CN111437435A

CN111437435A - Hydrogel cell scaffold and preparation method thereof

Info

Publication number: CN111437435A
Application number: CN202010424267.9A
Authority: CN
Inventors: 王明哲; 黄宇彬; 刘沙; 齐延新; 李晓媛; 周东方
Original assignee: Changchun Institute of Applied Chemistry of CAS
Current assignee: Changchun Institute of Applied Chemistry of CAS
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2020-07-24

Abstract

The invention provides a hydrogel cell scaffold and a preparation method thereof, wherein the preparation method comprises the following steps: a) blending a functional polymer material and polyvinyl alcohol, adding a metal ion crosslinking agent for reaction, and carrying out physical crosslinking to obtain hydrogel; the functional polymer material is a polymer material which can promote macrophage to be converted into M2 type; b) seeding macrophages of type M2 (M phi 2) onto the hydrogel obtained in step a) to obtain a hydrogel cell scaffold. The preparation method provided by the invention adopts the high molecular material capable of promoting the conversion of the macrophage to M2 type as the main raw material, and combines specific process steps and conditions to realize better overall interaction, so as to prepare the hydrogel cytoskeleton capable of supplementing and regulating the macrophage; the hydrogel cell scaffold not only can maintain the loaded M phi 2 phenotype unchanged, but also can promote the conversion of M1 type macrophages (M phi 1) at a chronic inflammation part to M2 type, and can be used for locally and effectively regulating a chronic inflammation microenvironment.

Description

Hydrogel cell scaffold and preparation method thereof

Technical Field

The invention relates to the technical field of high polymer medical materials, in particular to a hydrogel cell scaffold and a preparation method thereof.

Background

Chronic inflammation is the main cause of chronic diseases, such as diabetic foot ulcer, pressure sore, venous ulcer and the like which can not be cured for a long time. A large number of inflammatory cells (M phi 1, neutrophil leukocytes and the like) exist at the chronic inflammation part, and secrete a large number of inflammatory factors to form chemotactic gradient and promote more inflammatory cells to gather, so that the vicious circle is always in an inflammatory reaction stage, cannot transit to a proliferation stage and cannot be cured for a long time.

Research shows that macrophages play a great role in regulating inflammation, wherein M phi 1 can secrete inflammatory factors, generate a large amount of Reactive Oxygen Species (ROS), and enhance inflammatory response; and M phi 2 can secrete anti-inflammatory factors and growth factors, resist chronic inflammation, promote angiogenesis and promote tissue repair. Thus, macrophage phenotypic shift is key to promoting inflammation-proliferation shift. However, the chronic inflammation part has immunodeficiency, and the elimination of the chronic inflammation and the promotion of the repair are possibly limited by simply relying on the phenotype transformation of macrophages at the affected part, and the supplementation of exogenous M2 type macrophages can possibly effectively improve the tissue repair efficiency.

The three-dimensional network structure and high water content of the hydrogel are suitable for cell growth, and the hydrogel can effectively keep a moist environment of a wound, cool and relieve pain. The metal ions can be used as a cross-linking agent to stabilize the structure of the hydrogel and endow the hydrogel with new functions; for example, copper ions can promote angiogenesis, calcium ions can be beneficial to hemostasis, iron ions have an antibacterial function, and the like. Therefore, how to prepare hydrogel medical materials with better comprehensive performance by utilizing the crosslinking action of metal ions for regulating the microenvironment of chronic inflammation becomes a technical problem to be solved by technical personnel in the field.

Disclosure of Invention

In view of the above, the present invention aims to provide a hydrogel cell scaffold and a preparation method thereof, and the hydrogel cell scaffold prepared by the preparation method provided by the present invention not only can maintain the phenotype of the carried M2-type macrophages unchanged, but also can promote the conversion of the M1-type macrophages at chronic inflammation sites to M2-type macrophages, and can be used for locally and effectively regulating the microenvironment of chronic inflammation.

