CN113683787A

CN113683787A - Hydrogel material with secondary crosslinking characteristic and preparation method and application thereof

Info

Publication number: CN113683787A
Application number: CN202110892529.9A
Authority: CN
Inventors: 方慧敏; 汪振星; 孙家明; 陈雳风; 刘绍恺; 罗超; 牟珊; 侯金飞; 李嘉伦; 谢昕芳; 张郭; 孙谛; 王冰倩; 李志鹏; 赵阳; 姜文彬; 郭亚琪
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2021-08-04
Filing date: 2021-08-04
Publication date: 2021-11-23
Anticipated expiration: 2041-08-04
Also published as: CN113683787B

Abstract

The invention provides a hydrogel material with secondary crosslinking characteristic, a preparation method and application thereof. After the hydrogel material prepared by the invention is prepared into a solution, the photo-curing crosslinking can be realized by utilizing the photosensitive groups in the hydrogel molecules to prepare a specific shape, and the catechol groups in the hydrogel molecules can be utilized to crosslink again to realize the adhesion between the hydrogel and tissues or the splicing between hydrogels with different shapes. The hydrogel capable of being secondarily crosslinked has great application value in the fields of preparing wound dressings, conductive biosensors, tissue engineering scaffolds and the like.

Description

Hydrogel material with secondary crosslinking characteristic and preparation method and application thereof

Technical Field

The invention relates to the technical field of preparation of high-molecular hydrogel, in particular to a hydrogel material with secondary crosslinking characteristic and a preparation method and application thereof.

Background

Skin incisions caused by trauma or surgery are easy to generate hyperplastic or sunken scars after surgical suture, and the appearance is affected. The use of tissue glue instead of surgical sutures can reduce scarring of the skin. There are some products of suture-free tissue glue that are used to assist in the adhesion care during the suturing of surgical and cosmetic wounds. Such as blue Tissue Adhesive (Histoacryl Tissue Adhesive), 3M Tissue Adhesive (Vetbond), which is mainly composed of n-butyl 2-cyanoacrylate (enbuester), and stabilizer (hydroquinone, sulfur dioxide, phosphoric acid). Such tissue glues have certain disadvantages: firstly, doctors are required to manually coat the surfaces of the incisions, and uniform coating is difficult to realize manually; ② it takes several minutes to wait for the glue to change from solution to solid; thirdly, the biocompatibility of the matrix material of the glue is poor. Therefore, how to prepare a hydrogel material which can be formed by pre-crosslinking and can be tightly adhered to the skin again is a problem to be solved in clinic.

In the tissue engineering research, the uniform loading of seed cells can be realized by tissue engineering micro-tissues, and the micro-tissues are constructed in a hot emerging field, but in the current research, the micro-tissues are mostly directly placed at tissue defect parts, and the micro-tissues are not orderly assembled. Therefore, if the hydrogel can be firstly prepared into the micro-tissues and then the secondary crosslinking characteristics of the hydrogel are utilized to arrange different types of micro-tissues in order, the problem of assembling the micro-tissues is expected to be solved, and a new thought is provided for tissue engineering research.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a hydrogel material with secondary crosslinking characteristics, and a preparation method and application thereof.

The technical scheme provided by the invention is as follows: a hydrogel material with secondary crosslinking characteristics is prepared by simultaneously grafting catechol group and photosensitive group on any biomacromolecule of gelatin, alginate and hyaluronic acid to obtain catechol photosensitive macromolecules, dissolving and then carrying out photocrosslinking.

A preparation method of a hydrogel material with secondary crosslinking characteristics comprises the following steps:

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

(2) dissolving dopamine hydrochloride in a 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, oscillating in the dark, 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 activating the solution after the pH value 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 gelatin solution obtained in the step (3), placing the mixed solution in a constant-temperature shaking table, oscillating in the dark, dialyzing, and freeze-drying to obtain catechol photosensitive gelatin;

(5) preparing the catechol-based photosensitive gelatin obtained in the step (4) into an aqueous solution with the mass fraction of 5-20%, adding a photoinitiator, dropwise adding the solution into hydrogel molds with different shapes, and irradiating by using ultraviolet light to solidify the solution to obtain the sheet hydrogel dressing; or a three-dimensional light projection 3D printer is used for preparing hydrogel dressings or scaffolds with different shapes.

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

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

Further, the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide added in the step (1) and the step (3) is 5: 3-1: 1, 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 5-60 min.

