CN113683787B - A kind of hydrogel material with secondary crosslinking property and its preparation method and application - Google Patents

Abstract

The invention provides a hydrogel material with secondary crosslinking characteristic, a preparation method and application thereof, wherein the hydrogel material is prepared by grafting catechol groups and photosensitive groups on any biological macromolecule of gelatin, alginate and hyaluronic acid to obtain catechol photosensitive macromolecules, and carrying out photocrosslinking after solution preparation and related application. After the hydrogel material is prepared into a solution, the photo-curing crosslinking of the photosensitive groups in the hydrogel molecules can be firstly utilized to prepare a specific shape, and the catechol groups in the hydrogel molecules can be utilized to crosslink again to realize adhesion between the hydrogel and tissues or splicing between hydrogels with different shapes. The hydrogel capable of being crosslinked for the second time has great application value in the preparation of wound dressing, conductive biosensor, tissue engineering scaffold and other fields.

Description

A kind of hydrogel material with secondary crosslinking property and its preparation method and application

technical field

The invention relates to the technical field of polymer hydrogel preparation, in particular to a hydrogel material with secondary crosslinking properties, a preparation method and application thereof.

Background technique

Skin incisions caused by trauma or surgery are prone to hypertrophic or depressed scars after surgical suturing, which affects the appearance. Using tissue glue instead of surgical sutures can reduce scarring of the skin. At present, some seam-free tissue glue products have been used for auxiliary adhesion care when suturing surgical and cosmetic wounds. For example, Histoacryl Tissue Adhesive (Histoacryl Tissue Adhesive), 3M tissue glue (Vetbond), these tissue glues are mainly composed of 2-n-butyl cyanoacrylate (Enbuster), and stabilizers (hydroquinone, sulfur dioxide, phosphoric acid). This type of tissue glue has certain disadvantages: ① Doctors need to manually apply it on the incision surface, and it is difficult to achieve uniform coating by hand; ② It takes several minutes to wait for the glue to change from solution to solid; ③ The matrix material of the glue is not biocompatible. Therefore, how to prepare a hydrogel material that can be cross-linked in advance and can adhere tightly to the skin again is a clinical problem that needs to be solved urgently.

In tissue engineering research, since tissue-engineered micro-tissues can achieve uniform loading of seed cells, the construction of micro-tissues has become a hot emerging field. However, in current research, most of the micro-tissues are directly placed in the tissue defect site, and the orderly assembly of micro-tissues has not been realized yet. Therefore, if the hydrogel can be used to prepare microtissues first, and then the secondary crosslinking properties of the hydrogel can be used to arrange different types of microtissues in an orderly manner, it is expected to solve the problem of assembling microtissues and provide new ideas for tissue engineering research.

Contents of the invention

In view of the deficiencies of the above-mentioned prior art, the present invention provides a hydrogel material with secondary crosslinking properties and its preparation method and application. The prepared hydrogel material can be secondary crosslinked to realize the adhesion between hydrogel and tissue or the splicing between hydrogels of different shapes.

The technical solution provided by the present invention: a hydrogel material with secondary crosslinking properties, the hydrogel material is obtained by grafting catechol groups and photosensitive groups on any biomacromolecule of gelatin, alginate, and hyaluronic acid at the same time to obtain a catechol-based photosensitive macromolecule, which is obtained by photocrosslinking after dissolution.

A method for preparing a hydrogel material with secondary crosslinking properties, comprising the steps of:

(1) dissolving gelatin in PBS solution, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and adjusting the pH of the solution to 4.5-5.5 for activation;

(2) Dopamine hydrochloride is dissolved in PBS solution, the dopamine hydrochloride solution is added dropwise to the gelatin solution obtained in step (1), the mixed solution is placed in a constant temperature shaker, dialysis is obtained after shaking in the dark, and freeze-drying to obtain catechol-based gelatin;

(3) dissolving the catechol-based gelatin prepared in step (2) in the MES buffer solution, fully dissolving, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the solution, and adjusting the pH of the solution to 4.5-5.5 for activation;

