layer and RSF as a core layer from the aspects of material forming and structure design. The adopted silk fibroin is purely natural, is easy to obtain, has low price, simple preparation method and process, economy and high efficiency, and the obtained composite porous scaffold material has good conductivity and biocompatibility and has wide application prospect in the aspects of regeneration and repair of nervous tissues.

2. According to the invention, from the fiber forming process, GO is introduced into the RSF fiber or the support, so that the preparation steps are reduced, and the GO is reduced into RGO by using a hydrothermal method, so that the RSF/RGO composite porous fiber support with RGO as a skin layer and RSF as a core layer is obtained, and the RSF/RGO composite porous fiber support does not need a specific reducing agent for reduction, so that the cost is saved and chemical pollution is not easily generated.

3. Firstly, after RSF fibers enter the mixed solution A after electrostatic spinning, GO can be loaded on the surfaces of the RSF fibers to form a shell (GO) -core (RSF) structure. Secondly, ethanol can induce RSF crystallization and conformation change, plays a role in reinforcing RSF fibers, and compared with the traditional electrostatic spinning method, the method reduces the post-treatment process, so that the method is time-saving, simple and continuous.

Drawings

Fig. 1 is a comparative graph of laser confocal measurement after 4 days of schwann cells are seeded on a pure RSF fiber scaffold and a RSF/RGO composite porous fiber scaffold with a nucleocapsid structure, wherein (a) is a laser confocal measurement after 4 days of schwann cells are seeded on a pure RSF fiber scaffold, and (b) is a laser confocal measurement after 4 days of schwann cells are seeded on an RSF/RGO composite porous fiber scaffold with a nucleocapsid structure.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1:

boiling 0.5-1 wt% of sodium carbonate (Na)₂CO₃) Degumming the silkworm cocoons by using an aqueous solution, dissolving the degummed silkworm cocoons in a LiBr aqueous solution with the molar concentration of 9.0 +/-0.5 mol/L, and removing impurities and LiBr by centrifuging, filtering and dialyzing to obtain an RSF aqueous solution, wherein the RSF aqueous solution after centrifugal filtration is placed in a cellulose dialysis bag and is immersed in deionized water for dialysis after being sealed, the dialysis temperature is 6-12 ℃, the dialysis time is 72 hours, and the water changing period is 3-4 hours; and further concentrating the obtained RSF aqueous solution to obtain a solution S with the RSF mass fraction of 33% for later use.

Then, GO is ultrasonically dispersed in an ethanol water solution, which is marked as a mixed solution A, and water bath ultrasonic is used for dispersion in the embodiment, wherein the ultrasonic time is 5-10 min; transferring the solution S into an injector, treating the solution S by using the mixed solution A as a receiving electrode (in the embodiment, the size of GO is 1-5 microns, the GO concentration of the mixed solution A is 0.5 mg/mL, and the volume fraction of ethanol is 80%), carrying out electrostatic spinning, taking out and drying to obtain the RSF/GO composite fiber scaffold with GO as a skin layer and RSF as a core layer. The experimental parameters of the electrostatic spinning method are as follows: the relative humidity is 45 +/-5%, the temperature is 20 +/-5 ℃, the voltage is 20 kV, the receiving distance from the needle to the receiver (aluminum foil) is 10 cm, and the extrusion speed of the micro-injection pump is 1.2 mL/h.

Finally, carrying out hydrothermal treatment on the RSF/GO composite fiber scaffold, wherein the conditions of the hydrothermal method are as follows: the hydrothermal temperature is 120-180 ℃, the hydrothermal time is 3-8 h, in the embodiment, the RSF/GO composite fiber scaffold is subjected to hydrothermal treatment for 3h at 120 ℃, GO is reduced to RGO, and the RSF/RGO composite fiber scaffold with RGO as a skin layer and RSF as a core layer is prepared. At this time, the square resistance of the composite fiber scaffold was (1.0. + -. 0.6). times.10⁷ Ω/sq。

Example 2:

example 2 differs from example 1 in that example 2 controlled hydrothermal treatment for 6 hours at the last step, whereas example 1 was hydrothermal treatment for 3 hours, the other operations are the same as example 1, as follows:

