CN110028685B

CN110028685B - Preparation method of fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold

Info

Publication number: CN110028685B
Application number: CN201910288808.7A
Authority: CN
Inventors: 张艳; 周健; 陈振斐
Original assignee: Shanghai University of Engineering Science
Current assignee: Shanghai University of Engineering Science
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2022-02-11
Anticipated expiration: 2039-04-11
Also published as: CN110028685A

Abstract

The invention relates to a preparation method of a fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold, which comprises the following steps: (1) adding the porous graphene oxide solution into the silk fibroin solution to obtain a mixed solution A; (2) adding chitosan into acetic acid water solution, heating and stirring to prepare chitosan water solution; (3) centrifuging the chitosan aqueous solution in the step (2), and adding the chitosan aqueous solution into the mixed solution A in the step (1) to obtain a mixed solution B; (4) and (4) carrying out freeze drying treatment on the mixed solution B obtained in the step (3) to obtain a target product. Compared with the prior art, the method has the advantages of simple process and strong industrial feasibility, and the obtained three-dimensional scaffold material has the advantages of excellent mechanical property, high bioactivity and strong degradability.

Description

Preparation method of fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold

Technical Field

The invention belongs to the technical field of composite material preparation, and relates to a preparation method of a fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold.

Background

Silkworm silk fibroin is a natural polymer material which is prepared by dissolving and purifying silk and consists of eighteen amino acids such as alanine, alanine and serine, has rich sources and has the advantages of good biocompatibility, air permeability, moisture permeability, degradability and the like. In addition to being used in surgical sutures, cosmetics, food additives and other industries, more and more researchers have recently studied the possibility of applying the silk fibroin material to biomedical materials such as cell culture media, drug controlled release carriers and artificial skin. Although natural silk has excellent mechanical strength and toughness, due to the change of molecular secondary structure, after dissolution and regeneration, a scaffold material formed by silk fibroin usually becomes very brittle in a dry state and is difficult to process. The industry has addressed this problem by adding different inducers, in addition to mixing with other natural or synthetic polymers. In addition, a freeze-drying method is one of simple and effective methods for preparing the three-dimensional porous scaffold material, but for pure silk fibroin, fibroin molecules can be self-assembled into a lamellar structure in the freeze-drying process, so that the macroscopic pore structure of the silk fibroin is influenced to a certain extent, whether an external doping agent is added can improve the interaction among the silk fibroin molecules and inhibit the self-assembly of the silk fibroin molecules into a sheet shape is also one of the key problems.

Chitosan (CS) is a natural biological polysaccharide produced by deacetylation of chitin and capable of being degraded and absorbed in vivo, and is the only polysaccharide polymer material with positive charges in the nature. Due to its good biocompatibility, biodegradability and multifunctional group, chitosan has attracted a great deal of attention in a wide range of fields such as water treatment, separation membranes, food packaging, tissue engineering and drug delivery.

The graphene has excellent mechanical properties, large specific surface area and good biocompatibility, so that the graphene has great application potential in the field of biomedicine. The porous graphene oxide (HGO) is a derivative of graphene, and a plurality of oxygen-containing functional groups, such as carbonyl, carboxyl, hydroxyl and the like, are arranged on a porous graphene oxide sheet layer, so that the porous graphene oxide sheet layer can perform multiple covalent reactions with a plurality of silk protein peptide chains, and non-covalent actions such as ionic bonds, hydrogen bonds and the like also exist.

Therefore, if the silk fibroin, the chitosan and the graphene can be compounded to prepare the composite material combining the advantages of the three materials, the composite material has wide application prospect.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a preparation method of a fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold, and the fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold with better mechanical property and bioactivity is obtained by means of solution mixing, freeze drying and other measures.

The purpose of the invention can be realized by the following technical scheme:

a preparation method of a fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold comprises the following steps:

(1) adding the porous graphene oxide solution into the silk fibroin solution to obtain a mixed solution A;

(2) adding chitosan into acetic acid water solution, heating and stirring to prepare chitosan water solution;

(3) centrifuging the chitosan aqueous solution in the step (2), and adding the chitosan aqueous solution into the mixed solution A in the step (1) to obtain a mixed solution B;

(4) and (4) carrying out freeze drying treatment on the mixed solution B obtained in the step (3) to obtain a target product.

Further, in the step (1), the addition amounts of the porous graphene oxide solution and the silk fibroin solution satisfy: the mass ratio of the porous graphene oxide to the silk fibroin is 1-2: 1.

Further, in the step (1), the silk fibroin solution is a silk fibroin aqueous solution with a mass fraction of 3.5%;

the concentration of the porous graphene oxide solution is 0.1-0.3 g/L.

Further, in the step (1), the preparation process of the silk fibroin solution is as follows: placing fibrous silk fibroin obtained after degumming of silkworm cocoon in LiBr solution, adding Na₂CO₃Dissolving fibrous silk fibroin in the solution, and performing dialysis treatment to obtain the product.

