CN112126027A

CN112126027A - Hydrogel material and preparation method and application thereof

Info

Publication number: CN112126027A
Application number: CN202011014747.4A
Authority: CN
Inventors: 王山峰; 张阳; 全大萍
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2020-09-24
Filing date: 2020-09-24
Publication date: 2020-12-25
Anticipated expiration: 2040-09-24
Also published as: CN112126027B

Abstract

The invention discloses a hydrogel material and a preparation method and application thereof. Wherein, the two preparation raw materials of acrylic anhydride modified collagen and polyethylene glycol diacrylate are combined and matched to realize performance complementation; specifically, the hydrogel material can be endowed with biological activity by inducing acrylic anhydride to modify collagen in the polyethylene glycol diacrylate, and the existence of the polyethylene glycol diacrylate can overcome the defect of insufficient mechanical property when the collagen is used alone; and the modified collagen can be formed by ultraviolet crosslinking and curing, the forming process is simple, the three-dimensional structure is not limited, and the application range is wide. Through the mode, the hydrogel material disclosed by the invention has excellent mechanical properties and biological activity.

Description

Hydrogel material and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological materials, in particular to a hydrogel material and a preparation method and application thereof.

Background

Biomaterial science, as a rapidly developing interdisciplinary discipline, puts urgent demands on the performance and manufacturing processes of various biomaterials. Tissue engineering requires biomaterials with sufficient and appropriate mechanical properties to meet various applications, such as hard and soft tissue replacement. Meanwhile, the biological material also has good biocompatibility. Chemical crosslinking, as opposed to physical crosslinking, can produce sufficient structural stability to meet the various properties required for biomedical applications, where photocrosslinking has gained widespread use due to a number of advantages.

The natural biological material has the characteristics of rich source, excellent biocompatibility, good degradation performance and the like, and is widely applied to the field of tissue engineering. However, the requirements of tissue engineering on material properties are high, so that the available natural biomaterials are very limited. In a limited selection, collagen, which is a major component of the extracellular matrix, has specific functional peptide sequences that bind to receptors on the plasma membrane to direct cell differentiation and other behaviors, and is an ideal material for constructing the cell growth microenvironment. However, the use of collagen currently has two problems, namely that the crosslinking is dependent on toxic or expensive chemical crosslinking agents; and secondly, the mechanical property of the single-component collagen system is insufficient after crosslinking, and different use requirements are difficult to meet.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a hydrogel material, and a preparation method and application thereof.

The technical scheme adopted by the invention is as follows:

according to the first aspect of the invention, the hydrogel material is provided, and the preparation raw materials comprise, by mass, 0.1-1% of acrylic anhydride modified collagen, 5-40% of polyethylene glycol diacrylate, 0.03-0.3% of a photoinitiator, and the balance of a solvent.

Wherein the acrylic anhydride modified collagen is acrylic anhydride modified collagen, and the acrylic anhydride modification degree (measured by a TNBS method) of the modified collagen is generally 0.08-0.38 mmol/g. And polyethylene glycol diacrylate with the molecular weight of 0.2-20 k is generally adopted.

According to some embodiments of the invention, the acrylic anhydride modified collagen is acrylic anhydride modified type II collagen.

According to some embodiments of the invention, the acrylic anhydride-modified collagen is prepared by a preparation method comprising the steps of: dissolving collagen in a first solvent to obtain a first collagen solution; and then adjusting the pH value of the first collagen solution to 7.0-8.0, adding acrylic anhydride, uniformly mixing, fully reacting, dialyzing, and freeze-drying.

According to some embodiments of the invention, the first solvent is an aqueous hydrochloric acid solution or an aqueous acetic acid solution.

According to some embodiments of the present invention, the polyethylene glycol diacrylate is prepared by a preparation method comprising the steps of: dissolving polyethylene glycol and potassium carbonate in a second solvent to obtain a mixed solution; dissolving acryloyl chloride in a second solvent, and then dropwise adding the solution into the mixed solution for reaction; filtering and taking supernatant after the reaction is finished, and removing most of the second solvent by rotary evaporation; then ether precipitation and washing are adopted, and then drying treatment is carried out.

According to some embodiments of the invention, the second solvent is anhydrous dichloromethane. As the photoinitiator, lithium phenyl-2, 4, 6-trimethylbenzoylphosphite (LAP) can be used.

According to some embodiments of the invention, the solvent is aqueous hydrochloric acid.

In a second aspect of the present invention, there is provided a method for preparing any one of the hydrogel materials provided in the first aspect of the present invention, comprising the steps of:

s1, dissolving the acrylic anhydride modified collagen in a solvent, adding a photoinitiator, and uniformly mixing to obtain a collagen solution;

s2, adding polyethylene glycol diacrylate into the collagen solution, and stirring and dissolving the mixture in a dark place to obtain a mixed solution;

and S3, carrying out photocrosslinking reaction on the mixed solution under ultraviolet light.