(4) Dissolving 2-aminoethyl methacrylate hydrochloride in the MES solution, adding the 2-aminoethyl methacrylate hydrochloride solution dropwise to the catechol-based gelatin solution obtained in step (3), placing the mixed solution in a constant temperature shaker, dialysis after shaking in the dark, and freeze-drying to obtain catechol-based photosensitive gelatin;

(5) Formulate the catechol-based photosensitive gelatin obtained in step (4) into an aqueous solution with a mass fraction of 5-20%, add a photoinitiator, and then drop the solution into hydrogel molds of different shapes, and irradiate the solution with ultraviolet light to solidify the solution to obtain a sheet-shaped hydrogel dressing; or use a stereoscopic light projection 3D printer to prepare hydrogel dressings or brackets of different shapes.

Further, the mass fraction of the gelatin dissolved in the PBS solution in the step (1) is 0.5%-2%, the mass fraction of the catechol-based gelatin dissolved in the MES buffer solution obtained in the step (2) is 0.5%-2%, respectively, and the mass ratio of dopamine hydrochloride to gelatin in the step (2) is 0.2-1:1.

Further, the raw material gelatin used in the step (1) can be replaced with alginate or hyaluronic acid, the mass fractions of alginate or hyaluronic acid dissolved in PBS solution are 0.05%-0.5% and 0.01%-0.3%, respectively, and the mass fractions of catechol-based alginate or catechol-based hyaluronic acid dissolved in MES buffer solution obtained in step (2) are 0.05%-0.5% and 0.01%-0.3% respectively. %.

Further, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide added in the step (1) and step (3) is between 5:3-1:1, the mass concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the solution is 0.1%-1%, and the activation time of the step (1) and step (3) is 5-60min.

Further, in the step (2), the dopamine hydrochloride is prepared in a PBS solution with a mass concentration of 5%-10%, the temperature for shaking in the dark is 20-40°C, the shaking time is 5-24 hours, the pores of the dialysis bag are between 3000Da-1000000Da, and the dialysis time is 24-48h.

Further, in the step (4), 2-aminoethyl methacrylate hydrochloride is dissolved in the MES solution at a mass concentration of 5-10%, the mass ratio of 2-aminoethyl methacrylate hydrochloride to catechol-based gelatin is 0.2-1:1, the temperature for shaking in the dark is 20-40°C, the shaking time is 5-24 hours, the pores of the dialysis bag are between 3000Da-1000000Da, and the dialysis time is 24-48h .

Further, in the step (5), 365nm ultraviolet light is used for irradiation, and the irradiation time is between 5-30 seconds. The photoinitiator is LAP or I2959, and the mass concentration added is between 0.1-1%.

The secondary cross-linking method of the hydrogel material with secondary cross-linking properties obtained by the preparation method is to configure 0.1-1mM/L ferric chloride solution or 0.1-1mM/L sodium periodate solution, apply a thin layer of solution on the skin, and then stick the hydrogel material on the skin surface to achieve tight adhesion; or paste the two hydrogels together, drip ferric chloride solution or sodium periodate solution on the surface to achieve tight adhesion.

The application of a hydrogel material with secondary cross-linking properties, using photo-curing 3D printing technology, using hydrogel materials to prepare millimeter-scale hydrogel micro-units; using the secondary cross-linking characteristics of hydrogel to achieve the splicing between multiple hydrogel micro-units and the tight adhesion between hydrogel and tissue, to achieve precise repair of the defect; or using photo-curing 3D printing technology to pre-prepare the hydrogel material into a skin wound dressing of a specific shape, and use the secondary cross-linking characteristics of the hydrogel to form tight adhesion with the skin to cover the wound and promote skin repair. effect.

The hydrogel material prepared by the present invention has secondary crosslinking characteristics. After the material is made into a solution, the photosensitive group in the hydrogel molecule can be used to realize photocuring and crosslinking to prepare a specific shape, and the catechol group in the hydrogel molecule can also be used to crosslink again to realize the adhesion between the hydrogel and the tissue or the splicing between hydrogels of different shapes. This type of secondary cross-linkable hydrogel has great application value in the preparation of wound dressings, conductive biosensors, tissue engineering scaffolds and other fields.