The invention provides a preparation method of a hydrogel cell scaffold, which comprises the following steps:

a) blending a functional polymer material and polyvinyl alcohol, adding a metal ion crosslinking agent for reaction, and carrying out physical crosslinking to obtain hydrogel; the functional polymer material is a polymer material which can promote macrophage to be converted into M2 type;

b) seeding M2 type macrophages on the hydrogel obtained in step a) to obtain a hydrogel cell scaffold.

Preferably, the polymer material capable of promoting the conversion of macrophages to M2 in step a) comprises one or more of gelatin, keratin, chitosan, hyaluronic acid, alginate, modified cellulose and polylactic acid.

Preferably, the mass ratio of the functional polymer material to the polyvinyl alcohol in the step a) is 1: (4-10).

Preferably, the metal ion crosslinking agent in step a) includes one or more of a compound containing iron ions, a compound containing copper ions, a compound containing zinc ions, and a compound containing calcium ions.

Preferably, the pH value of the reaction in the step a) is 6-8.

Preferably, the physical crosslinking in step a) is performed by freeze-thaw cycle crosslinking and/or lyophilization crosslinking.

Preferably, the process of freeze-thaw cycle crosslinking specifically comprises the following steps:

freezing the reacted metal ion crosslinked hydrogel at-80 to-15 ℃ for 20 to 24 hours, and then melting at 4 to 30 ℃ for 1 to 3 hours to complete one freeze-thaw cycle; the reaction is carried out for 1 to 4 times.

Preferably, the freeze-drying and cross-linking mode is vacuum freeze-drying; the temperature of the vacuum freeze drying is-70 ℃ to-50 ℃, and the time is 24h to 60 h.

Preferably, the inoculation process in step b) is specifically as follows:

placing the cell suspension of M2 type macrophages on the hydrogel obtained in the step a), and culturing for 23-25 h at 36-38 ℃ to obtain the hydrogel cell scaffold.

The invention also provides a hydrogel cell scaffold prepared by the preparation method of the technical scheme.

The invention provides a hydrogel cell scaffold and a preparation method thereof, wherein the preparation method comprises the following steps: a) blending a functional polymer material and polyvinyl alcohol, adding a metal ion crosslinking agent for reaction, and carrying out physical crosslinking to obtain hydrogel; the functional polymer material is a polymer material which can promote macrophage to be converted into M2 type; b) seeding M2 type macrophages on the hydrogel obtained in step a) to obtain a hydrogel cell scaffold. Compared with the prior art, the preparation method provided by the invention adopts the high molecular material capable of promoting the conversion of the macrophage to M2 type as the main raw material, and combines specific process steps and conditions to realize better overall interaction, so as to prepare the hydrogel cytoskeleton capable of supplementing and regulating the macrophage; the hydrogel cytoskeleton not only can maintain the phenotype of the carried M2 type macrophage unchanged, but also can promote the conversion of the M1 type macrophage at a chronic inflammation part to M2 type, and can be used for locally and effectively regulating a chronic inflammation microenvironment.

In addition, the preparation method provided by the invention combines metal ion crosslinking and physical crosslinking, and the prepared hydrogel is soft and elastic, has a three-dimensional network structure with uniform pore diameter, high water content and good biocompatibility, and is suitable for carrying cells; in addition, the size and the shape of the material can be changed according to the mould, and the material can be made into a sheet-shaped attaching material and a block-shaped filling material, so that the material has wide application prospect.

Drawings

FIG. 1 is a schematic view of a M Φ 2-loaded hydrogel cell scaffold prepared by the preparation method provided in example 1 of the present invention;

FIG. 2 is the HA/PVA prepared_1FTHydrogel (a), HA/PVA_4FTHydrogel (b), Cu-HA/PVA_1FTHydrogel (c), Cu-HA/PVA_4FTHydrogel (d), HA/PVA_1FT+FDHydrogel (e) and Cu-HA/PVA_1FT+FDThe rheological profile of hydrogel (f);

FIG. 3 is the HA/PVA prepared_1FT+FDHydrogel (a) and Cu-HA/PVA_1FT+FDThe degradation curve of hydrogel (b);

FIG. 4 shows PVA hydrogel (a) and Cu-HA/PVA_1FT+FDSEM photograph of hydrogel (b);