In a third aspect of the invention, there is provided a use of any one of the hydrogel materials provided in the first aspect of the invention in 3D printing.

The embodiment of the invention has the beneficial effects that:

the embodiment of the invention provides a hydrogel material, which can realize performance complementation by combining and matching two preparation raw materials of acrylic anhydride modified collagen and polyethylene glycol diacrylate. Specifically, the hydrogel material can be endowed with biological activity by introducing acrylic anhydride modified collagen into polyethylene glycol diacrylate, and the existence of the polyethylene glycol diacrylate can solve the problem of insufficient mechanical property when the collagen is used alone; in addition, the collagen modified by the acrylic anhydride (namely the collagen modified by the acrylic anhydride) can overcome the defect that the crosslinking of the collagen depends on toxic or expensive chemical crosslinking agents, and the modified collagen can be crosslinked and cured by ultraviolet light, so that the forming process is simple, the three-dimensional structure is not limited, and the application range is wide. Through the mode, the hydrogel material has excellent mechanical property and biological activity, and is expected to be used as a tissue engineering material for regeneration of bones, cartilages, skins, nerves and the like.

Drawings

FIG. 1 is a schematic diagram illustrating the synthesis of a hydrogel material according to one embodiment of the present invention;

FIG. 2 is a schematic diagram of electrophoresis of type II collagen used in the preparation of hydrogel materials in accordance with one embodiment of the present invention;

FIG. 3 shows the polyethylene glycol diacrylate prepared in examples 6 to 8 of the present invention¹H nuclear magnetic analysis chart;

FIG. 4 is a graph showing the infrared analysis of the polyethylene glycol diacrylate prepared in examples 6 to 8 of the present invention;

FIG. 5 is a graph showing photo-crosslinking performance analysis of a collagen solution in example 9 of the present invention;

FIG. 6 is a graph showing shear modulus analysis of hydrogel materials according to examples 9 to 12 of the present invention;

FIG. 7 is a graph showing cytotoxicity analysis of hydrogel materials according to examples 9 to 12 of the present invention;

FIG. 8 is a diagram showing cell proliferation analysis of hydrogel materials according to examples 9 to 12 of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

The invention provides a preparation method of a hydrogel material, a schematic preparation flow diagram of which is shown in figure 1, and the preparation method specifically comprises the following steps:

preparation of (I) acrylic anhydride modified type II collagen

The method is characterized in that II type collagen extracted from pig articular cartilage is taken as a raw material, the extracted II type collagen is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the purity and the molecular weight of the extracted II type collagen are characterized, and the obtained result is shown in figure 2.

As can be seen from FIG. 2, the obtained type II collagen has beta chain and gamma chain, only one alpha chain, and no staining band below 100kDa, which accords with the characteristics of type II collagen, and the purity of the separated type II collagen is high.

The specific method for modifying the type II collagen by the acrylic anhydride comprises the following steps:

s1, dissolving 0.25g type II Collagen (Collagen) in 100mL of 10mM HCl solution, and stirring at 4 ℃ until the Collagen is dissolved to obtain a first Collagen solution;

s2, adding 0.2M Na into the first collagen solution in a divided manner and slowly₂HPO₄Adjusting the pH value of the solution to 7.5;

s3, adding acrylic anhydride into the first collagen solution processed in the step S2 according to the addition amount shown in the table 1, uniformly stirring, and reacting for 8 hours;

s4, after the reaction is finished, the reaction solution is filled into a dialysis bag and dialyzed in 10mM HCl solution at 4 ℃ for three days, and the dialysis solution is replaced every 12 h.

And S5, and freeze-drying the obtained solution after dialysis to obtain white sponge-like solid, namely the acrylic anhydride modified type II Collagen (Collagen-AA).

The degree of substitution of the amino groups of collagen was measured by TNBS (2,4,6-Trinitrobenzenesulfonic acid) method for the acrylic anhydride-modified type II collagen synthesized as described above.

The TNBS method is specifically performed as follows;

1. solution to be tested: several samples to be tested and standard samples were dissolved in 10mM hydrochloric acid solution, 0.1M borate buffer solution with pH 8 was added, and 1M sodium hydroxide was used to adjust pH 8 to prepare a solution to be tested of 2.5 mg/mL.

2. Working solution: accurately weigh 60mg glycine and formulate 100mL of 8mM glycine solution with 0.1M borate buffered solution at pH 8; then, 0, 0.5mL, 1mL, 1.5mL, and 2mL of the solution were diluted to 10mL and prepared into glycine solutions with concentrations of 0, 0.4mM, 0.8mM, 1.2mM, and 1.6mM, respectively, for use.

