阅读说明：本技术 一种高力学强度聚乙烯醇-丙烯酰胺-琼脂糖水凝胶的制备方法 (Preparation method of polyvinyl alcohol-acrylamide-agarose hydrogel with high mechanical strength ) 是由 张伟 李津铭 李姮 吴承伟 于 2021-08-27 设计创作，主要内容包括：本发明属于关节软骨替代材料领域,提供了一种高力学强度聚乙烯醇-丙烯酰胺-琼脂糖水凝胶的制备方法。以聚乙烯醇和丙烯酰胺为基质材料,加入少量琼脂糖。丙烯酰胺通过化学交联剂先形成第一网络,聚乙烯醇在冷冻循环中物理交联形成第二网络。琼脂糖可以与聚乙烯醇和丙烯酰胺的分子链形成氢键,进一步提高水凝胶的机械强度。此外,琼脂糖的引入使水凝胶能够粘附软骨细胞并促进软骨细胞增殖。该方法制备的水凝胶有望应用于关节软骨置换。(The invention belongs to the field of articular cartilage substitute materials, and provides a preparation method of polyvinyl alcohol-acrylamide-agarose hydrogel with high mechanical strength. Polyvinyl alcohol and acrylamide are used as matrix materials, and a small amount of agarose is added. Acrylamide forms a first network through a chemical cross-linking agent, and polyvinyl alcohol forms a second network through physical cross-linking in a refrigeration cycle. The agarose can form hydrogen bonds with molecular chains of polyvinyl alcohol and acrylamide, so that the mechanical strength of the hydrogel is further improved. Furthermore, the introduction of agarose enables the hydrogel to adhere to chondrocytes and promote chondrocyte proliferation. The hydrogel prepared by the method is expected to be applied to articular cartilage replacement.)

1. A preparation method of polyvinyl alcohol-acrylamide-agarose hydrogel with high mechanical strength is characterized by comprising the following steps:

firstly, adding polyvinyl alcohol, acrylamide, agarose and a chemical cross-linking agent N, N' -methylene bisacrylamide into deionized water according to a proportion, heating in a constant-temperature water bath at 90-99 ℃, and continuously stirring until the polyvinyl alcohol is completely dissolved and the rest substances are uniformly mixed to obtain a mixed solution A; in the mixed solution A, the mass fraction of polyvinyl alcohol is 10-20 wt.%, the mass fraction of acrylamide is 10-20 wt.%, the mass fraction of N, N' -methylene bisacrylamide is 0.1-0.5 wt.%, and the mass fraction of agarose is 0.5-5 wt.%;

step two, placing the mixed solution A at room temperature, continuously stirring, and cooling to 20-50 ℃ for later use; adding tetramethylethylenediamine into the mixed solution A, continuously stirring until the mixture is uniformly mixed, and then adding a saturated ammonium persulfate aqueous solution in an ice-water bath environment to obtain a mixed solution B; adding 100-400 mu L of tetramethylethylenediamine and 3-6 mL of saturated ammonium persulfate solution into every 100mL of the mixed solution A;

and step three, pouring the mixed solution B into a mold, and performing freezing and unfreezing circulation on the mixed solution B to obtain the polyvinyl alcohol-acrylamide-agarose hydrogel.

2. The preparation method according to claim 1, wherein the freeze-thaw cycle is to take out the mixed solution B after freezing and forming, and to thaw the mixed solution B, and to repeat the process for a plurality of times until the PVA-AAm-AG hydrogel is formed; the freezing temperature is-18 to-25 ℃, the freezing time is 12 to 20 hours, the unfreezing temperature is 15 to 25 ℃, and the unfreezing time is 6 to 12 hours; circulating for more than 2 times.

Technical Field

The invention belongs to the field of biological materials, particularly relates to the field of substitute materials for articular cartilage injury, and particularly relates to a preparation method of polyvinyl alcohol-acrylamide-agarose hydrogel with high mechanical strength.

Background

The hydrogel material has the characteristics of high water content, low friction coefficient, good chemical stability and the like, and is expected to be used for repairing biological tissue damage. However, the traditional hydrogel is usually composed of a single network of hydrophilic polymers, has poor mechanical properties, is difficult to meet the requirement of high mechanical strength of articular cartilage, and limits the application of the hydrogel in the field of articular cartilage substitute materials. At the same time, it must also have good biocompatibility as an articular cartilage replacement material. The biocompatibility of the hydrogel material can be effectively improved by adding a large amount of bioactive components (such as gelatin, collagen, growth factors and the like) into the hydrogel, but the introduction of the materials generally causes the mechanical properties of the hydrogel to be greatly reduced. The development of hydrogel materials with both excellent mechanical properties and good biocompatibility remains a great challenge.