FIG. 5 shows macrophage seeded PVA hydrogel (a) and macrophage seeded Cu-HA/PVA_1FT+FDSEM photograph of hydrogel (b);

FIG. 6 is a control plot of fluorescence intensity after macrophage seeding of each hydrogel scaffold.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention firstly mixes functional polymer material and polyvinyl alcohol, then adds metal ion cross-linking agent to react, and then obtains hydrogel through physical cross-linking. In the invention, the functional polymer material is a polymer material which can promote macrophage to be converted into M2 type; the polymer material capable of promoting the macrophage to convert to M2 type preferably comprises one or more of gelatin, keratin, chitosan, hyaluronic acid, alginate, modified cellulose and polylactic acid, and more preferably gelatin, hyaluronic acid or alginate. The source of the functional polymer material is not particularly limited in the present invention, and commercially available products known to those skilled in the art may be used.

The polyvinyl alcohol (PVA) is not particularly limited in its source in the present invention, and commercially available products known to those skilled in the art may be used. In the present invention, the mass ratio of the functional polymer material to the polyvinyl alcohol is preferably 1: (4-10), more preferably 1: (5-9).

In the present invention, the blending process is preferably specifically:

mixing the functional polymer material with water, and magnetically stirring until the functional polymer material is completely dissolved; mixing polyvinyl alcohol with water, and stirring at 70-90 ℃ until the polyvinyl alcohol is completely dissolved; and uniformly mixing the two solutions to obtain a blending system.

In the present invention, the metal ion crosslinking agent preferably includes one or more of a compound containing an iron ion, a compound containing a copper ion, a compound containing a zinc ion, and a compound containing a calcium ion, and more preferably a compound containing a copper ion, a compound containing a zinc ion, or a compound containing a calcium ion. In a preferred embodiment of the invention, the metal ion crosslinking agent is copper sulfate, and is introduced into the blending system through a copper sulfate aqueous solution; in another preferred embodiment of the present invention, the metal ion crosslinking agent is calcium chloride, and is introduced into the blending system through a calcium chloride aqueous solution; in another preferred embodiment of the present invention, the metal ion crosslinking agent is zinc chloride, and the blending system is introduced by an aqueous solution of zinc chloride.

In the present invention, the amount of the metal ion crosslinking agent added is preferably 0.1 to 0.5% by mass of the functional polymer material.

In the invention, the reaction mode is metal ion crosslinking, so as to obtain the metal ion crosslinking hydrogel. In the invention, the pH value of the reaction is preferably 6-8, and more preferably 6.5-7.5; the pH value is adjusted by adding 1M NaOH aqueous solution or 1M HCl aqueous solution.

In the present invention, the physical crosslinking is preferably freeze-thaw cycle crosslinking and/or lyophilization crosslinking, and more preferably freeze-thaw cycle crosslinking and lyophilization crosslinking. In a preferred embodiment of the present invention, the physical crosslinking is performed by first performing freeze-thaw cycle crosslinking and then performing freeze-drying crosslinking to obtain a freeze-dried gel.

In the present invention, the freeze-thaw cycle crosslinking process is preferably specifically:

freezing the reacted metal ion crosslinked hydrogel at-80 to-15 ℃ for 20 to 24 hours, and then melting at 4 to 30 ℃ for 1 to 3 hours to complete one freeze-thaw cycle; the treatment is carried out for 1 to 4 times;

more preferably:

freezing the reacted metal ion crosslinked hydrogel at-20 ℃ for 22h, and then melting at 25 ℃ for 2h to complete one freeze-thaw cycle; this was done 1 time.

The freeze-thaw cross-linked container is not particularly limited by the invention, and a six-hole plate and moulds with various structures which are well known by the technicians in the field can be adopted; therefore, the size and the shape of the prepared hydrogel can be changed according to the mould, and the hydrogel can be made into a sheet-shaped attaching material and a block-shaped filling material, and has wide application prospect.

In the present invention, the freeze-drying and cross-linking means is preferably vacuum freeze-drying; the temperature of the vacuum freeze drying is preferably-70 ℃ to-50 ℃, and more preferably-60 ℃; the time of the vacuum freeze drying is preferably 24-60 h; obtaining the freeze-dried gel.