Finally, carrying out hydrothermal treatment on the RSF/GO composite fiber scaffold, wherein the conditions of the hydrothermal method are as follows: the hydrothermal temperature is 120-180 ℃, the hydrothermal time is 3-8 h, in the embodiment, the RSF/GO composite fiber scaffold is subjected to hydrothermal treatment for 6h at 120 ℃, GO is reduced to RGO, and the RSF/RGO composite fiber scaffold with RGO as a skin layer and RSF as a core layer is prepared. At this time, the square resistance of the composite fiber scaffold was (1.8. + -. 0.6). times.10⁷ Ω/sq。

Example 3:

example 3 differs from example 2 in controlling the GO solution concentration of mixed solution a to be 2.0mg/mL, whereas example 2 controls the GO solution concentration of mixed solution a to be 0.5 mg/mL, the other operations being the same as in example 2, as follows:

Then, GO is ultrasonically dispersed in an ethanol water solution, which is marked as a mixed solution A, and water bath ultrasonic is used for dispersion in the embodiment, wherein the ultrasonic time is 5-10 min; transferring the solution S into an injector, treating the solution S by using the mixed solution A as a receiving electrode (in the embodiment, the size of GO is 1-5 microns, the concentration of the GO solution of the mixed solution A is 2.0mg/mL, and the volume fraction of ethanol is 80%), carrying out electrostatic spinning, taking out, and drying to obtain the RSF/GO composite fiber scaffold with GO as a skin layer and RSF as a core layer. The experimental parameters of the electrostatic spinning method are as follows: the relative humidity is 45 +/-5%, the temperature is 20 +/-5 ℃, the voltage is 20 kV, the receiving distance from the needle to the receiver (aluminum foil) is 10 cm, and the extrusion speed of the micro-injection pump is 1.2 mL/h.

Finally, carrying out hydrothermal treatment on the RSF/GO composite fiber scaffold, wherein the conditions of the hydrothermal method are as follows: the hydrothermal temperature is 120-180 ℃, the hydrothermal time is 3-8 h, in the embodiment, the RSF/GO composite fiber scaffold is subjected to hydrothermal treatment for 6h at 120 ℃, GO is reduced to RGO, and the RSF/RGO composite fiber scaffold with RGO as a skin layer and RSF as a core layer is prepared. At this time, the square resistance of the composite fiber scaffold was (7.2. + -. 0.5). times.10⁵Ω/sq。

Example 4:

example 4 differs from example 3 in controlling the GO solution concentration of mixed solution a to be 5.0mg/mL, whereas example 3 controls the GO solution concentration of mixed solution a to be 2.0mg/mL, the other operations being the same as in example 3, as follows:

Then, ultrasonically dispersing GO in an ethanol water solution, and marking as a mixed solution A, wherein water bath ultrasound is used for dispersion in the embodiment, and the ultrasonic time is 5-10 min; transferring the solution S into an injector, taking the mixed solution A as a receiving electrode (in the embodiment, the size of GO is 1-5 μm, the concentration of the GO solution of the mixed solution A is 5.0mg/mL, and the volume fraction of ethanol is 80%), processing the solution S by an electrostatic spinning method, taking out and drying to obtain the RSF/GO composite fiber scaffold taking GO as a skin layer and RSF as a core layer. The experimental parameters of the electrostatic spinning method are as follows: the relative humidity is 45 +/-5%, the temperature is 20 +/-5 ℃, the voltage is 20 kV, the receiving distance from the needle to the receiver (aluminum foil) is 10 cm, and the extrusion speed of the micro-injection pump is 1.2 mL/h.

Finally, performing hydrothermal treatment on the RSF/GO composite fiber scaffold by a hydrothermal methodThe conditions are as follows: the hydrothermal temperature is 120-180 ℃, the hydrothermal time is 3-8 h, in the embodiment, the RSF/GO composite fiber scaffold is subjected to hydrothermal treatment for 6h at 120 ℃, GO is reduced to RGO, and the RSF/RGO composite fiber scaffold with RGO as a skin layer and RSF as a core layer is prepared. At this time, the square resistance of the composite fiber scaffold was (2.3. + -. 0.5). times.10²Ω/sq。

See table 1 below: according to the block resistance test results of the RSF/RGO composite fiber scaffolds obtained in the embodiments 1 to 4, the block resistance test of the RSF/RGO composite fiber scaffolds generated in the embodiments 1 to 4 respectively has the GO concentration of 0.5-5.0mg/mL and the hydrothermal time of 3-8 h, and the block resistance test of the RSF/RGO composite fiber scaffolds generated in the embodiments 1 and 4 respectively has the GO concentration of 0.5 mg/mL and the hydrothermal time of 3h and 6 h; examples 2 to 4 are the sheet resistance values when the concentrations of GO were 0.5 mg/mL, 2.0mg, 5.0mg/mL, respectively, for a hydrothermal time of 6 hours and the hydrothermal times were all 6 hours