Further, the concentration of the LiBr solution was 9M, Na₂CO₃The concentration of the solution was 0.02M and the temperature of the solution preparation was 60 ℃.

Further, in the step (2), the mass fraction of the acetic acid aqueous solution is 2%, and the conditions of heating and stirring are as follows: stirring the mixture for 1 hour in a water bath at the temperature of 100 ℃ at the rotating speed of 600r/min to prepare a chitosan water solution with the mass fraction of 2 percent.

Further, in the step (3), the mass ratio of the chitosan aqueous solution to the mixed solution A is 0.1-1: 1.

Further, in the step (4), the temperature of freeze drying is-20 ℃ to-60 ℃, and the drying time is 10-15 h.

The silk fibroin as a natural polymer has good biocompatibility, but has high crystallinity and high brittleness, the crystallinity of the silk fibroin can be improved by adding chitosan, the porous graphene oxide can be well combined with the silk fibroin and the chitosan, and a better porous network structure is provided for the three-dimensional scaffold. In the preparation process of the silk fibroin solution, LiBr is needed for effective degumming, and compared with a ternary solvent for dissolution, the silk fibroin solution is convenient and fast to use single lithium bromide and high in degumming rate. If the silk fibroin is added too much, the sheet structure of the stent is increased; if HGO is not added, the SF/CS forms a sheet structure mainly with small holes, which seriously influences the overall structure of the three-dimensional bracket and influences subsequent adsorption and the like.

Compared with the prior art, the invention has the following advantages:

1) the silk fibroin, the chitosan and the porous graphene oxide are contacted with each other to generate cross-linking and supermolecule self-assembly, a cross-linking agent is not required to be added, the PH is not required to be adjusted, the silk fibroin can be changed from a random conformation to a regular structure due to the existence of the graphene oxide, the addition of chemical reagents such as methanol and the like can be reduced, the process is simple, and the industrial production can be realized;

2) the obtained three-dimensional scaffold has excellent mechanical property, high bioactivity and strong biodegradability;

3) has wide application prospect: can be used as tissue biological material, artificial organ, facial mask, filtering and separating material, etc.

Drawings

FIG. 1 is a flow chart of the manufacturing process of the present invention;

FIG. 2 is a scanning electron micrograph of a three-dimensional scaffold prepared in example 1

Fig. 3 is a scanning electron microscope image of the silk fibroin/chitosan composite scaffold prepared in example 4.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.

Example 1

A preparation method of a fibroin, chitosan and porous graphene oxide composite three-dimensional scaffold comprises the following specific steps:

1) using 0.02mol/L Na₂CO₃Degumming silkworm cocoons by using the solution to obtain silk fibroin, dissolving the silk fibroin in 9mol/L LiBr solution for 4h at 60 ℃, dialyzing for 72h by using a dialysis membrane to obtain silk fibroin aqueous solution with the mass volume fraction of 6-7 w/v%, adding proper water to prepare silk fibroin solution with the mass fraction of 3.5%, and storing the silk fibroin solution in a refrigerator at 4 ℃ for later use;

2) adding 0.1g/l of porous graphene oxide solution into the fibroin solution obtained in the step 1) to obtain a mixed solution A, wherein the content of graphene oxide: the mass ratio of the silk fibroin is 2: 1;

3) placing 2.5g of chitosan into 2% acetic acid water solution, magnetically stirring for 1h in water bath at 100 ℃ at 600r/min to prepare chitosan solution with the mass fraction of 2%, and centrifuging before use;

4) centrifuging the chitosan solution obtained in the step 3), adding the centrifuged chitosan solution into the mixed solution A obtained in the step 2) to obtain a mixed solution B, wherein the mass ratio of chitosan to the mixed solution A is 0.1:1, and ultrasonically oscillating the mixed solution B for 15-20 min to enable the porous graphene, the silk fibroin and the chitosan to be crosslinked and fully combined;

5) freezing the mixed solution B obtained in the step 4) at-20 ℃ overnight, and drying for 10h by using a freeze dryer to obtain the fibroin, chitosan and porous graphene oxide composite three-dimensional scaffold material.

Example 2

1) preparing a silk fibroin solution with the mass fraction of 3.5% according to the method of the embodiment 1, and storing the silk fibroin solution in a refrigerator at 4 ℃ for later use;

2) adding 0.2g/l of porous graphene oxide solution into the fibroin solution obtained in the step 1) to obtain a mixed solution A, wherein the content of graphene oxide: the mass ratio of the silk fibroin is 1.5: 1;

3) a chitosan solution with a mass fraction of 2% was prepared according to the method of example 1, and centrifuged before use;

4) centrifuging the chitosan solution obtained in the step 3), adding the centrifuged chitosan solution into the mixed solution A obtained in the step 2) to obtain a mixed solution B, wherein the mass ratio of chitosan to the mixed solution A is 0.6:1, and ultrasonically oscillating the mixed solution B for 15-20 min to enable the porous graphene, the silk fibroin and the chitosan to be crosslinked and fully combined;

5) freezing the mixed solution B obtained in the step 4) at-40 ℃ overnight, and drying for 12h by using a freeze dryer to obtain the fibroin, chitosan and porous graphene oxide composite three-dimensional scaffold material.