After obtaining the lyophilized gel, the present invention preferably further comprises:

and (4) imbibing the freeze-dried gel by using a phosphate buffer solution to obtain the hydrogel. The above change is a change in the form of the gel, which is intended to facilitate the subsequent inoculation process, and the present invention is not particularly limited thereto.

The preparation method provided by the invention preferably combines metal ion crosslinking and physical crosslinking, and the prepared hydrogel is soft and elastic, has a three-dimensional network structure with uniform pore diameter, high water content and good biocompatibility, and is suitable for carrying cells.

After obtaining the hydrogel, the invention inoculates M2 type macrophage on the obtained hydrogel to obtain the hydrogel cell scaffold. In the present invention, the inoculation process is preferably specifically:

placing the cell suspension of M2 type macrophage on the obtained hydrogel, and culturing for 23-25 h at 36-38 ℃ to obtain a hydrogel cell scaffold;

more preferably:

and (3) placing the cell suspension of the M2 type macrophage on the obtained hydrogel, and culturing at 37 ℃ for 24h to obtain the hydrogel cell scaffold. By adopting the inoculation process, the cells can be attached to the gel, and the cells can be carried.

The invention also provides a hydrogel cell scaffold prepared by the preparation method of the technical scheme. The preparation method provided by the invention adopts the high molecular material capable of promoting the conversion of the macrophage to M2 type as the main raw material, and combines specific process steps and conditions to realize better overall interaction, so as to prepare the hydrogel cytoskeleton capable of supplementing and regulating the macrophage; the hydrogel cytoskeleton not only can maintain the phenotype of the carried M2 type macrophage unchanged, but also can promote the conversion of the M1 type macrophage at a chronic inflammation part to M2 type, and can be used for locally and effectively regulating a chronic inflammation microenvironment.

To further illustrate the present invention, the following examples are provided for illustration.

Example 1

(1) Adding 100mg Hyaluronic Acid (HA) into 25ml deionized water, and magnetically stirring until the HA is completely dissolved; adding 900mg of polyvinyl alcohol (PVA) into 25ml of deionized water, and stirring at 80 ℃ until the PVA is completely dissolved; and uniformly mixing the two solutions, adding 52 mu l of copper sulfate aqueous solution (3mg/ml), adding 1M NaOH or 1M HCl to adjust the pH value to 6.5-7.5, and uniformly stirring to obtain the copper ion crosslinked hydrogel (Cu-HA/PVA, the crosslinking degree is 0.5%).

(2) Injecting the Cu-HA/PVA hydrogel obtained in the step (1) into a six-hole plate (2 ml/hole) for one freeze-thaw cycle (one-freeze-thaw cycle, 1FT) (-20 ℃ freeze-22 h, 25 ℃ thaw 2h), and then freeze-drying (freeze-dry, FD) to obtain the freeze-dried gel.

(3) 3ml of phosphate buffer (PBS, pH 7.4) was added to the wells of the lyophilized gel, and the lyophilized gel was imbibed to form a hydrogel (Cu-HA/PVA)_1FT+FD) (ii) a Placing the M2 type macrophage (M phi 2) cell suspension on the gel, culturing at 37 deg.C for 24h to attach the cells to the gel to obtain M phi 2-loaded hydrogel cell scaffold (Cu-HA/PVA)_1FT+FD@ M Φ 2, FIG. 1).

As a control, hydrogel cell scaffolds (Cu-HA/PVA) carrying M1 type macrophages (M Φ 1) were prepared according to the preparation method provided in example 1 above_1FT+FD@ M Φ 1), cell-free hydrogel scaffold (Cu-HA/PVA)_1FT+FD) Hydrogel scaffolds (HA/PVA) that are not cell-loaded nor ion-crosslinked_1FT+FD) And pure PVA without HA_1FT+FDA hydrogel scaffold; meanwhile, a hydrogel bracket (HA/PVA) which is only subjected to one-time freeze-thaw cycle crosslinking is prepared_1FT) Hydrogel scaffolds (HA/PVA) crosslinked only by four freeze-thaw cycles_4FT) Hydrogel scaffold (Cu-HA/PVA) crosslinked by copper ions and subjected to one-time freeze-thaw cycle_1FT) Hydrogel scaffold (Cu-HA/PVA) crosslinked by copper ions and four times of freeze-thaw cycles_4FT)。