TABLE 1 Square resistance test results of the RSF/RGO composite fiber scaffolds obtained in examples 1-4 according to the present invention

Referring to the attached drawing 1, fig. 1 is a graph showing the laser confocal contrast of schwann cells seeded on a pure RSF fiber scaffold and a RSF/RGO composite porous fiber scaffold with a nucleocapsid structure for 4 days, wherein (a) is a graph showing the laser confocal contrast of schwann cells seeded on a pure RSF fiber scaffold for 4 days, and (b) is a graph showing the laser confocal contrast of schwann cells seeded on a RSF/RGO composite porous fiber scaffold with a nucleocapsid structure for 4 days. As can be seen, the cells in graph (a) are mostly in an aggregated state and have a round shape, while the cells in graph (b) are mostly in a fusiform shape on the RSF/RGO scaffold, and have a better spreading state, which indicates that the adhesion and growth of the cells on the composite scaffold are facilitated.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose of the embodiments is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A preparation method of a conductive polymer-based graphene composite porous scaffold is characterized by comprising the following steps:

step 1) dissolving degummed silk in a lithium bromide aqueous solution, removing impurities and lithium bromide through centrifugation, filtration and dialysis to obtain a regenerated silk fibroin aqueous solution, and further concentrating until the mass percentage of the regenerated silk fibroin reaches a certain percentage to form a solution S;

step 2) ultrasonically dispersing graphene oxide in an ethanol water solution, marking as a mixed solution A, wherein the concentration of the graphene oxide in the mixed solution A is 0.5-5 mg/mL, carrying out electrostatic spinning on the solution S in the step 1) by taking the mixed solution A as a receiving electrode, taking out and drying to obtain a regenerated silk fibroin/graphene oxide composite fiber scaffold taking the graphene oxide as a skin layer and the regenerated silk fibroin as a core layer;

and 3) treating the regenerated silk fibroin/graphene oxide composite fiber scaffold obtained in the step 2) by adopting a hydrothermal method, reducing graphene oxide on the surface of the regenerated silk fibroin/graphene oxide composite fiber scaffold into reduced graphene oxide, taking out the reduced graphene oxide and the reduced graphene oxide to be dried to finally obtain the regenerated silk fibroin/reduced graphene oxide composite porous fiber scaffold taking the reduced graphene oxide as a skin layer and the regenerated silk fibroin as a core layer.

2. The preparation method of the conductive polymer-based graphene composite porous scaffold as claimed in claim 1, wherein the regenerated silk fibroin of the solution S in the step 1) is 27-35% by mass.

3. The method for preparing the conductive polymer-based graphene composite porous scaffold according to claim 1, wherein the molar concentration of the lithium bromide aqueous solution in the step 1) is 9.0 ± 0.5 mol/L.

4. The preparation method of the conductive polymer-based graphene composite porous scaffold as claimed in claim 1, wherein the graphene oxide in the step 2) has a size of 1-5 μm, and the volume fraction of the ethanol aqueous solution is 70-80%.

5. The preparation method of the conductive polymer-based graphene composite porous scaffold as claimed in claim 1, wherein the ultrasonic dispersion treatment in step 2) is performed by using water bath ultrasound, and the ultrasound time is 5-10 min.

6. The method for preparing the conductive polymer-based graphene composite porous scaffold according to claim 1, wherein the electrostatic spinning method in the step 2) is performed under the following conditions: the environment temperature is 15-25 ℃, the humidity is 40-60 RH%, the voltage is 16-20 kV, the receiving distance from the needle head to the receiver is 10-15 cm, and the extrusion speed of the micro-injection pump is 0.6-1.2 mL/h.

7. The method for preparing the conductive polymer-based graphene composite porous scaffold according to claim 1, wherein the hydrothermal method in the step 3) has the following conditions: the hydrothermal temperature is 120-180 ℃, and the hydrothermal time is 3-8 h.

8. The preparation method of the conductive polymer-based graphene composite porous scaffold as claimed in claim 7, wherein a medium for treating the regenerated silk fibroin/graphene oxide composite fiber scaffold by a hydrothermal method is an ethanol aqueous solution with a volume fraction of 80-100%.