The previous research shows that the double-network structure can effectively improve the mechanical property of the hydrogel. The polyvinyl alcohol hydrogel has relatively high mechanical properties, and acrylamide contains a large amount of acylamino in the structure, so that hydrogen bonds are easily formed, and the acrylamide is suitable to be used as a component of a covalent cross-linking network, so that a polyvinyl alcohol-acrylamide (PVA-AAm) double-network hydrogel system becomes a hotspot in the field of developing high-mechanical-strength hydrogels. Although the double-network structure can improve the mechanical properties of the hydrogel material, the mechanical properties of the conventional PVA-AAm double-network hydrogel are still different from those of articular cartilage (Journal of Materials Science,2019,54, 3368-3382; Journal of Materials Chemistry A,2020, 8, 6776-6784; Materials Letters,2018,207, 53-56). As a substitute material of articular cartilage materials, the mechanical property needs to simultaneously meet the following conditions: compressive modulus >0.53MPa, compressive strength >4MPa, tensile strength >0.8 MPa. Furthermore, the biocompatibility of these polyvinyl alcohol-acrylamide hydrogel materials reported so far is still unclear.

The invention introduces Agarose (AG) into a polyvinyl alcohol-acrylamide hydrogel system, and the effect of the Agarose (AG) comprises two aspects: (1) the agarose can generate strong hydrogen bond action with polyvinyl alcohol and acrylamide macromolecular chains in the hydrogel, so that the mechanical property of the hydrogel is improved, and (2) the agarose has good biocompatibility, so that the adhesion capability of the hydrogel to cells can be improved by introducing the agarose, and the biocompatibility of the hydrogel is promoted. The hydrogel disclosed by the invention has excellent mechanical properties in both compression and tension, has a compression modulus of 0.9MPa and a compression limit of 8.17MPa, and simultaneously has a Young modulus of 0.97MPa and a tension limit of 2.45MPa, so that the performance requirements of the articular cartilage substitute material are met. In addition, cell experiments show that the developed hydrogel has good chondrocyte adhesion and the capacity of promoting cell proliferation. The hydrogel is expected to be applied to substitute materials for articular cartilage injuries.

Disclosure of Invention

Aiming at the problems of poor mechanical property, poor biocompatibility and the like of polyvinyl alcohol-acrylamide hydrogel, the invention provides a preparation method of polyvinyl alcohol-acrylamide-agarose (PVA-AAm-AG) hydrogel. Polyvinyl alcohol and acrylamide are used as matrix materials, and the gel is formed by combining chemical crosslinking and a physical crosslinking freezing and thawing method. The agarose can generate strong hydrogen bond action with the macromolecular chains of the polyvinyl alcohol and the acrylamide, and the mechanical property of the hydrogel is improved. Moreover, because the natural polysaccharide is introduced into the hydrogel, the prepared hydrogel has good biocompatibility and is expected to be applied to the field of articular cartilage substitute materials.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a preparation method of polyvinyl alcohol-acrylamide-agarose hydrogel with high mechanical strength comprises the following steps:

firstly, adding polyvinyl alcohol, acrylamide, agarose and a chemical cross-linking agent N, N' -methylene bisacrylamide (NNMBA) into deionized water according to a proportion, heating in a constant-temperature water bath at 90-99 ℃, and continuously stirring until the polyvinyl alcohol is completely dissolved and other substances are uniformly mixed to obtain a mixed solution A; in the mixed solution A, the mass fraction of polyvinyl alcohol is 10-20 wt.%, the mass fraction of acrylamide is 10-20 wt.%, the mass fraction of N, N' -methylene bisacrylamide is 0.1-0.5 wt.%, and the mass fraction of agarose is 0.5-5 wt.%;

step two, placing the mixed solution A at room temperature, continuously stirring, and cooling to 20-50 ℃ for later use; adding Tetramethylethylenediamine (TEMED) into the mixed solution A, continuously stirring until the mixture is uniformly mixed, and then adding a saturated Ammonium Persulfate (APS) aqueous solution into the mixed solution A in an ice-water bath environment to obtain a mixed solution B; adding 100-400 mu L of tetramethylethylenediamine and 3-6 mL of saturated ammonium persulfate solution into every 100mL of the mixed solution A;

pouring the mixed solution B into a mold, and performing freezing and thawing circulation on the mixed solution B to obtain polyvinyl alcohol-acrylamide-agarose hydrogel; the freezing and unfreezing cycle means that the mixed solution B is taken out and unfrozen after being frozen and formed, and is repeated for many times until PVA-AAm-AG hydrogel is formed; the freezing temperature is-18 to-25 ℃, the freezing time is 12 to 20 hours, the unfreezing temperature is 15 to 25 ℃, and the unfreezing time is 6 to 12 hours; circulating for more than 2 times.