3. And (3) absorbance measurement: respectively adding 1mL of solution to be detected or working solution and 1mL of 1% TNBS solution into each centrifuge tube with a plug, and reacting for 2h at 37 ℃; after the reaction is finished, adding 0.5mL of 1M hydrochloric acid solution into each centrifuge tube to stop the reaction, and adding 1mL of 10% sodium dodecyl sulfate solution to prevent the solution from precipitating; taking a plurality of solutions to be detected or working solutions in each centrifugal tube, and measuring absorbance at 345nm by using an enzyme-labeling instrument; and drawing a working curve of the amino content and the absorbance of the working solution, and calculating the amino content in the sample to be detected according to the absorbance of the solution to be detected, thereby indirectly calculating the amino substitution degree, wherein the obtained result is shown in table 1.

TABLE 1

Preparation of (di) polyethylene glycol diacrylate

The specific method comprises the following steps:

s1, drying polyethylene glycol (PEG) at 50 ℃ in vacuum overnight, K₂CO₃Grinding, vacuum drying at 100 deg.C overnight, and cooling in vacuum;

s2, 50g of PEG was dissolved in 100mL of anhydrous dichloromethane in a 500mL three-necked flask, and K was added in the amount shown in Table 2₂CO₃Fully stirring and dispersing;

s3, dissolving acryloyl chloride in anhydrous dichloromethane (1:10v/v) according to the addition amount shown in the table 2, dropwise and slowly adding the solution into a three-neck flask, and reacting at room temperature for 24 hours under the protection of nitrogen;

s4, filtering the mixture to remove solids, performing rotary evaporation on the supernatant at 37 ℃ to remove most of the solvent, fully precipitating with diethyl ether, washing with diethyl ether for 3-5 times, and finally completely drying in vacuum to obtain the product, namely the polyethylene glycol diacrylate (PEGDA).

TABLE 2

Sample (I)	Molecular weight of PEG	K₂CO₃Mass/g	Acryloyl chloride volume/mL
				Example 6	1.5k	13.8	8.1
Example 7	4k	5.2	3.0
				Example 8	10k	2.1	1.2

Dissolving the polyethylene glycol diacrylate prepared in examples 6 to 8 in deuterated chloroform¹The H NMR spectrum was characterized, and the obtained results are shown in FIG. 3. As can be seen from fig. 3, the chemical shifts can be well assigned to the corresponding protons, ═ 6.5, 6.2, 5.9(6H, H)₂C ═ CH-), 3.6(PEG chain protons).

In addition, a certain amount of the polyethylene glycol diacrylate prepared in the above examples 6 to 8 and potassium bromide ground tablets were subjected to Fourier infrared analysis and characterization, and the obtained results are shown in FIG. 4. The infrared absorption peak is 2883cm as can be seen from FIG. 4^-1(-CH_2-)，1724cm^-1(-C＝O)，1635cm^-1(H₂C＝CH-)。

(III) preparation of hydrogel Material

The preparation method comprises the following steps:

s1, adopting the acrylic anhydride modified II type collagen prepared in the embodiment 3, and violently stirring the modified II type collagen at 4 ℃ and dissolving the modified II type collagen in 20mM HCl to prepare a collagen solution with the mass concentration of 1%;

s2, 1mL of 1% collagen solution is added with 20mM HCl and 0.3% LAP solution accounting for 1/10 of the volume of the final solution to be diluted into collagen solutions with different concentrations (shown in Table 3);

s3, adding 200mg of the 1.5k polyethylene glycol diacrylate with the molecular weight prepared in the example 6 into the prepared collagen solution, and stirring and dissolving the mixture at 4 ℃ in dark to ensure that the mass concentration of the mixture is 10 percent;

s4, transferring the uniform transparent bubble-free mixed solution to silica gelPlacing in a mold, and placing in ultraviolet light (365nm, 4800 μ w/cm)²) Irradiating for 10min, and taking out the cross-linked slice after cooling to room temperature;

s5, soaking the hydrogel obtained by crosslinking in a PBS solution for two days, and changing the PBS one day to obtain the colorless transparent neutral hydrogel (PEGDA/Col-AA hydrogel).

TABLE 3

The photo-crosslinking performance characterization of the collagen solution with the above concentration of 0.25% was performed, and the obtained results are shown in fig. 5, where G' is the storage modulus and G ″ is the loss modulus. As can be seen from fig. 5, the collagen solution at this concentration can be crosslinked in response to ultraviolet light, and it can be understood that the acrylic anhydride-modified type II collagen at higher concentration in examples 10 to 16 can also be crosslinked in response to ultraviolet light.

