CN111320767B - A preparation method of thixotropic hydrogel for 3D bioprinting - Google Patents

Abstract

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the steps of: A. dissolving protein powder, polyphenol powder and polysaccharide powder in deionized water to obtain protein solutions with the concentration of 3.0-10.0wt.% respectively; a polyphenol solution having a concentration of 0.10-0.75 wt.%; a polysaccharide solution having a concentration of 2.0-8.0 wt.%; B. adding the protein solution and the polyphenol solution prepared in the step A into a reactor according to the proportion of 1-10 to 1, and magnetically stirring for 1-3 hours under the water bath condition of 40-50 ℃ to obtain a protein-polyphenol binary compound solution; C. and (3) adding the polysaccharide solution into the compound solution obtained in the step (B), adding the polysaccharide solution into the compound solution in a volume ratio of 2-4. The hydrogel prepared by the method is used as a 3D biological printing material, and has excellent biocompatibility and injectability.

Description

A preparation method of thixotropic hydrogel for 3D bioprinting

technical field

The invention relates to a preparation method of thixotropic hydrogel for 3D bioprinting.

Background technique

3D bioprinting is a model based on medical image conversion design. Under the precise control of the computer, the bioprinting "ink" (biocompatible materials and cells) is printed layer by layer, and a three-dimensional structure and microenvironment similar to that in the body is constructed in vitro. The three-dimensional structure and microenvironment obtained by 3D bioprinting provide a bionic three-dimensional cell culture condition, in which the cells are properly cultured and finally cultivated into functional species for in vivo replacement and regeneration as well as in vitro drug screening and disease research. Tissue/Organ. At present, there are three common 3D bioprinting methods: inkjet printing, laser-assisted and extrusion (Li J, Chen M, Fan X, et al.Recent advances in bioprinting techniques: approaches, applications and future prospects. Journal of Translational Medicine, 2016, 14(1): 271.). Among them, extrusion-type 3D bioprinting uses mechanical force or air pressure to extrude "bio-ink" (usually a hydrogel material carrying cells) from a nozzle to construct a three-dimensional structure (Zhang Z, Wang B, Hui D, et al. 3D bioprinting of soft materials-based regenerative vascular structures and tissues. Composites Part B: Engineering, 2017.), which has the characteristics of easy operation, economy, mild conditions and printing higher density cells; at the same time, it can avoid Use harsh working conditions such as high temperature and high pressure.

Although the operation and principle of extrusion-type 3D bioprinting are simple, extrusion-type 3D bioprinting technology has high requirements on the performance of "bio-ink" materials, which require materials to have good injectability and moldability, so that they can be processed at a low temperature. Under the action of strong mechanical force, the "bio-ink" can be ejected smoothly, and can form a shape quickly; at the same time, it must have excellent biocompatibility, which can protect and regulate the cells wrapped in hydrogel. Cells provide a three-dimensional environment close to the extracellular matrix to ensure cell migration, growth and differentiation (N.E.Fedorovich, J.R.De Wijn, A.J.Verbout, J.Alblas, W.J.Dhert, Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing, Tissue Eng Part A14(1)(2008) 127-33).

If the "bio-ink" material wants to be extruded smoothly and protect the cells wrapped in it from the shear force as small as possible, it needs to have a small viscosity; and the viscosity becomes smaller and the molding performance will be seriously reduced, making the printed three-dimensional structure collapse. Thixotropic hydrogel materials can take advantage of their characteristics of "thinning under force and recovery after force removal" (You Chen, a Yihan Wang, Qian Yang, et al. A novel thixotropic magnesium phosphate-based bioink with excellent printability for application in 3Dprinting, journal of materials chemistry B, 2018, 6, 4502-4513), but the existing thixotropic materials are often chemically synthesized materials, and the biocompatibility is not excellent; and the literature The reported thixotropic materials used in the biomedical field have a long recovery time after thixotropy (You Chen, a Yihan Wang, Qian Yang, et al. Anovelthixotropic magnesium phosphate-based bioink with excellent printability for application in 3D printing, journal of materials chemistry B, 2018, 6, 4502-4513), leading to poor formability after printing, it is difficult to continue printing on the printing layer faster, which limits its application in 3D bioprinting. Therefore, the current development of 3D bioprinting urgently needs a "bioink" hydrogel material with good biocompatibility and high thixotropy (fast recovery time).