Further, in the step (2), the dopamine hydrochloride is prepared into 5-10% mass concentration in PBS solution, the temperature of vibration in a dark place is 20-40 ℃, the vibration time is 5-24 hours, the pore space of a dialysis bag is 3000Da-1000000Da, and the dialysis time is 24-48 hours.

Further, in the step (4), the 2-aminoethyl methacrylate hydrochloride is dissolved in MES solution to prepare a mixture with a mass concentration of 5-10%, and the mass ratio of the 2-aminoethyl methacrylate hydrochloride to the catechol gelatin is 0.2-1: 1, the temperature of vibration in dark is 20-40 ℃, the vibration time is 5-24 hours, the pore space of the dialysis bag is 3000Da-1000000Da, and the dialysis time is 24-48 hours.

Further, 365nm ultraviolet light is adopted for irradiation 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.

The secondary crosslinking using method of the hydrogel material with the secondary crosslinking characteristic obtained by the preparation method comprises the steps of preparing 0.1-1mM/L ferric chloride solution or 0.1-1mM/L sodium periodate solution, thinly coating a layer of solution on the skin, and then attaching the hydrogel material to the surface of the skin, so that the tight adhesion can be realized; or the two hydrogels are stuck together, and ferric chloride solution or sodium periodate solution is dripped on the surfaces of the two hydrogels to realize tight adhesion.

The application of the hydrogel material with the secondary crosslinking characteristic utilizes a photocuring 3D printing technology to prepare a millimeter-scale hydrogel microcell by using the hydrogel material; by utilizing the secondary crosslinking characteristic of the hydrogel, splicing among a plurality of hydrogel microcells and tight adhesion between the hydrogel and tissues are achieved, and accurate repair of the defect part is realized; or by utilizing a photocuring 3D printing technology, the hydrogel material is prepared into the skin wound dressing with a specific shape in advance, and the hydrogel is tightly adhered to the skin by utilizing the secondary crosslinking characteristic of the hydrogel, so that the effect of covering the wound and promoting skin repair is achieved.

The hydrogel material prepared by the invention has secondary crosslinking characteristics, and after the material is prepared into a solution, the material can firstly realize photocuring crosslinking by utilizing photosensitive groups in hydrogel molecules to prepare a specific shape, and can also realize adhesion between the hydrogel and tissues or splicing between hydrogels with different shapes by utilizing catechol groups in the hydrogel molecules to perform crosslinking again. The hydrogel capable of being secondarily crosslinked has great application value in the fields of preparing wound dressings, conductive biosensors, tissue engineering scaffolds and the like.

Drawings

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

FIG. 2 is a physical diagram of the chemical synthesis steps and corresponding products of the present invention;

FIG. 3 is a graph showing the results of NMR spectroscopy of hydrogel materials of the present invention;

FIG. 4 is a graph of Fourier transform infrared spectra of hydrogel materials of the present invention;

FIG. 5 is a diagram of a tissue engineering chamber model of hydrogel material printed by a 3D printer according to the present invention;

FIG. 6 is a graph showing the effect of the hydrogel material of the present invention after secondary crosslinking;

FIG. 7 is a graph showing the effect of adhesion of the hydrogel material of the present invention to tissues;

FIG. 8 is an electron micrograph of hydrogel materials prepared using dialysis bags of different molecular weights in accordance with the present invention;

FIG. 9 is a graph of the in vitro degradation rates of hydrogel materials prepared using dialysis bags of different molecular weights in accordance with the present invention;

FIG. 10 is a graph showing the results of measurement of swelling of hydrogel materials in physiological saline;

FIG. 11 is a graph showing the results of hardness measurements of hydrogel materials;

FIG. 12 is a graph showing the results of elasticity measurements of hydrogel materials;

FIG. 13 is a cytooptic microscope photograph of photocrosslinking of a hydrogel material after mixing with bone marrow mesenchymal stem cells (BMSCs);

FIG. 14 is a cytooptic photograph of photocrosslinking of hydrogel material after mixing with Human Umbilical Vein Endothelial Cells (HUVEC);

FIG. 15 is a graph showing the survival of cells observed after 1 day and 5 days of incubation after photocrosslinking of hydrogel material mixed with HUVEC;

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

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

Dissolving gelatin in PBS solution to obtain a solution with a mass fraction of 2%, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the solution after fully dissolving, wherein the mass of EDC/NHS is 5/3, the mass concentration of EDC in the solution is 0.5%, adjusting the pH of the solution to 5, and activating for 30 minutes. Preparing dopamine hydrochloride into a solution with the mass fraction of 10% in a PBS (phosphate buffer solution), dropwise adding the dopamine hydrochloride solution into a gelatin solution, wherein 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 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size 3500Da) for 36 hours and changed 6 times. And (4) freeze-drying to obtain the catechol gelatin.