The hydrogel materials prepared in examples 9 to 12 were prepared into disks with a diameter of 20mm and a thickness of 1mm, and the shear modulus was measured at 37 ℃ and γ of 1% at 0.1 to 100rad/s on a rotational rheometer, specifically, the storage modulus G' and the loss modulus G ″ were measured as a function curve of frequency, and the results are shown in fig. 6, where PEGDA indicates no collagen was added in fig. 6. As can be seen from fig. 6, the shear modulus of the hydrogel significantly increased as the content of the collagen component increased.

The hydrogel materials prepared in examples 9-12 were prepared into disks with a diameter of 5mm and a thickness of 1mm for cytotoxicity experiments. Specifically, by culturing murine mesenchymal stem cells in a 24-well plate at a density of about 20000 cells per square centimeter, the hydrogel was soaked in 1mL of the medium by trans-well without contact with the cells. The same density of cell seeding wells without hydrogel served as positive controls. CCK-8 assays were performed on days 1, 4 and 7 after inoculation. Absorbance at 450nm was measured by a microplate reader. Cell viability was calculated using the following formula: cell viability (%) ═ (OD)_{Test specimen}/OD_{Control sample}) X 100%. The results are shown in FIG. 7, in which the 0% group represents the PEGDA group to which no collagen was added. From FIG. 7As can be seen, none of the hydrogel materials prepared in examples 9-12 above were cytotoxic.

Cell proliferation experiments were performed on the hydrogel materials obtained in examples 9 to 12, and specifically, murine bone marrow mesenchymal stem cells were seeded on the hydrogel surface in a 48-well plate at a density of about 20000 cells per square centimeter. CCK-8 assays were performed at 4h and 1, 4 and 7 days after inoculation. Specifically, absorbance at 450nm was measured using a microplate reader, and the cell proliferation rate was calculated using the following formula: cell proliferation rate (%) ═ (OD)_sample/OD_original) X 100% where OD_originalIs the absorbance, OD, corresponding to 4h of culture on the surface of the hydrogel_sampleThe absorbance of the cells was measured for 1, 4 and 7 days after culturing on the hydrogel surface. The results are shown in FIG. 8, in which the 0% group represents the PEGDA group without collagen addition. As can be seen from fig. 8, the cells proliferated more in the collagen-containing group at 4 days and 7 days, compared to the collagen-free PEGDA group.

From the above, the hydrogel material prepared by the above method of the present invention has excellent mechanical properties and biological activity, and is expected to be used as a tissue engineering material for bone, cartilage, skin, nerve regeneration, etc.; and can be used as 3D printing ink or used for preparing 3D printing ink, and further used for 3D printing.

Claims

1. The hydrogel material is characterized by comprising the following preparation raw materials in percentage by mass: 0.1-1% of acrylic anhydride modified collagen, 5-40% of polyethylene glycol diacrylate, 0.03-0.3% of photoinitiator and the balance of solvent.

2. The hydrogel material of claim 1, wherein the acrylic anhydride modified collagen is acrylic anhydride modified type II collagen.

3. The hydrogel material according to claim 1, wherein the modification degree of the acrylic anhydride-modified collagen is 0.08 to 0.38 mmol/g.

4. The hydrogel material according to claim 1, wherein the acrylic anhydride modified collagen is prepared by a preparation method comprising the steps of: dissolving collagen in a first solvent to obtain a first collagen solution; and then adjusting the pH value of the first collagen solution to 7.0-8.0, adding acrylic anhydride, uniformly mixing, fully reacting, dialyzing, and freeze-drying.

5. The hydrogel material of claim 4, wherein the first solvent is an aqueous hydrochloric acid solution or an aqueous acetic acid solution.

6. The hydrogel material of claim 1, wherein the polyethylene glycol diacrylate is prepared by a preparation method comprising the steps of: dissolving polyethylene glycol and potassium carbonate in a second solvent to obtain a mixed solution; dissolving acryloyl chloride in a second solvent, and then dropwise adding the solution into the mixed solution for reaction; filtering and taking supernatant after the reaction is finished, and removing most of the second solvent by rotary evaporation; then ether precipitation and washing are adopted, and then drying treatment is carried out.

7. The hydrogel material of claim 6, wherein the second solvent is anhydrous dichloromethane.

8. The hydrogel material according to any one of claims 1 to 7, wherein the solvent is an aqueous hydrochloric acid solution.

9. The method for preparing a hydrogel material according to any one of claims 1 to 8, comprising the steps of:

10. Use of the hydrogel material of any one of claims 1 to 8 in 3D printing.