Contents of the invention

The object of the present invention is to provide a kind of preparation method of the thixotropic hydrogel that is used for 3D bioprinting, and the technology of this method is simple, preparation cost is low; The thixotropic hydrogel prepared is used as 3D bioprinting material, and its The injection performance and molding performance are good, and it has excellent biocompatibility. The cell activity after printing is high, which can provide cells with a growth environment closer to the extracellular matrix components and promote the regeneration of tissues and organs.

The technical solution adopted by the present invention to realize the purpose of the invention is a preparation method of a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A, stock solution preparation: protein powder, polyphenol powder and polysaccharide powder are respectively dissolved in deionized water to obtain a protein solution with a concentration of 3.0-10.0wt.%; a polyphenol solution with a concentration of 0.10-0.75wt.%. 2.0-8.0wt.% polysaccharide solution;

B. Synthesis of protein-polyphenol binary complex: Add the protein solution and polyphenol solution prepared in step A into the reactor at a ratio of 1-10:1, and magnetically stir in a water bath at 40-50°C for 1- 3h, obtain the protein-polyphenol binary complex solution;

C. Preparation of gel: Add the polysaccharide solution configured in step A to the protein-polyphenol binary complex solution in step B, wherein the volume ratio of the polysaccharide solution to the protein-polyphenol binary complex solution is 2-4 : 1, under the condition of 40-50 DEG C of water bath, stir magnetically until the color of the solution is uniform without delamination.

Reaction principle and mechanism of the present invention are:

First, the protein solution and the polyphenol solution are subjected to a water bath reaction. After the phenolic hydroxyl group in the polyphenol solution is oxidized and loses H- ^, the vacancy on the benzene ring is covalently bonded to the α-amino group in the amino acid residue of the protein. The polyphenol solution The unoxidized phenolic hydroxyl group in the protein forms an intermolecular hydrogen bond with a single atom in the side chain group of the amino acid residue of the protein, thereby generating a protein-polyphenol binary complex.

Then the polysaccharide solution is added to the protein-polyphenol binary complex solution for gel reaction, the α-amino group of the protein amino acid residue in the complex is positively charged and the carboxyl group of the negatively charged polysaccharide forms electrostatic adsorption, and the protein- Polyphenol-polysaccharide-based ternary complex hydrogels.

The using method of thixotropic (protein-polyphenol-polysaccharide base) hydrogel of the present invention is:

The prepared thixotropic hydrogel and cells are configured into a bioprinting "ink", which is then placed in an extrusion 3D printer slurry cartridge for 3D bioprinting. The printed scaffold model is placed in cell culture medium, and then cultured in a 37°C, 5% CO ₂ incubator. After cell growth and proliferation, it will be used for in vivo replacement and regeneration, in vitro drug screening and disease research. Tissues/organs.

Compared with prior art, the beneficial effect of the present invention is:

1. The thixotropy of the protein-polyphenol-polysaccharide-based thixotropic hydrogel prepared in the present invention mainly comes from the hydrogen bonding between protein and polyphenol, and the electrostatic interaction between protein and polysaccharide. During extrusion, these two interaction forces are destroyed by the extrusion force, and the hydrogel undergoes shear thinning and becomes a sol; when it is used as a 3D bioprinting material, it has excellent injectability, and the extrusion required for printing The output is small, the damage to cells is small, the activity of cells is high, and it can better promote the regeneration of tissues and organs. When the external force is removed (molding after extrusion), hydrogen bonds will be quickly re-formed and electrostatic interactions will occur between the components, returning to the gel state (thixotropy); it can maintain the gel structure for a long time after printing stable and has excellent formability.

Experiments have proved that the hydrogel prepared by the present invention and cells are printed into a three-dimensional tissue (organ) scaffold by using an extrusion 3D bioprinter. The injection pressure during printing is only 1N, the printing process is smooth, there is no clogging of nozzles and stoppage phenomenon, and the printed three-dimensional scaffold has a stable structure, no collapse phenomenon, excellent formability, and good shape fidelity.