The invention provides a gelling mechanism of polyvinyl alcohol-acrylamide-agarose hydrogel, which comprises the following steps: after polyvinyl alcohol and acrylamide are mixed, heated and dissolved, acrylamide forms a first network through a chemical cross-linking agent, polyvinyl alcohol is physically cross-linked in a refrigeration cycle to form a second network, and the two matrixes form an interpenetrating network after being gelatinized. Agarose is a natural polysaccharide, can form hydrogen bonds with macromolecular chains of polyvinyl alcohol and acrylamide, promotes aggregation of hydrogel molecular chains, and improves the mechanical strength of the material. Through repeated freezing and thawing cycles, the structure of the hydrogel is further optimized, and the mechanical property of the hydrogel is greatly improved.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a preparation method of PVA-AAm-AG hydrogel, which enhances the mechanical property of the hydrogel and improves the biocompatibility of the hydrogel by introducing agarose. The method provided by the invention is simple to operate, has an obvious effect of improving the mechanical property of the polyvinyl alcohol-acrylamide hydrogel, and meanwhile, the hydrogel prepared by the method can be adhered to chondrocytes to promote the proliferation of the chondrocytes, thereby being beneficial to the application of the hydrogel in the field of articular cartilage substitute materials.

Drawings

FIG. 1 is a flow chart of PVA-AAm-AG hydrogel preparation;

FIG. 2 is a scanning electron microscope topography of PVA-AAm-AG hydrogel;

FIG. 3 is a graph of the compression modulus of PVA-AAm-AG hydrogel;

FIG. 4 shows the adsorption rate of PVA-AAm-AG hydrogel to chondrocytes

FIG. 5 is a graph showing the proliferation rate of PVA-AAm-AG hydrogel-inoculated cells.

Detailed Description

The following describes in detail embodiments of the invention with reference to fig. 1, 2 and 3 of the technical implementation and the accompanying description.

FIG. 1 is a flow chart of PVA-AAm-AG hydrogel preparation, which can be prepared according to the operation of the flow chart. FIG. 2 shows the microscopic morphology of the hydrogel, which was sliced and freeze-dried under vacuum to observe the cross-sectional structure of the hydrogel. FIG. 3 is a graph showing the compressive properties of the hydrogels prepared, and the unconfined uniaxial compression test was performed on a cylindrical hydrogel sample (diameter: 15.3mm) with a strain rate of 30%/min. The thickness of each sample was measured with a vernier caliper before testing. Before testing, the top and bottom platforms were lubricated with saline to approximate a pure sliding condition, and strain 0.1-0.2 was selected to calculate secant compressive modulus. As can be seen from the figure, compared with the pure PVA hydrogel and the pure acrylamide hydrogel with the same concentration, the compression modulus of the PVA-AAm-AG hydrogel prepared by the method is greatly improved, and the mechanical property required by the articular cartilage substitute material can be met. FIG. 4 is a graph of chondrocyte adhesion rate normalized to the adhesion of agarose-free hydrogel after 4 hours. Resuspending articular chondrocytes of a rat in a culture medium, inoculating the articular chondrocytes on hydrogel, taking out the hydrogel after culturing for 4 hours, washing the surface of the hydrogel, and detecting the content of cells adhered to the surface of the hydrogel by a CCK-8 method. The hydrogel inoculated with rat chondrocytes was further cultured in a carbon dioxide incubator for 7 days, the hydrogel was taken out at different time intervals, and the proliferation of the cells in the hydrogel was examined by the CCK-8 method. As can be seen from the figure, the ability of the hydrogel to adhere to cells was greatly increased after agarose addition. Fig. 5 is a graph showing the proliferation rate of chondrocytes, and it can be found that the proliferation rate of chondrocytes in hydrogel is significantly increased after agarose is added, using the proliferation rate of chondrocytes after 1 day of agarose-free hydrogel as a normalization standard.