Description of drawings

Fig. 1 is the flow chart of preparation method of the present invention;

Fig. 2 is the physical figure of chemical synthesis step of the present invention and corresponding product;

Fig. 3 is the H NMR spectrum result figure of the hydrogel material in the present invention;

Fig. 4 is the Fourier transform infrared spectrum result figure of the hydrogel material in the present invention;

Fig. 5 is a model diagram of a tissue engineering room printed by a 3D printer of the hydrogel material in the present invention;

Fig. 6 is the effect diagram after secondary crosslinking of the hydrogel material in the present invention;

Fig. 7 is an effect diagram of the adhesion of the hydrogel material of the present invention to the tissue;

Fig. 8 is the electron micrograph of the hydrogel material prepared using dialysis bags of different molecular weights in the present invention;

Fig. 9 is an in vitro degradation rate diagram of hydrogel materials prepared using dialysis bags of different molecular weights in the present invention;

Fig. 10 is a diagram of the swelling detection results in physiological saline of the hydrogel material;

Fig. 11 is the hardness detection result figure of hydrogel material;

Fig. 12 is the elastic detection result figure of hydrogel material;

Fig. 13 is the cell light micrograph of photocrosslinking after the hydrogel material is mixed with bone marrow mesenchymal stem cells (BMSC);

Fig. 14 is a light micrograph of cells photocrosslinked after the hydrogel material is mixed with human umbilical vein endothelial cells (HUVEC);

Figure 15 is a picture of the hydrogel material mixed with HUVEC after photocrosslinking, and observing the cell survival after 1 day and 5 days of culture;

Figure 16 is a flow chart of the application of the hydrogel material of the present invention;

Detailed ways

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

Example 1

Dissolve gelatin in PBS solution to form a solution with a mass fraction of 2%. After fully dissolving, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the solution, wherein the mass of EDC/NHS is 5/3, and the mass concentration of EDC in the solution is 0.5%. The pH of the solution is adjusted to 5 and activated for 30 minutes. Take dopamine hydrochloride in PBS solution to prepare a solution with a mass fraction of 10%, add the dopamine hydrochloride solution drop by drop to the gelatin solution, and the mass ratio of gelatin to dopamine hydrochloride in the mixed solution is 2:1. The mixed solution was placed in a constant temperature shaker and shaken overnight at 25°C in the dark. The solution was dialyzed for 36 hours using a dialysis bag (with a pore size of 3500 Da), and the solution was changed 6 times. Catechol-based gelatin was prepared after freeze-drying.

The prepared catechol-based gelatin was dissolved in MES buffer solution to form a solution with a mass fraction of 1%. After fully dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the solution, wherein the mass of EDC/NHS was 5/3, and the mass concentration of EDC in the solution was 1%. The pH of the solution was adjusted to 5, and activated for 30 minutes. Take 2-aminoethyl methacrylate hydrochloride (AEMA) and prepare a solution with a mass fraction of 5% in MES solution, add 2-aminoethyl methacrylate hydrochloride drop by drop to the catechol-based gelatin solution, and the mass ratio of 2-aminoethyl methacrylate hydrochloride to catechol-based gelatin is 0.5:1. The mixed solution was placed in a constant temperature shaker and shaken overnight at 25°C in the dark. The solution was dialyzed for 36 hours using a dialysis bag (pore size at 3500 Da), and the solution was changed every 6 hours. Mixed molecular weight catechol-based photosensitive gelatin was prepared after freeze-drying.

Add 15% catechol-based photosensitive gelatin solution dropwise to hydrogel molds of different shapes, add 0.5% photoinitiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and irradiate with 365nm ultraviolet light for 20 seconds to cure the solution to obtain sheet-shaped hydrogel dressings; or use a stereoscopic light projection 3D printer to prepare hydrogel dressings or brackets of different shapes.