2. The raw materials of the present invention are proteins, polyphenols and polysaccharides existing in animals and plants, and have excellent biocompatibility. And the three components are similar to the extracellular matrix components, which can better provide an ideal environment for cell growth.

3. The preparation operations are all carried out in liquid state at normal temperature, with mild conditions, simple process and low preparation cost.

Further, the protein in step A of the present invention may be one or a mixture of gelatin, fibrinogen, fibrin, silk fibroin, hyaluronic acid or collagen.

Further, the polyphenol in step A of the present invention is one or more mixtures of tannic acid, tea polyphenols, caffeic acid, catechin, gallic acid, ellagic acid, arbutin, and anthocyanins .

Further, the polysaccharide in step A of the present invention is sodium alginate, chitosan, gellan gum, dextran, agarose or linseed gum.

The above protein powder, polyphenol powder and polysaccharide powder are substances that exist in animals and plants, and have high biocompatibility; and the components of the three substances are similar to the components of the extracellular matrix, which can promote new bone formation and better serve cells. Provides an ideal environment for growth.

The present invention will be further described in detail below in combination with specific embodiments.

detailed description

Example 1

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A, stock solution preparation: gelatin powder, tannic acid powder and sodium alginate powder are respectively dissolved in deionized water to obtain a gelatin solution with a concentration of 6.0wt.% respectively; a tannic acid solution with a concentration of 0.25wt.%; 3.0wt.% sodium alginate solution;

B. Synthesis of gelatin-tannic acid binary complex: add the gelatin solution and tannic acid solution prepared in step A into the reactor at a ratio of 1:1, and stir magnetically for 2 hours in a water bath at 40°C to obtain gelatin - tannic acid binary complex solution;

C, preparation of gel: the sodium alginate solution configured in step A is added to the gelatin-tannic acid binary complex solution of B step, wherein the sodium alginate solution and the gelatin-tannic acid binary complex solution The volume ratio is 3:1, and the color of the magnetically stirred solution is uniform under the condition of a water bath at 50°C, and there is no layering.

Example 2

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A, stoste preparation: fibrinogen powder, tea polyphenol powder and chitosan powder are respectively dissolved in deionized water, respectively to obtain a concentration of 3wt.% fibrinogen solution; concentration of 0.10wt.% tea polyphenol Solution; concentration is 2.0wt.% chitosan solution;

B. Synthesis of fibrinogen-tea polyphenol binary complex: add the fibrinogen solution and tea polyphenol solution prepared in step A into the reactor at a ratio of 5:1, and stir magnetically in a water bath at 45°C 1h, obtain the fibrinogen-tea polyphenol binary complex solution;

C. Preparation of gel: Add the chitosan solution configured in step A to the fibrinogen-tea polyphenol binary complex solution in step B, wherein the chitosan solution and the fibrinogen-tea polyphenol binary The volume ratio of the complex solution is 2:1, and the color of the solution is uniform under magnetic stirring under the condition of a water bath at 45° C. without stratification.

Example 3

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A, stock solution preparation: respectively dissolve fibrin powder, caffeic acid powder and gellan gum powder in deionized water to obtain respectively a fibrin solution with a concentration of 10.0wt.%; a caffeic acid solution with a concentration of 0.75wt.%; 8.0wt.% gellan gum solution;

B. Synthesis of fibrin-caffeic acid binary complex: Add the fibrin solution and caffeic acid solution prepared in step A into the reactor at a ratio of 10:1, and stir magnetically for 3 hours in a water bath at 50°C to obtain fibers Protein-caffeic acid binary complex solution;

C, preparation of gel: the gellan gum solution configured in step A is added to the fibrin-caffeic acid binary complex solution of B step, wherein the gellan gum solution and the fibrin-caffeic acid binary complex solution The volume ratio is 4:1, and the color of the magnetically stirred solution is uniform under the condition of a water bath at 40°C, and there is no layering.