And (3) performance characterization:

1) characterization of the rheological properties of the hydrogel:

FIG. 2 is the HA/PVA prepared_1FTHydrogel (a), HA/PVA_4FTHydrogel (b), Cu-HA/PVA_1FTHydrogel (c), Cu-HA/PVA_4FTHydrogel (d), HA/PVA_1FT+FDHydrogel (e) and Cu-HA/PVA_1FT+FDRheological Curve (37 ℃ C., angular frequency 1 r) of hydrogel (f)ad/s). As can be seen from FIG. 2, the freeze-dried crosslinked HA/PVA_1FT+FDHydrogel and Cu-HA/PVA_1FT+FDThe modulus (G ', G') of the hydrogel is much higher than that of a hydrogel that has not been crosslinked by lyophilization; therefore, the mechanical property of the hydrogel is greatly improved by freeze-drying crosslinking, and the hydrogel is more suitable for serving as a cell scaffold.

2) Degradation of the hydrogel: placing hydrogel with known mass in a centrifuge tube filled with PBS 7.4, shaking at constant temperature of 37 ℃, taking out the hydrogel at a preset time point, freeze-drying, weighing, and calculating the degradation rate.

FIG. 3 is the HA/PVA prepared_1FT+FDHydrogel (a) and Cu-HA/PVA_1FT+FDDegradation curve of hydrogel (b). As can be seen from FIG. 3, HA/PVA that was not crosslinked by copper ions_1FT+FDThe quality of the hydrogel is rapidly reduced by 10% within 2h, and since the copper ions mainly have a crosslinking effect on HA, the 10% quality reduction is caused by rapid HA loss; and Cu-HA/PVA crosslinked by copper ions_1FT+FDIn the hydrogel, the HA is crosslinked by the copper ions, so that the mass reduction rate of the hydrogel is slower, the residence time of the HA at an action part is increased, and the effect of the HA is better exerted.

3) Characterization of the hydrogel morphology:

FIG. 4 shows PVA hydrogel (a) and Cu-HA/PVA_1FT+FDSEM photograph (scale: 20 μm) of hydrogel (b). As can be seen from FIG. 4, the obtained hydrogel has a three-dimensional network structure and uniform pore size; after the HA is added, the pore size of the gel is obviously increased, so that the gel is favorable for cell adhesion and growth in the gel, and is more suitable for serving as a cell supporting bracket.

4) Characterization of cell adhesion properties of the hydrogel: macrophages were seeded onto the hydrogel surface and after 24h the gel was freeze dried and SEM photographed.

FIG. 5 shows macrophage seeded PVA hydrogel (a) and macrophage seeded Cu-HA/PVA_1FT+FDSEM photograph (scale: 20 μm) of hydrogel (b). As can be seen in FIG. 5, the macrophages on the PVA hydrogel are piled up together, like a sphere, and do not cling to the hydrogel; on the HA/PVA hydrogel, macrophages are uniformly distributed and tightly adhered to the surface of the hydrogel; thus the addition of HA increased the hydrogelCell adhesion properties of (2).

5) Effect of hydrogel scaffold on macrophage phenotype, macrophage (M phi) was inoculated into 6-well plates, lipopolysaccharide (L PS, 1. mu.g/M L) or interleukin-4 (I L-4, 40ng/M L) were added to stimulate M1 or M2 types, respectively, Cu-HA/PVA of the same size was added to stimulate the cells to M1 or M2 types_1FT+FDPlacing hydrogel at the bottom of 6-well plate, inoculating M phi 1 or M phi 2 after conversion stimulation on the surface, taking the cell hole without hydrogel as blank control, incubating at 37 deg.C for 72h, blowing down the cell, and placing in new 6-well plate to adhere to the wall again; fixation with 4% paraformaldehyde, 5% albumin blocking, addition of CD206(M Φ 2-labeled protein) primary antibody, addition of FITC secondary antibody, final DAPI nuclear staining, observation of cell fluorescence intensity with confocal laser microscopy and quantification of fluorescence by ImageJ (fig. 6).