Example 2

Dissolve gelatin in PBS solution to form a solution with a mass fraction of 2%. After fully dissolving, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the solution, wherein the mass of EDC/NHS is 5/3, and the mass concentration of EDC in the solution is 0.5%. The pH of the solution is adjusted to 5 and activated for 30 minutes. Take dopamine hydrochloride in PBS solution to prepare a solution with a mass fraction of 10%. Add the dopamine hydrochloride solution drop by drop to the gelatin solution, and the mass ratio of gelatin to dopamine hydrochloride in the mixed solution is 2:1. The mixed solution was placed in a constant temperature shaker and shaken overnight at 25°C in the dark. The solution was dialyzed for 36 hours using a dialysis bag (with a pore size of 1,000,000 Da), and the solution was changed 6 times. Catechol-based gelatin was prepared after freeze-drying.

The prepared catechol-based gelatin was dissolved in MES buffer solution to form a solution with a mass fraction of 1%. After fully dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the solution, wherein the mass of EDC/NHS was 5/3, and the mass concentration of EDC in the solution was 1%. The pH of the solution was adjusted to 5, and activated for 30 minutes. Take 2-aminoethyl methacrylate hydrochloride (AEMA) and prepare a solution with a mass fraction of 5% in MES solution, add 2-aminoethyl methacrylate hydrochloride drop by drop to the catechol-based gelatin solution, and the mass ratio of 2-aminoethyl methacrylate hydrochloride to catechol-based gelatin is 0.5:1. The mixed solution was placed in a constant temperature shaker and shaken overnight at 25°C in the dark. The solution was dialyzed for 36 hours using a dialysis bag (porosity at 1,000,000 Da), and the solution was changed every 6 hours. After freeze-drying, a large molecular weight catechol-based photosensitive gelatin was prepared.

Add 15% catechol-based photosensitive gelatin solution dropwise to hydrogel molds of different shapes, add 0.5% photoinitiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and irradiate with 365nm ultraviolet light for 20 seconds to cure the solution to obtain a sheet-shaped hydrogel dressing; or use a stereoscopic light projection 3D printer to prepare hydrogel dressings or brackets of different shapes.

Catechol-based photosensitive gelatins with different molecular weights were prepared in Examples 1 and 2, that is, 3500Da and 1000kDa GDMA in the experimental test results

Example 3

Dissolve gelatin in PBS solution to form a solution with a mass fraction of 2%. After fully dissolving, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the solution, wherein the mass of EDC/NHS is 5/3, and the mass concentration of EDC in the solution is 0.5%. The pH of the solution is adjusted to 5 and activated for 30 minutes. Take dopamine hydrochloride in PBS solution to prepare a solution with a mass fraction of 10%, add the dopamine hydrochloride solution drop by drop to the gelatin solution, and the mass ratio of gelatin to dopamine hydrochloride in the mixed solution is 3:1. The mixed solution was placed in a constant temperature shaker and shaken overnight at 25°C in the dark. The solution was dialyzed for 36 hours using a dialysis bag (with a pore size of 3500 Da), and the solution was changed 6 times. Catechol-based gelatin was prepared after freeze-drying.

The prepared catechol-based gelatin was dissolved in MES buffer solution to form a solution with a mass fraction of 1%. After fully dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the solution, wherein the mass of EDC/NHS was 5/3, and the mass concentration of EDC in the solution was 1%. The pH of the solution was adjusted to 5, and activated for 30 minutes. Take 2-aminoethyl methacrylate hydrochloride (AEMA) and prepare a solution with a mass fraction of 5% in MES solution, add 2-aminoethyl methacrylate hydrochloride drop by drop to the catechol-based gelatin solution, and the mass ratio of 2-aminoethyl methacrylate hydrochloride to catechol-based gelatin is 0.3:1. The mixed solution was placed in a constant temperature shaker and shaken overnight at 25°C in the dark. The solution was dialyzed for 36 hours using a dialysis bag (pore size at 3500 Da), and the solution was changed every 6 hours. After freeze-drying, a large molecular weight catechol-based photosensitive gelatin was prepared. Add 15% catechol-based photosensitive gelatin solution dropwise to hydrogel molds of different shapes, add 0.5% photoinitiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and irradiate with 365nm ultraviolet light for 20 seconds to cure the solution to obtain a sheet-shaped hydrogel dressing; or use a stereoscopic light projection 3D printer to prepare hydrogel dressings or brackets of different shapes.