Dissolving the prepared catechol gelatin in MES buffer solution to prepare a solution with the mass fraction of 1%, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the solution after full dissolution, wherein the mass of the EDC/NHS is 5/3, the mass concentration of the EDC in the solution is 1%, adjusting the pH of the solution to 5, and activating for 30 minutes. Preparing 2-aminoethyl methacrylate hydrochloride (AEMA) into a MES solution to obtain a solution with the mass fraction of 5%, dropwise adding the 2-aminoethyl methacrylate hydrochloride into a catechol gelatin solution, wherein the mass ratio of the 2-aminoethyl methacrylate hydrochloride to the catechol gelatin is 0.5: 1. the mixed solution was placed in a constant temperature shaker and shaken overnight at 25 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size 3500Da) for 36 hours, with changes every 6 hours. And (3) freeze-drying to prepare the catechol-based photosensitive gelatin with mixed molecular weight.

Dripping 15% of catechol-based photosensitive gelatin solution into hydrogel molds of different shapes, adding 0.5% of photoinitiator phenyl-2, 4, 6-trimethyl benzoyl lithium phosphite (LAP), irradiating by 365nm ultraviolet light for 20 seconds, and solidifying the solution to obtain a sheet hydrogel dressing; or a three-dimensional light projection 3D printer is used for preparing hydrogel dressings or scaffolds with different shapes.

Example 2

Dissolving gelatin in PBS solution to obtain a solution with a mass fraction of 2%, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the solution after fully dissolving, wherein the mass of EDC/NHS is 5/3, the mass concentration of EDC in the solution is 0.5%, adjusting the pH of the solution to 5, and activating for 30 minutes. And preparing the dopamine hydrochloride into a solution with the mass fraction of 10% in a PBS solution. Dropwise adding the dopamine hydrochloride solution into the gelatin solution, wherein 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 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size 1000000Da) for 36 hours and changed 6 times. And (4) freeze-drying to obtain the catechol gelatin.

Dissolving the prepared catechol gelatin in MES buffer solution to prepare a solution with the mass fraction of 1%, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the solution after full dissolution, wherein the mass of the EDC/NHS is 5/3, the mass concentration of the EDC in the solution is 1%, adjusting the pH of the solution to 5, and activating for 30 minutes. Preparing 2-aminoethyl methacrylate hydrochloride (AEMA) into a MES solution to obtain a solution with the mass fraction of 5%, dropwise adding the 2-aminoethyl methacrylate hydrochloride into a catechol gelatin solution, wherein the mass ratio of the 2-aminoethyl methacrylate hydrochloride to the catechol gelatin is 0.5: 1. the mixed solution was placed in a constant temperature shaker and shaken overnight at 25 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size 1000000Da) for 36 hours, with changes every 6 hours. And freeze-drying to obtain the high-molecular-weight catechol-based photosensitive gelatin.

Dripping 15% of catechol-based photosensitive gelatin solution into hydrogel molds with different shapes, adding 0.5% of photoinitiator phenyl-2, 4, 6-trimethyl benzoyl lithium phosphite (LAP), irradiating by using 365nm ultraviolet light for 20 seconds, and solidifying the solution to obtain a sheet hydrogel dressing; or a three-dimensional light projection 3D printer is used for preparing hydrogel dressings or scaffolds with different shapes.

In examples 1 and 2, o-catechol-based photosensitive gelatin of different molecular weights, i.e., 3500Da and 1000kDa GDMA in experimental detection results, was prepared

Example 3

Dissolving gelatin in PBS solution to obtain a solution with a mass fraction of 2%, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the solution after fully dissolving, wherein the mass of EDC/NHS is 5/3, the mass concentration of EDC in the solution is 0.5%, adjusting the pH of the solution to 5, and activating for 30 minutes. Preparing dopamine hydrochloride into a solution with the mass fraction of 10% in PBS (phosphate buffer solution), dropwise adding the dopamine hydrochloride solution into a gelatin solution, wherein 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 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size 3500Da) for 36 hours and changed 6 times. And (4) freeze-drying to obtain the catechol gelatin.