In FIG. 6, a1 and a2 are fluorescence intensities of M.PHI.1 stimulated with L PS (negative control) and M.PHI.2 stimulated with I L-4 (positive control), respectively, and b1 and c1 are blank control and Cu-HA/PVA, respectively_1FT+FDThe fluorescence intensity of the hydrogel group after being inoculated with M phi 1 and cultured for 72 h; b2 and c2 are blank control and Cu-HA/PVA, respectively_1FT+FDFluorescence intensity of hydrogel group inoculated with M phi 2 after 72h of culture.

As can be seen from FIG. 6, M.PHI.1 is in Cu-HA/PVA_1FT+FDAfter culturing on the hydrogel for 72h, the fluorescence intensity is obviously enhanced compared with that of a negative control group, while the fluorescence intensity of a blank control group is only slightly enhanced; illustrating the Cu-HA/PVA_1FT+FDThe hydrogel promotes the transformation from M phi 1 to M phi 2; m phi 2 in Cu-HA/PVA_1FT+FDAfter culturing on the hydrogel for 72h, the fluorescence intensity is basically the same as that of the positive control group, while the fluorescence intensity of the blank control group is obviously reduced, which indicates that the fluorescence intensity of Cu-HA/PVA is obviously reduced_1FT+FDThe hydrogel can maintain the M phi 2 phenotype unchanged; the HA in the hydrogel can not only promote the transformation of M phi 1 to M2 type, but also maintain the M phi 2 phenotype unchanged. Thus, M.phi.2-supporting Cu-HA/PVA_1FT+FDThe @ M phi 2 hydrogel cell scaffold can not only maintain the phenotype of the loaded M phi 2 unchanged, but also promote the conversion of the inflammatory site M phi 1 to M phi 2.

Example 2

Adding 100mg of Sodium Alginate (SA) into 12.5ml of deionized water, and magnetically stirring until the Sodium Alginate (SA) is completely dissolved; adding 500mg of polyvinyl alcohol (PVA) into 12.5ml of deionized water, and stirring at 80 ℃ until the PVA is completely dissolved; and uniformly mixing the two solutions, adding 74 mu l of calcium chloride aqueous solution (5mg/ml), adding 1M NaOH or 1M HCl to adjust the pH value to 6.5-7.5, and uniformly stirring to obtain the copper ion crosslinked hydrogel (Ca-SA/PVA, the crosslinking degree is 1%).

(2) And (2) injecting the Ca-SA/PVA hydrogel obtained in the step (1) into a mould, performing primary freeze-thaw cycle (freezing at the temperature of minus 20 ℃ for 22 hours, and melting at the temperature of 25 ℃ for 2 hours), and then performing freeze-drying to obtain the freeze-dried gel.

(3) Adding appropriate amount of phosphate buffer solution (PBS, pH 7.4) into the lyophilized gel hole, and allowing the lyophilized gel to absorb liquid to obtain hydrogel (Ca-SA/PVA)_1FT+FD) (ii) a Then placing the M phi 2 cell suspension on the gel, culturing at 37 ℃ for 24h to attach the cells to the gel to obtain the M phi 2-loaded hydrogel cell scaffold (Ca-SA/PVA)_1FT+FD@MΦ2)。

As a control, M.phi.1-supporting hydrogel cell scaffolds (Ca-SA/PVA) were prepared according to the preparation method provided in example 2 above_1FT+FD@ M Φ 1), cell-free hydrogel scaffold (Ca-SA/PVA)_1FT+FD) Hydrogel scaffolds that do not support cells nor undergo ionic crosslinking (SA/PVA)_1FT+FD) And simple PVA without SA_1FT+FDA hydrogel scaffold; meanwhile, a hydrogel scaffold (SA/PVA) which is only subjected to one-time freeze-thaw cycle crosslinking is prepared_1FT) Hydrogel scaffolds (SA/PVA) crosslinked only by four freeze-thaw cycles_4FT) Hydrogel scaffold (Ca-SA/PVA) crosslinked by calcium ions and one-time freeze-thaw cycle_1FT) Hydrogel scaffold (Ca-SA/PVA) crosslinked by calcium ions and four times of freeze-thaw cycles_4FT)。