Example 4

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A. Stock solution preparation: dissolve silk fibroin powder, catechin powder and dextran powder in deionized water respectively to obtain a silk fibroin solution with a concentration of 7wt.% and a catechin solution with a concentration of 0.50wt.%. Concentration is the dextran solution of 5.0wt.%.

B. Synthesis of silk fibroin-catechin binary complex: Add the silk fibroin solution and catechin solution prepared in step A into the reactor at a ratio of 4:1, and stir magnetically under the condition of a water bath at 47°C 2.5h, obtain the silk fibroin-catechin binary complex solution;

C. Preparation of gel: Add the dextran solution configured in step A to the silk fibroin-catechin binary complex solution in step B, wherein the dextran solution and silk fibroin-catechin binary complex solution The volume ratio is 4:1, and the color of the magnetically stirred solution is uniform under the condition of a water bath at 46°C, and there is no layering.

Example 5

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A. Stock solution preparation: dissolve silk fibroin powder, catechin powder and dextran powder in deionized water respectively to obtain a silk fibroin solution with a concentration of 8.0wt.%; a catechin solution with a concentration of 0.45wt.% ; Concentration is 5.0wt.% dextran solution;

B. Synthesis of silk fibroin-catechin binary complex: add the silk fibroin solution and catechin solution prepared in step A into the reactor at a ratio of 1:1, and stir magnetically in a water bath at 45°C 2h, obtain the silk fibroin-catechin binary complex solution;

C. Preparation of gel: Add the dextran solution configured in step A to the silk fibroin-catechin binary complex solution in step B, wherein the dextran solution and silk fibroin-catechin binary complex solution The volume ratio is 2:1, and the color of the magnetically stirred solution is uniform under the condition of a water bath at 50°C, and there is no layering.

Example 6

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A. Stock solution preparation: respectively dissolve hyaluronic acid powder, gallic acid powder and agarose powder in deionized water to obtain a hyaluronic acid solution with a concentration of 4.0wt.% and a gallic acid solution with a concentration of 0.75wt.%. Concentration is 2.0wt.% agarose solution;

B. Synthesis of hyaluronic acid-gallic acid binary complex: Add the hyaluronic acid solution and gallic acid solution prepared in step A into the reactor at a ratio of 6:1, and magnetically stir for 2 hours in a water bath at 48°C. Obtain hyaluronic acid-gallic acid binary complex solution;

C, preparation of the gel: the agarose solution configured in step A is added to the hyaluronic acid-gallic acid binary complex solution of step B, wherein the agarose solution and the hyaluronic acid-gallic acid binary complex solution The volume ratio is 3:1, and the color of the magnetically stirred solution is uniform under the condition of a water bath at 42°C, and there is no layering.

Example 7

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A, stock solution preparation: respectively dissolve hyaluronic acid and collagen powder, ellagic acid powder and linseed gum powder in deionized water, and obtain the hyaluronic acid and collagen solution that concentration is 3.0wt.% respectively; Concentration is 0.5wt. % ellagic acid solution; concentration is 5.0wt.% linseed gum solution;

B. Synthesis of hyaluronic acid and collagen-ellagic acid binary complex: Add the hyaluronic acid, collagen solution and ellagic acid solution prepared in step A into the reactor at a ratio of 6:1, and put them in a water bath at 40°C Under the condition of magnetic stirring for 3 hours, the solution of hyaluronic acid and collagen-ellagic acid binary complex is obtained;

C. Preparation of gel: Add the linseed gum solution configured in step A to the hyaluronic acid and collagen-ellagic acid binary complex solution in step B, wherein the linseed gum solution is mixed with hyaluronic acid and collagen-tanned The volume ratio of the flower acid binary complex solution is 2:1, and the color of the solution is uniform under magnetic stirring in a water bath at 40°C without stratification.