Dissolving the prepared catechol gelatin in MES buffer solution to prepare a solution with the mass fraction of 1%, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the solution after full dissolution, wherein the mass of the EDC/NHS is 5/3, the mass concentration of the EDC in the solution is 1%, adjusting the pH of the solution to 5, and activating for 30 minutes. Preparing 2-aminoethyl methacrylate hydrochloride (AEMA) into a MES solution to prepare a solution with the mass fraction of 5%, dropwise adding the 2-aminoethyl methacrylate hydrochloride into a catechol gelatin solution, wherein the mass ratio of the 2-aminoethyl methacrylate hydrochloride to the catechol gelatin is 0.3: 1. the mixed solution was placed in a constant temperature shaker and shaken overnight at 25 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size 3500Da) for 36 hours, with changes every 6 hours. And freeze-drying to obtain the high-molecular-weight catechol-based photosensitive gelatin. Dripping 15% of catechol-based photosensitive gelatin solution into hydrogel molds with different shapes, adding 0.5% of photoinitiator phenyl-2, 4, 6-trimethyl benzoyl lithium phosphite (LAP), irradiating by using 365nm ultraviolet light for 20 seconds, and solidifying the solution to obtain a sheet hydrogel dressing; or a three-dimensional light projection 3D printer is used for preparing hydrogel dressings or scaffolds with different shapes.

Example 3 the concentration of dopamine hydrochloride and 2-aminoethyl methacrylate hydrochloride was reduced compared to example 1 and the grafting yield of the reaction product was reduced.

Example 4

Dissolving sodium alginate in PBS solution to obtain 0.2 wt% solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) after fully dissolving, wherein the mass of EDC/NHS is 3/2, the concentration of EDC in the solution is 0.2%, adjusting the pH of the solution to 5, and activating for 10 min. Dopamine hydrochloride is prepared into 8% concentration in PBS solution, and the dopamine hydrochloride solution is dropwise added into the alginate solution. The mixed solution was placed in a constant temperature shaker and shaken overnight at 35 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size at 3000Da) for 48 hours, with changes every 6 hours. And (5) freeze-drying to obtain the o-catechol sodium alginate.

Dissolving the prepared catechol sodium alginate in MES buffer solution to prepare a solution with the mass fraction of 0.2%, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the solution after full dissolution, wherein the mass of the EDC/NHS is 3/2, the concentration of the EDC in the solution is 1%, adjusting the pH of the solution to 5, and activating for 50 minutes. Preparing 5 mass percent solution of 2-aminoethyl methacrylate hydrochloride (AEMA) in MES solution, and dropwise adding the 2-aminoethyl methacrylate hydrochloride solution into the o-catechol alginate solution. The mixed solution was placed in a constant temperature shaker and shaken overnight at 35 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size at 3000Da) for 48 hours, with changes every 6 hours. And (4) freeze-drying to obtain the catechol photosensitive alginate.

Dripping a catechol sodium alginate solution with the concentration of 3% into hydrogel molds with different shapes, adding a photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP) with the concentration of 0.5%, irradiating by using 365nm ultraviolet light for 30 seconds, and solidifying the solution to obtain a sheet hydrogel dressing; or a three-dimensional light projection 3D printer is used for preparing hydrogel dressings or scaffolds with different shapes.

Example 5

Dissolving hyaluronic acid in PBS solution to obtain 0.1 wt% solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) after fully dissolving, wherein the mass of EDC/NHS is 1/1, the concentration of EDC in the solution is 0.2%, adjusting the pH of the solution to 4.5, and activating for 40 min. Dopamine hydrochloride is taken to be prepared into a PBS solution with the concentration of 10 percent, and the dopamine hydrochloride solution is dropwise added into the hyaluronic acid solution. The mixed solution was placed in a constant temperature shaker and shaken overnight at 20 ℃ in the dark. The solution was dialyzed using dialysis bags (pore size between 3000Da) for 24 hours, with changes every 6 hours. And (4) freeze-drying to obtain the catechol hyaluronic acid.