Compared with Example 1, Example 3 reduces the concentration of dopamine hydrochloride and 2-aminoethyl methacrylate hydrochloride, and the grafting rate of the reaction product decreases.

Example 4

Dissolve alginic acid sodium salt in PBS solution to make a solution with a mass fraction of 0.2%. After fully dissolving, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the solution, wherein the mass of EDC/NHS is 3/2, and the concentration of EDC in the solution is 0.2%. The pH of the solution is adjusted to 5, and activated for 10 minutes. Dopamine hydrochloride was prepared in PBS solution to a concentration of 8%, and the dopamine hydrochloride solution was added drop by drop to the alginate solution. The mixed solution was placed in a constant temperature shaker and shaken overnight at 35°C in the dark. The solution was dialyzed for 48 hours using a dialysis bag (pore size at 3000 Da), and the solution was changed every 6 hours. After freeze-drying, catechol-based sodium alginate was prepared.

The prepared catechol-based sodium alginate was dissolved in MES buffer solution to form a solution with a mass fraction of 0.2%. After fully dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the solution, wherein the mass of EDC/NHS was 3/2, and the concentration of EDC in the solution was 1%. The pH of the solution was adjusted to 5, and activated for 50 minutes. 2-Aminoethyl methacrylate hydrochloride (AEMA) was prepared into a solution with a mass fraction of 5% in the MES solution, and the 2-aminoethyl methacrylate hydrochloride solution was added drop by drop to the catechol-based alginate solution. The mixed solution was placed in a constant temperature shaker and shaken overnight at 35°C in the dark. The solution was dialyzed for 48 hours using a dialysis bag (pore size at 3000 Da), and the solution was changed every 6 hours. The catechol-based photosensitive alginate was prepared after freeze-drying.

Add 3% catechol-based sodium alginate solution dropwise to hydrogel molds of different shapes, add 0.5% photoinitiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and irradiate with 365nm ultraviolet light for 30 seconds to cure the solution to obtain a sheet-shaped hydrogel dressing; or use a stereoscopic light projection 3D printer to prepare hydrogel dressings or brackets of different shapes.

Example 5

Dissolve hyaluronic acid in PBS solution to make a solution with a mass fraction of 0.1%. After fully dissolving, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the solution, wherein the mass of EDC/NHS is 1/1, and the concentration of EDC in the solution is 0.2%. The pH of the solution is adjusted to 4.5 and activated for 40 minutes. The dopamine hydrochloride was prepared in PBS solution to a concentration of 10%, and the dopamine hydrochloride solution was added drop by drop to the hyaluronic acid solution. The mixed solution was placed in a constant temperature shaker and shaken overnight at 20°C in the dark. The solution was dialyzed for 24 hours using a dialysis bag (pore size between 3000 Da), and the solution was changed every 6 hours. Catechol-based hyaluronic acid was prepared after freeze-drying.

Dissolve the prepared catechol-based hyaluronic acid in 100mM/L MES buffer solution to form a solution with a concentration of 0.1%. After fully dissolving, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the solution, wherein the mass of EDC/NHS is 1/1, and the concentration of EDC in the solution is 0.2%. Adjust the pH of the solution to 4.5 and activate for 40 minutes . 2-Aminoethyl methacrylate hydrochloride (AEMA) was prepared into a solution with a mass fraction of 10% in MES solution, and the 2-aminoethyl methacrylate hydrochloride solution was added drop by drop to the catechol-based hyaluronic acid solution. The mixed solution was placed in a constant temperature shaker and shaken overnight at 20°C in the dark. The solution was dialyzed for 24 hours using a dialysis bag (pore size 10000 Da), and the solution was changed every 6 hours. The catechol-based photosensitive hyaluronic acid was prepared after freeze-drying.

Add 2% catechol-based photosensitive hyaluronic acid solution dropwise to hydrogel molds of different shapes, add 0.5% photoinitiator phenyl-2,4,6-trimethylbenzoyllithium phosphite (LAP), and irradiate with 365nm ultraviolet light for 20 seconds to cure the solution to obtain a sheet-shaped hydrogel dressing; or use a stereoscopic light projection 3D printer to prepare hydrogel dressings or brackets of different shapes.