The experimental results show that: freeze-dried crosslinked hydrogels (SA/PVA)_1FT+FDAnd Ca-SA/PVA_1FT+FD) The modulus of the hydrogel is much higher than that of the hydrogel which is not subjected to freeze-drying crosslinking; therefore, the mechanical property of the hydrogel is greatly improved by freeze-drying crosslinking, and the hydrogel is more suitable for serving as a cell scaffold. Due to the calcium ion crosslinking of SA, the residence time of SA at the action part is increased, and the SA is more beneficial to play the action effect. The obtained hydrogel has a three-dimensional network structure and uniform pore diameter; after SA is added, the pore diameter of the gel is obviously increasedIs favorable for cell adhesion and growth to the interior of the gel, and is more suitable for serving as a cell supporting bracket. PVA (polyvinyl alcohol)_1FT+FDMacrophages on the hydrogel are piled together and are similar to a ball shape and are not attached to the hydrogel; while in Ca-SA/PVA_1FT+FDOn the hydrogel, macrophages are uniformly distributed and tightly adhered to the surface of the hydrogel; the addition of SA therefore increases the cell adhesion properties of the hydrogel. M phi 1 in Ca-SA/PVA_1FT+FDAfter culturing on the hydrogel for 72h, the fluorescence intensity is obviously enhanced compared with that of a negative control group, while the fluorescence intensity of a blank control group is only slightly enhanced; description of Ca-SA/PVA_1FT+FDThe hydrogel promotes the transformation from M phi 1 to M phi 2; m phi 2 in Ca-SA/PVA_1FT+FDAfter 72h of culture on the hydrogel, the fluorescence intensity is basically the same as that of the positive control group, while the fluorescence intensity of the blank control group is obviously reduced, which indicates that the Ca-SA/PVA concentration is reduced_1FT+FDThe hydrogel can maintain the M phi 2 phenotype unchanged; the SA present in the hydrogel can not only promote the transformation of M phi 1 to M2 type, but also maintain the M phi 2 phenotype unchanged.

Example 3

Adding 100mg gelatin (Gel) into 12.5ml deionized water, and magnetically stirring at 40 deg.C until completely dissolved; adding 600mg of polyvinyl alcohol (PVA) into 12.5ml of deionized water, and stirring at 80 ℃ until the PVA is completely dissolved; and uniformly mixing the two solutions, adding 53 mu l of zinc chloride aqueous solution (5mg/ml), adding 1M NaOH or 1M HCl to adjust the pH value to 6.5-7.5, and uniformly stirring to obtain the copper ion crosslinked hydrogel (Zn-Gel/PVA, the crosslinking degree is 0.5%).

(2) And (2) injecting the Zn-Gel/PVA hydrogel obtained in the step (1) into a mould, performing one-time freeze-thaw cycle (freezing at the temperature of minus 20 ℃ for 22 hours, and melting at the temperature of 25 ℃ for 2 hours), and then performing freeze-drying to obtain the freeze-dried Gel.

(3) Adding a proper amount of phosphate buffer solution (PBS, pH 7.4) into the freeze-dried Gel hole, and enabling the freeze-dried Gel to absorb liquid to become hydrogel (Zn-Gel/PVA)_1FT+FD) (ii) a Then placing the M phi 2 cell suspension on the Gel, culturing for 24h at 37 ℃ to attach the cells to the Gel to obtain the M phi 2-loaded hydrogel cell scaffold (Zn-Gel/PVA)_1FT+FD@MΦ2)。