The prepared catechol hyaluronic acid was dissolved in 100mM/L MES buffer solution to prepare a 0.1% solution, after sufficient dissolution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the solution, wherein the mass of EDC/NHS was 1/1, the concentration of EDC in the solution was 0.2%, the pH of the solution was adjusted to 4.5, and activation was carried out for 40 minutes. Preparing a solution with the mass fraction of 10% by taking 2-aminoethyl methacrylate hydrochloride (AEMA) in a MES solution, and dropwise adding the 2-aminoethyl methacrylate hydrochloride solution into the catechol hyaluronic acid solution. The mixed solution was placed in a constant temperature shaker and shaken overnight at 20 ℃ in the dark. The solution was dialyzed using dialysis bags (pore 10000Da) for 24 hours, changing every 6 hours. And (3) freeze-drying to obtain the catechol-based photosensitive hyaluronic acid.

Dripping a catechol-based photosensitive hyaluronic acid solution with the concentration of 2% into hydrogel molds with different shapes, adding a photoinitiator phenyl-2, 4, 6-trimethyl benzoyl lithium phosphite (LAP) with the concentration of 0.5%, irradiating by using 365nm ultraviolet light for 20 seconds, and solidifying the solution to obtain a sheet hydrogel dressing; or a three-dimensional light projection 3D printer is used for preparing hydrogel dressings or scaffolds with different shapes.

GD-MA hydrogel material detection

Dissolving raw materials and products in heavy water, scanning hydrogen spectrum (instrument model: Bruker 600MHz) with a nuclear magnetic resonance spectrometer to obtain absorption peaks of different materials, wherein the absorption peaks of GD-MA are overlapped with dopamine and gelatin, which shows that GD is synthesized by the two raw materials, namely, catechol groups are grafted on the side chain of the gelatin; the characteristic peak shows that the hydrogel is successfully prepared; the result of GD-MA NMR is shown in FIG. 3.

Grinding and tabletting raw materials and products in potassium bromide, and performing Fourier infrared spectrum detection (instrument model: Thermo Scientific Nicolet 6700), wherein characteristic peaks show that a catechol group and a methacrylic acid ester bond on a gelatin side chain are grafted by an amido bond reaction. The GD-MA Fourier transform infrared spectrum is shown in FIG. 4, and the peak at 1640cm-1 indicates the formation of the amide bond (HNCO). The absorption peak at 1300nm may be changed by the deformation vibration of the C-H stretching vibration of 3500-3100N-H stretching vibration 1680-1630C ═ O stretching vibration 1655-H bending vibration 3100-3000 alkene, and the deformation vibration of the methyl group.

The GD-MA has excellent photosensitive characteristics, tissue engineering chamber models with different shapes can be prepared by using a three-dimensional light projection 3D printer, as shown in figure 5 (instrument: engineering for life photocuring biological 3D printer BP8600), and the GD-MA has good printing performance and can print precise structures.

As shown in fig. 6, after the GD-MA is photocured, a sodium periodate solution is dripped on the hydrogel, the GD-MA hydrogel turns orange yellow, and the contact surface is tightly adhered; after the ferric oxide solution is dripped into the hydrogel, the GD-MA hydrogel turns blue, and the contact surface is tightly adhered. The GD-MA hydrogel is shown to be capable of realizing secondary crosslinking through a catechol group on a side chain of the GD-MA hydrogel after photocrosslinking.

As shown in fig. 7, the adhesion of GD-MA hydrogel to tissue was tightly adhered by secondary crosslinking, tight adhesion of hydrogel and tissue was demonstrated by twist folding, tight adhesion assembly between hydrogels was demonstrated by stacking hydrogel sheets, soaking in PBS for 30 minutes, and tight assembly of hydrogel was demonstrated by twist folding.

As shown in FIG. 8, GD-MA materials with two molecular weights are prepared by using dialysis bags with different molecular weights, and are prepared into hydrogels with different concentrations for electron microscope observation, and the GD-MA hydrogel with large molecular weight prepared by using 1000kDa dialysis has larger and more uniform pores and is probably more favorable for cell growth

As shown in fig. 9, in vitro degradation was seen upon PBS soaking at 37 ℃, 3w of data was recorded, and it was judged from the data that the in vitro degradation rate of large molecular weight GD-MA was faster, possibly associated with high porosity. (failure to weigh at week 4 due to hydrogel degradation)

As shown in FIG. 10, when the GD-MA hydrogel was tested for swelling in physiological saline, the hydrogel was not swollen significantly, and the shape was maintained well.