Example 3

2) adding 0.3g/l of porous graphene oxide solution into the fibroin solution obtained in the step 1) to obtain a mixed solution A, wherein the content of graphene oxide: the mass ratio of the silk fibroin is 1: 1;

4) centrifuging the chitosan solution obtained in the step 3), adding the centrifuged chitosan solution into the mixed solution A obtained in the step 2) to obtain a mixed solution B, wherein the mass ratio of chitosan to the mixed solution A is 1:1, and ultrasonically oscillating the mixed solution B for 15-20 min to enable the porous graphene, the silk fibroin and the chitosan to be crosslinked and fully combined;

5) freezing the mixed solution B obtained in the step 4) at-60 ℃ overnight, and drying for 15h by using a freeze dryer to obtain the fibroin, chitosan and porous graphene oxide composite three-dimensional scaffold material.

The fibroin, chitosan and porous graphene oxide composite three-dimensional scaffold material prepared in this example 1 was subjected to morphology characterization. FIG. 1 is a flow chart of a preparation process of a composite three-dimensional scaffold material, and FIG. 2 is a scanning electron microscope image of the three-dimensional scaffold material, as shown in the figure, the composite three-dimensional scaffold prepared by the invention has high porosity, the aperture is about 20-40 um, and the pores are communicated with the pores, and the scaffold material has good porous structure and bioactivity and can be used as a tissue biomaterial, an artificial organ, a facial mask and a filtering separation material.

Example 4

A preparation method of a fibroin and chitosan composite scaffold comprises the following specific steps:

2) a chitosan solution with a mass fraction of 2% was prepared according to the method of example 1, and centrifuged before use;

3) centrifuging the chitosan solution obtained in the step 2), adding the centrifuged chitosan solution into the silk fibroin solution obtained in the step 1) to obtain a mixed solution A, wherein the mass ratio of chitosan to silk fibroin is 1:1, and ultrasonically oscillating the mixed solution A for 15-20 min to enable the silk fibroin and the chitosan to be crosslinked and fully combined;

4) freezing the mixed solution A obtained in the step 3) at-60 ℃ overnight, and drying for 15h by using a freeze dryer to obtain the fibroin/chitosan composite scaffold material.

The silk fibroin/chitosan composite scaffold prepared in this example 4 was subjected to morphology characterization. Fig. 3 is a scanning electron microscope image of the composite scaffold, and as shown in the figure, the composite scaffold mainly has a flaky small-pore structure without adding porous graphene oxide, and has high brittleness and poor mechanical properties.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. a preparation method of silk fibroin/chitosan/porous graphene oxide composite three-dimensional support, is characterized in that, comprises the following steps:

(1) get porous graphene oxide solution and join in silk fibroin solution, obtain mixed solution A;

(2) take chitosan and add it to the acetic acid aqueous solution, heat and stir to prepare the chitosan aqueous solution;

(3) centrifuging the aqueous chitosan solution in step (2) and adding it to the mixed solution A in step (1) to obtain mixed solution B;

(4) freeze-drying the obtained mixed solution B of step (3) to obtain the target product;

In step (1), the addition amount of the porous graphene oxide solution and the silk fibroin solution satisfies: the mass ratio of the porous graphene oxide to the silk fibroin is 1-2:1;

In step (1), the silk fibroin solution is a silk fibroin aqueous solution with a mass fraction of 3.5%;

The concentration of the porous graphene oxide solution is 0.1-0.3g/L;

In step (2), the mass fraction of the acetic acid aqueous solution is 2%, and the heating and stirring conditions are as follows: stirring at a speed of 600 r/min in a 100° C. water bath for 1 h, the mass fraction of the prepared chitosan aqueous solution is 2%;

In step (3), the mass ratio of the chitosan aqueous solution to the mixed solution A is 0.1-1:1.

2. the preparation method of a kind of silk fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold according to claim 1, is characterized in that, in step (1), the preparation process of silk fibroin solution is: The fibrous silk fibroin obtained after degumming is placed in a LiBr solution, a Na ₂ CO ₃ solution is added to dissolve the fibrous silk fibroin, and then the dialysis treatment is performed to complete the process.

3. the preparation method of a kind of silk fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold according to claim 2, is characterized in that, the concentration of LiBr solution is 9M, and the concentration of Na ₂ CO ₃ solution is 0.02M , the temperature of solution preparation is 60 ℃.

4. the preparation method of a kind of silk fibroin/chitosan/porous graphene oxide composite three-dimensional scaffold according to claim 1, is characterized in that, in step (4), the temperature of freeze-drying is -20 ℃～-60 ℃ ℃, drying time is 10-15h.