GD-MA hydrogel material detection

The raw materials and products were dissolved in heavy water, and the hydrogen spectrum was scanned on a nuclear magnetic resonance spectrometer (instrument model: Bruker600MHz), and the absorption peaks of different materials were obtained. The absorption peaks of GD-MA overlapped with dopamine and gelatin, indicating that GD was synthesized from these two raw materials, that is, catechol groups were grafted on the side chain of gelatin; the characteristic peaks showed that the hydrogel was successfully prepared;

The raw materials and products were grinded in potassium bromide and pressed into tablets, and then detected by Fourier transform infrared spectroscopy (instrument model: Thermo Scientific Nicolet 6700). The characteristic peaks showed that the catechol groups and methacrylate bonds on the gelatin side chains were grafted by amide bonds. The results of GD-MA Fourier transform infrared spectroscopy are shown in Fig. 4, and the peak at 1640cm-1 illustrates the formation of an amide bond (HNCO). 3500-3100N-H stretching vibration 1680-1630C=O stretching vibration 1655-1590N-H bending vibration 3100-3000 The C-H stretching vibration of alkenes caused 1640 or the stretching vibration of carbon-carbon double bond The deformation vibration of methyl group changed the absorption peak at 1300nm.

GD-MA has excellent photosensitive properties, and can use stereoscopic light projection 3D printers to prepare tissue engineering room models of various shapes, as shown in Figure 5 (instrument: engineering for life photocurable biological 3D printer BP8600), GD-MA has good printing performance, and can print out precise structures.

As shown in Figure 6, after GD-MA was photocured, sodium periodate solution was added to the hydrogel, and the GD-MA hydrogel turned orange-yellow, and the contact surface was tightly adhered; after adding iron oxide solution to the hydrogel, the GD-MA hydrogel turned blue, and the contact surface was tightly adhered. It shows that the GD-MA hydrogel can also achieve secondary crosslinking through the catechol groups on its side chains after photocrosslinking.

As shown in Figure 7, the adhesion of the GD-MA hydrogel to the tissue can be tightly adhered to the tissue through the secondary cross-linking, and the twisting and folding show the tight adhesion between the hydrogel and the tissue.

As shown in Figure 8, GD-MA materials with two molecular weights were prepared by using dialysis bags with different molecular weights. After making hydrogels with different concentrations, they were observed under an electron microscope. It was found that the large molecular weight GD-MA hydrogels prepared by 1000kDa dialysis had larger pores and more uniformity, which may be more conducive to cell growth.

As shown in Figure 9, the in vitro degradation was observed by soaking in PBS at 37°C, and the data of 3w was recorded. Judging from the data, the degradation rate of GD-MA with large molecular weight in vitro is faster, which may be related to the high porosity. (Unable to weigh at week 4 due to hydrogel degradation)

As shown in Figure 10, the swelling test of the GD-MA hydrogel in physiological saline shows that the swelling of the hydrogel is not obvious, and the shape is maintained well.

As shown in Figure 11 and Figure 12, the hardness of the hydrogel increases with the increase of GD-MA concentration, and the 12% GD-MA material has better elasticity.

As shown in Figures 13-15, GD-MA has good biocompatibility.

The hydrogel prepared by the present invention has printability and bioadhesive effect; as shown in Figure 16, the millimeter-scale hydrogel tissue engineering chamber is prepared using the above-mentioned materials using the DLP light-curing 3D printing technology; then the tissue engineering microtissue cultivated in the bioreactor is assembled and filled in the first stage to make it a microunit; finally, the bioadhesion of the hydrogel is used to perform secondary assembly and splicing of multiple microunits in the tissue engineering chamber, which can realize precise repair of the defect.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention shall be covered within the scope of protection of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (7)

1. A secondary crosslinking application method of a hydrogel material with secondary crosslinking characteristics is characterized in that 0.1-1mM/L ferric chloride solution or 0.1-1mM/L sodium periodate solution is prepared, a layer of solution is coated on skin, and then the hydrogel material is stuck on the surface of the skin, so that tight adhesion can be realized; or the two hydrogels are stuck together, and ferric chloride solution or sodium periodate solution is added dropwise to the surfaces of the hydrogels, so that tight adhesion can be realized;

the preparation method of the hydrogel material comprises the following steps:

(1) Dissolving gelatin in PBS solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adjusting the pH of the solution to 4.5-5.5, and activating;

(2) Dissolving dopamine hydrochloride in PBS (phosphate buffer solution), dropwise adding the dopamine hydrochloride solution into the gelatin solution obtained in the step (1), placing the mixed solution in a constant-temperature shaking table, shaking in a dark place, dialyzing, and freeze-drying to obtain catechol gelatin;

(3) Dissolving the catechol gelatin prepared in the step (2) in MES buffer solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the solution after full dissolution, and performing activation after the pH of the solution is adjusted to 4.5-5.5;

(4) Dissolving 2-aminoethyl methacrylate hydrochloride in MES solution, dropwise adding the 2-aminoethyl methacrylate hydrochloride solution into the catechol-based gelatin solution obtained in the step (3), placing the mixed solution in a constant temperature shaking table, dialyzing after shaking in a dark place, and freeze-drying to obtain catechol-based photosensitive gelatin;

(5) Preparing catechol-based photosensitive gelatin obtained in the step (4) into an aqueous solution with the mass fraction of 5-20%, adding a photoinitiator, then dripping the solution into hydrogel molds with different shapes, and using ultraviolet light irradiation to cure the solution to obtain a sheet-shaped hydrogel dressing; or using a stereoscopic light projection 3D printer to prepare hydrogel dressings or stents of different shapes.

2. The method for using the hydrogel material with the secondary crosslinking property according to claim 1, wherein the mass fraction of the gelatin dissolved in the PBS solution in the step (1) is 0.5% -2%, the mass fraction of the catechol-based gelatin dissolved in the MES buffer solution obtained in the step (2) is 0.5% -2%, and the mass ratio of the dopamine hydrochloride to the gelatin in the mixed solution is 0.2-1:1.

3. the method according to claim 1, wherein the raw material gelatin used in the step (1) is replaced with alginate or hyaluronic acid, the mass fraction of the alginate or hyaluronic acid dissolved in the PBS solution is 0.05% -0.5% and 0.01% -0.3%, respectively, and the mass fraction of the catechol alginate or catechol hyaluronic acid dissolved in the MES buffer solution obtained in the step (2) is 0.05% -0.5% and 0.01% -0.3%, respectively.

4. The method for using the secondary crosslinking of the hydrogel material with the secondary crosslinking property according to claim 1 or 3, wherein the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride added in the step (1) and the step (3) to the N-hydroxysuccinimide is 5:3-1:1, wherein the mass concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the solution is 0.1-1%, and the activation time of the step (1) and the step (3) is 5-60min.

5. The method for using the hydrogel material with the secondary crosslinking property according to claim 1 or 3, wherein the dopamine hydrochloride in the step (2) is prepared into a PBS solution with a mass concentration of 5% -10%, the temperature of light-proof oscillation is 20-40 ℃, the oscillation time is 5-24 hours, the pore size of a dialysis bag is 3000Da-1000000Da, and the dialysis time is 24-48 hours.

6. The method according to claim 1 or 3, wherein in the step (4), the 2-aminoethylmethacrylate hydrochloride is dissolved in a MES solution having a mass concentration of 5 to 10%, and the mass ratio of the 2-aminoethylmethacrylate hydrochloride to catechol gelatin in the mixed solution is 0.2 to 1:1, the temperature of light-shielding oscillation is 20-40 ℃, the oscillation time is 5-24 hours, the pore space of the dialysis bag is 3000Da-1000000Da, and the dialysis time is 24-48 hours.

7. The method for using the secondary crosslinking of the hydrogel material with the secondary crosslinking property according to claim 1 or 3, wherein 365nm ultraviolet light is adopted in the step (5), the irradiation time is between 5 and 30 seconds, the photoinitiator is LAP or I2959, and the added mass concentration is between 0.1 and 1 percent.