As shown in FIGS. 11 and 12, the GD-MA concentration increased, the hydrogel stiffness also increased, and the 12% GD-MA material had better elasticity.

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

The hydrogel prepared by the invention has printability and biological adhesion effect; as shown in fig. 16, a millimeter-sized hydrogel tissue engineering chamber was then prepared using the above materials using DLP photocuring 3D printing techniques; then the tissue engineering micro-tissue cultured by the bioreactor is subjected to first-level assembly and filling to form a micro-unit; finally, the biological adhesiveness of the hydrogel is utilized to carry out two-stage assembly and splicing on the plurality of tissue engineering room micro units, so that the precise repair of the defect part can be realized

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A hydrogel material having secondary crosslinking characteristics, characterized in that: the hydrogel material is prepared by grafting catechol group and photosensitive group on any biomacromolecule of gelatin, alginate and hyaluronic acid simultaneously to obtain catechol photosensitive macromolecules, dissolving and then carrying out photo-crosslinking.

2. A preparation method of a hydrogel material with secondary crosslinking characteristics is characterized by comprising the following steps:

3. The method for preparing a hydrogel material with secondary crosslinking characteristics as claimed in claim 2, 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 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.

4. the method for preparing a hydrogel material with secondary crosslinking characteristics as claimed in claim 2, wherein: the raw material gelatin adopted in the step (1) can be replaced by alginate or hyaluronic acid, the mass fractions of the alginate or the hyaluronic acid dissolved in the PBS solution are 0.05% -0.5% and 0.01% -0.3%, and the mass fractions of the catechol-based alginate or the catechol-based hyaluronic acid dissolved in the MES buffer solution obtained in the step (2) are 0.05% -0.5% and 0.01% -0.3%, respectively.

5. The method for preparing a hydrogel material with secondary crosslinking characteristics according to claim 2 or 4, wherein: the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide added in the steps (1) and (3) is 5: 3-1: 1, 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 5-60 min.

6. The method for preparing a hydrogel material with secondary crosslinking characteristics according to claim 2 or 4, wherein: in the step (2), the dopamine hydrochloride is prepared into 5-10% mass concentration in PBS solution, the temperature of vibration in a dark place is 20-40 ℃, the vibration time is 5-24 hours, the pore space of a dialysis bag is 3000Da-1000000Da, and the dialysis time is 24-48 hours.

7. The method for preparing a hydrogel material with secondary crosslinking characteristics according to claim 2 or 4, wherein: in the step (4), the 2-aminoethyl methacrylate hydrochloride is dissolved in MES solution to prepare a mixture with a mass concentration of 5-10%, and the mass ratio of the 2-aminoethyl methacrylate hydrochloride to the catechol gelatin in the mixed solution is 0.2-1: 1, the temperature of vibration in dark is 20-40 ℃, the vibration time is 5-24 hours, the pore space of the dialysis bag is 3000Da-1000000Da, and the dialysis time is 24-48 hours.

8. The method for preparing a hydrogel material with secondary crosslinking characteristics according to claim 2 or 4, wherein: 365nm ultraviolet light is adopted for irradiation in the step (5), the irradiation time is 5-30 seconds, the photoinitiator is LAP or I2959, and the added mass concentration is 0.1-1%.

9. The method for using a hydrogel material having secondary crosslinking characteristics obtained by the production method according to claim 2 or 4, wherein: preparing 0.1-1mM/L ferric chloride solution or 0.1-1mM/L sodium periodate solution, thinly coating a layer of solution on the skin, and then attaching the hydrogel material on the surface of the skin to realize tight adhesion; or the two hydrogels are stuck together, and ferric chloride solution or sodium periodate solution is dripped on the surfaces of the two hydrogels to realize tight adhesion.

10. The use of a hydrogel material having secondary crosslinking properties according to claim 1, wherein: utilizing a photocuring 3D printing technology to prepare a millimeter-scale hydrogel microcell by using a hydrogel material; by utilizing the secondary crosslinking characteristic of the hydrogel, splicing among a plurality of hydrogel microcells and tight adhesion between the hydrogel and tissues are achieved, and accurate repair of the defect part is realized; or by utilizing a photocuring 3D printing technology, the hydrogel material is prepared into the skin wound dressing with a specific shape in advance, and the hydrogel is tightly adhered to the skin by utilizing the secondary crosslinking characteristic of the hydrogel, so that the effect of covering the wound and promoting skin repair is achieved.