Example 8

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A, stock solution preparation: the mixed powder of collagen powder, arginin fruit and anthocyanin and sodium alginate powder are dissolved in deionized water respectively, obtain the collagen solution that concentration is 3.0wt.% respectively; Concentration is 0.5wt.% Arginin fruit and A mixed solution of anthocyanins; a sodium alginate solution with a concentration of 5.0wt.%;

B. Synthesis of collagen-arginin fruit and anthocyanin binary complex: Add the collagen solution prepared in step A and the mixed solution of arginin fruit and anthocyanin in a ratio of 6:1 to the reactor, and put them in a water bath at 40°C Magnetically stirred for 3 hours to obtain a collagen-arginin and anthocyanin binary complex solution;

C. Preparation of gel: Add the sodium alginate solution configured in step A to the collagen-arginin and anthocyanin binary complex solution in step B, wherein the sodium alginate solution is mixed with collagen-arginin and anthocyanin binary The volume ratio of the complex solution is 2:1, and the color of the solution is uniform under magnetic stirring under the condition of a water bath at 40° C. without stratification.

Example 9

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A, stoste preparation: gelatin powder, arginin fruit powder and chitosan powder are dissolved in deionized water respectively, and concentration is respectively obtained the gelatin solution of 3.0wt.%; Concentration is the arginin fruit solution of 0.5wt.%; Concentration is 5.0wt. % chitosan solution;

B. Synthesis of gelatin-arginin binary compound: add the gelatin solution and arginin solution prepared in step A into the reactor at a ratio of 6:1, and stir magnetically for 3 hours in a water bath at 40°C to obtain the gelatin-arginin binary compound Complex solution;

C, preparation of gel: the chitosan solution configured in step A is added in the gelatin-arginin binary complex solution of B step, wherein the volume ratio of chitosan solution and gelatin-arginin binary compound solution is 2 : 1, under the condition of 40 DEG C of water bath, the magnetically stirred solution has uniform color and no stratification, to obtain final product.

Example 10

A method for preparing a thixotropic hydrogel for 3D bioprinting, comprising the following steps:

A. Stock solution preparation: Dissolve collagen powder, anthocyanin powder and gellan gum powder in deionized water respectively to obtain a collagen solution with a concentration of 3.0wt.%; a mixed solution of anthocyanins with a concentration of 0.5wt.% ; Concentration is 5.0wt.% gellan gum solution;

B. Synthesis of collagen-anthocyanin binary complex: Add the collagen solution and anthocyanin solution prepared in step A into the reactor at a ratio of 10:1, and stir magnetically for 2 hours in a water bath at 45°C to obtain collagen - anthocyanin binary complex solution;

C, preparation of the gel: the gellan gum solution configured in step A is added to the collagen-anthocyanin binary complex solution in step B, wherein the gellan gum solution and the collagen-anthocyanin binary complex solution The volume ratio is 2:1, and the color of the magnetically stirred solution is uniform under the condition of a water bath at 50°C, and there is no layering.

Claims (3)

1. A preparation method of thixotropic hydrogel for 3D bioprinting comprises the following steps:

A. preparing stock solution: dissolving protein powder, polyphenol powder and polysaccharide powder in deionized water respectively to obtain protein solutions with the concentration of 3.0-10.0 wt.%; a polyphenol solution having a concentration of 0.10-0.75 wt.%; a polysaccharide solution having a concentration of 2.0-8.0 wt.%;

the polysaccharide is sodium alginate, chitosan, gellan gum, dextran, agarose or flaxseed gum;

B. synthesis of protein-polyphenol binary complex: adding the protein solution and the polyphenol solution prepared in the step A into a reactor according to the proportion of 1-10 to 1, and magnetically stirring for 1-3 hours under the water bath condition of 40-50 ℃ to obtain a protein-polyphenol binary compound solution;

C. preparation of gel: and (3) adding the polysaccharide solution prepared in the step (A) into the protein-polyphenol binary compound solution prepared in the step (B), wherein the volume ratio of the polysaccharide solution to the protein-polyphenol binary compound solution is 2-4.

2. The method of preparing the thixotropic hydrogel for 3D bioprinting according to claim 1, wherein: the protein in the step A is a mixture of more than one of gelatin, fibrinogen, fibrin, silk fibroin or collagen.

3. The method of preparing the thixotropic hydrogel for 3D bioprinting according to claim 1, wherein: the polyphenol in the step A is a mixture of more than one of tannin, tea polyphenol, caffeic acid, gallic acid, ellagic acid, arbutin or anthocyanin.