Example 1

a) Adding 10g of PVA, 10g of AAm, 0.5g of agarose and 0.1g of N, N' -methylenebisacrylamide into 79mL of deionized water, heating in a constant-temperature water bath at 90 ℃, and stirring until the PVA is completely dissolved to obtain a mixed solution A.

b) And (3) placing the mixed solution A at room temperature, continuously stirring, and cooling to 20 ℃ for later use. And (3) dropwise adding 100 mu L of tetramethylethylenediamine into the mixed solution A, adding 3mL of ammonium persulfate saturated solution in an ice-water bath environment, and stirring to obtain a mixed solution B.

c) And pouring the mixed solution B into a mold, and performing circulating freezing and thawing, wherein the freezing temperature is-18 ℃, the freezing time is 12 hours, the thawing temperature is 15 ℃, and the thawing time is 6 hours. The cycle number is 2 times, and the polyvinyl alcohol-acrylamide hydrogel is obtained.

Based on the PVA-AAm-AG hydrogel prepared by the steps, the experiments of appearance observation, mechanical property and biocompatibility are carried out. The method comprises the following specific steps:

1) and (3) appearance observation: and freezing the prepared hydrogel by using liquid nitrogen, quenching, carrying out freeze vacuum drying treatment, and observing the appearance of the quenched section by using a scanning electron microscope.

2) Mechanical property experiment: a cylindrical hydrogel sample (diameter: 15.3mm) prepared was subjected to an unconfined uniaxial compression test at a strain rate of 30%/min. The thickness of each sample was measured with a vernier caliper before testing.

3) Biocompatibility experiment: resuspending articular chondrocytes of a rat in a culture medium, inoculating the articular chondrocytes on hydrogel, taking out the hydrogel after culturing for 4 hours, washing the surface of the hydrogel, and detecting the content of cells adhered to the surface of the hydrogel by a CCK-8 method. The hydrogel inoculated with rat chondrocytes was further cultured in a carbon dioxide incubator for 7 days, the hydrogel was taken out at different time intervals, and the proliferation of the cells in the hydrogel was examined by the CCK-8 method.

Example 2

a) Adding 20g of PVA, 20g of AAm, 5g of agarose and 0.5g of N, N' -methylenebisacrylamide into 55mL of deionized water, heating in a constant-temperature water bath at 99 ℃, and stirring until the PVA is completely dissolved to obtain a mixed solution A.

b) And (4) placing the mixed solution A at room temperature, continuously stirring, and cooling to 50 ℃ for later use. And (3) dropwise adding 200 mu L of tetramethylethylenediamine into the mixed solution A, adding 6mL of ammonium persulfate saturated solution in an ice-water bath environment, and stirring to obtain a mixed solution B.

c) And pouring the mixed solution B into a mold, and performing circulating freezing and thawing, wherein the freezing temperature is-25 ℃, the freezing time is 20 hours, the thawing temperature is 25 ℃, and the thawing time is 12 hours. The cycle number is 4 times, and the polyvinyl alcohol-acrylamide hydrogel is obtained.

Based on the PVA-AAm-AG hydrogel prepared by the steps, morphology observation, mechanical property and biocompatibility experiments are carried out, and the specific steps are as in example 1.

Example 3

a) Adding 15g of PVA, 15g of AAm, 2.5g of agarose and 0.25g of N, N' -methylenebisacrylamide into 67mL of deionized water, heating in a constant-temperature water bath at 95 ℃, and stirring until the PVA is completely dissolved to obtain a mixed solution A.

b) And placing the mixed solution A at room temperature, continuously stirring, and cooling to 35 ℃ for later use. And (3) dropwise adding 450 mu L of tetramethylethylenediamine into the mixed solution A, adding 4.5mL of ammonium persulfate saturated solution in an ice-water bath environment, and stirring to obtain a mixed solution B.

c) And pouring the mixed solution B into a mold, and performing circulating freezing and thawing, wherein the freezing temperature is-22 ℃, the freezing time is 16 hours, the thawing temperature is 20 ℃, and the thawing time is 9 hours. The cycle number is 3 times, and the polyvinyl alcohol-acrylamide hydrogel is obtained.

Based on the PVA-AAm-AG hydrogel prepared by the steps, morphology observation, mechanical property and biocompatibility experiments are carried out, and the specific steps are as in example 1.

The above examples merely represent embodiments of the present invention and are not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.