As a control, M.phi.1-supporting hydrogel cell scaffolds (Zn) were prepared according to the preparation method provided in example 3 above-Gel/PVA_1FT+FD@ M Φ 1), cell-free hydrogel scaffold (Zn-Gel/PVA)_1FT+FD) Hydrogel scaffolds that are cell-free and ion-crosslinked (Gel/PVA)_1FT+FD) And pure PVA without Gel_1FT+FDA hydrogel scaffold; meanwhile, a hydrogel scaffold (Gel/PVA) which is only subjected to one-time freeze-thaw cycle crosslinking is prepared_1FT) Hydrogel scaffolds (Gel/PVA) crosslinked only by four freeze-thaw cycles_4FT) Hydrogel scaffold (Zn-Gel/PVA) crosslinked by zinc ions and once freeze-thaw cycle_1FT) Hydrogel scaffold (Zn-Gel/PVA) crosslinked by zinc ions and four times of freeze-thaw cycles_4FT)。

The experimental results show that: freeze-dried crosslinked hydrogels (Gel/PVA)_1FT+FDAnd Zn-Gel/PVA_1FT+FD) The modulus of the hydrogel is much higher than that of the hydrogel which is not subjected to freeze-drying crosslinking; therefore, the mechanical property of the hydrogel is greatly improved by freeze-drying crosslinking, and the hydrogel is more suitable for serving as a cell scaffold. The zinc ions cross-link the Gel, so that the retention time of the Gel at the action part is increased, and the Gel is more favorable for exerting the action effect. The obtained hydrogel has a three-dimensional network structure and uniform pore diameter; after Gel is added, the pore diameter of the Gel is obviously increased, cell adhesion and growth to the interior of the Gel are facilitated, and the Gel is more suitable for serving as a cell supporting bracket. PVA (polyvinyl alcohol)_1FT+FDMacrophages on the hydrogel are piled together and are similar to a ball shape and are not attached to the hydrogel; in Zn-Gel/PVA_1FT+FDOn the hydrogel, macrophages are uniformly distributed and tightly adhered to the surface of the hydrogel; the addition of Gel therefore increases the cell adhesion properties of the hydrogel. M phi 1 in Zn-Gel/PVA_1FT+FDAfter culturing on the hydrogel for 72h, the fluorescence intensity is obviously enhanced compared with that of a negative control group, while the fluorescence intensity of a blank control group is only slightly enhanced; description of Zn-Gel/PVA_1FT+FDThe hydrogel promotes the transformation from M phi 1 to M phi 2; m phi 2 in Zn-Gel/PVA_1FT+FDAfter culturing on the hydrogel for 72h, the fluorescence intensity is basically the same as that of the positive control group, while the fluorescence intensity of the blank control group is obviously reduced, which indicates that Zn-Gel/PVA_1FT+FDThe hydrogel can maintain the M phi 2 phenotype unchanged; the Gel in the hydrogel can not only promote the transformation of M phi 1 to M2 type, but also maintain the M phi 2 phenotype unchanged.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a hydrogel cell scaffold comprises the following steps:

2. The method according to claim 1, wherein the polymer material capable of promoting the conversion of macrophages to M2 in step a) comprises one or more of gelatin, keratin, chitosan, hyaluronic acid, alginate, modified cellulose and polylactic acid.

3. The method according to claim 1, wherein the mass ratio of the functional polymer material to the polyvinyl alcohol in the step a) is 1: (4-10).

4. The method according to claim 1, wherein the metal ion crosslinking agent in step a) comprises one or more of a compound containing iron ions, a compound containing copper ions, a compound containing zinc ions, and a compound containing calcium ions.

5. The method according to claim 1, wherein the reaction in step a) has a pH of 6 to 8.

6. The method of claim 1, wherein the physical crosslinking in step a) is performed by freeze-thaw cycle crosslinking and/or lyophilization crosslinking.

7. The preparation method according to claim 6, wherein the freeze-thaw cycle crosslinking process is specifically:

8. The method of claim 6, wherein the lyophilization and crosslinking is vacuum lyophilization; the temperature of the vacuum freeze drying is-70 ℃ to-50 ℃, and the time is 24h to 60 h.

9. The method according to claim 1, wherein the inoculation in step b) is specifically performed by:

10. A hydrogel cell scaffold prepared by the method according to any one of claims 1 to 9.