Cell super-infiltration hydrogel scaffold as well as preparation method and application thereof

Abstract

The invention relates to a cell super-infiltration hydrogel scaffold as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The invention provides a preparation method of a cell super-infiltration hydrogel scaffold. The preparation method comprises the following steps: preparing gelatin into gelatin microspheres by using a water-in-oil-emulsification method; the preparation method comprises the following steps: mixing chitosan, procyanidine and an acetic acid solution to obtain a chitosan-procyanidine solution; and mixing the gelatin microspheres with a chitosan-procyanidine solution, placing the mixture in a template, and carrying out freeze drying, alkali treatment and heating to obtain the cell ultra-infiltration hydrogel scaffold. According to the preparation method, gelatin microspheres and a chitosan-procyanidine solution are mixed, an ice crystal template can be formed in hydrogel through freeze drying on the basis of the collaborative construction process of template leaching and freeze drying, ice crystals are melted to form a small-hole structure after heating treatment, and the gelatin microspheres can be subjected to in-situ crosslinking through heating treatment, so that the gelatin microspheres are fixed on the inner walls of holes; the mechanical property of the stent is enhanced while a macroporous structure is formed in the hydrogel stent.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a cell super-infiltrating hydrogel scaffold, its preparation method, and its application. Background Technology

[0002] Tissue engineering is a primary means of tissue repair and regeneration. Its core lies in the synergistic and effective integration of biomaterial scaffolds, seed cells, and the host microenvironment. This process requires tissue-engineered scaffolds to construct an ideal three-dimensional microenvironment for the infiltration, adhesion, proliferation, and differentiation of seed cells. Their structural characteristics lie in creating porous structures that facilitate cell adhesion. Compared to synthetic polymers, natural polymers possess excellent biocompatibility and biodegradability. Currently, methods for constructing three-dimensional porous scaffolds include foaming, sol-gel methods, freeze-drying, phase separation, and template leaching. Although these methods achieve pore sizes ranging from tens to hundreds of micrometers, achieving super-infiltration and adhesion of seed cells remains challenging. The challenges include: 1. The staggered design of suitable pore sizes on the bioscaffold to balance the needs of cell migration and nutrient diffusion; 2. The construction of surface properties of the pore walls to promote seed cell adhesion; 3. The balance between material pore size and scaffold mechanical properties (high porosity / macropore structures often lead to decreased scaffold strength or even structural collapse).

[0003] Traditional porous scaffold fabrication methods (such as foaming, sol-gel, freeze-drying, and phase separation) often suffer from uneven pore size distribution, resulting in disordered hierarchical nested pore structures with uncontrollable pore sizes. This interwoven pore distribution often hinders cell migration and infiltration into the scaffold. In contrast, template leaching, by introducing size-controllable template materials, can precisely control the pore size and three-dimensional spatial distribution of the macroporous structure within the scaffold, and can also regulate its porosity, enabling the precise construction of a pre-defined pore network. However, this method has limitations in constructing secondary micron-scale micropore structures, while subcellular-scale pores play a crucial role in promoting initial cell adhesion, migration, and extracellular matrix deposition.

[0004] Therefore, an ideal tissue engineering scaffold needs to integrate macroscopic guidance of large pore channels (to achieve high porosity regulation) and microscopic regulation of small pore structures (to promote cell infiltration and adhesion) while taking into account the mechanical and structural properties of the material, so as to optimize the cell infiltration process through the synergistic effect of multi-scale pores. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a cell super-infiltrating hydrogel scaffold that not only has certain material mechanical and structural properties, but also has macroscopic macropores and microscopic micropores, as well as its preparation method and application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing a cell-super-infiltrating hydrogel scaffold, comprising the following steps: S1. Gelatin microspheres were prepared from gelatin using a water-in-oil emulsion method. S2. Mix chitosan, proanthocyanidins and acetic acid solution to obtain chitosan-proanthocyanidin solution; S3. The gelatin microspheres described in step S1 are mixed with the chitosan-proanthocyanidin solution described in step S2 and placed in a template. The mixture is then freeze-dried, treated with alkali, and heated to obtain a cell-super-infiltrating hydrogel scaffold.

[0007] This invention involves mixing gelatin microspheres with a chitosan-proanthocyanidin solution and constructing the scaffold using a synergistic process of template leaching and freeze-drying. Freeze-drying sublimates the ice crystal template formed in the hydrogel, creating a microporous structure. Further heat treatment dissolves the gelatin microspheres and allows for in-situ cross-linking with proanthocyanidins without cross-linking agents, fixing the gelatin molecules to the inner walls of the pores. This results in a macroporous structure within the hydrogel scaffold while enhancing its mechanical properties. Furthermore, the gelatin molecules possess RGD sequences, which synergistically promote the adhesion and growth of infiltrating cells with proanthocyanidins.

[0008] The hydrogel scaffold of this invention is obtained by mixing gelatin microspheres and chitosan-proanthocyanidin solution, placing them in a template, freeze-drying, alkali treatment, and heating, thus obtaining a high-porosity, hierarchical hydrogel scaffold. Proanthocyanidins, as polyphenolic compounds, possess the property of crosslinking gelatin molecules, therefore, in-situ deposition and crosslinking of gelatin molecules on the pore walls can be achieved without the addition of additional crosslinking agents. Simultaneously, the hydrogen bonds between chitosan molecules and the crosslinking of chitosan and gelatin molecules by proanthocyanidins enable the hydrogel scaffold to maintain a large-pore structure while possessing good mechanical stability, effectively balancing the contradiction between large-pore structure and mechanical stability.

[0009] In a preferred embodiment of the preparation method described in this invention, in step S1, the average particle size of the gelatin microspheres is 200-600 μm. The collected gelatin microspheres are then screened to obtain microspheres of a specific size, facilitating the subsequent construction of macroporous structures with different pore sizes. The particle size screening can be performed using a sieve with a specific mesh size, or by collecting the microspheres using gravity sedimentation.

[0010] In a preferred embodiment of the preparation method of the present invention, in step S1, the average particle size of the gelatin microspheres is 300-500 μm. The average particle size of the gelatin microspheres of the present invention may also be at least one of 200-300 μm, 300-400 μm, 400-500 μm, and 500-600 μm.

[0011] As a preferred embodiment of the preparation method described in this invention, in step S1, the water-in-oil emulsion method mainly involves adding a 26-30 wt% gelatin solution to liquid paraffin, stirring, allowing it to settle, sieving, and washing to obtain gelatin microspheres.

[0012] In a preferred embodiment of the preparation method described in this invention, in step S1, the 26-30 wt% gelatin solution in the water-in-oil emulsion method is obtained by dissolving gelatin in water at 70-80°C. Since gelatin is solid at room temperature, it needs to be heated to completely dissolve in water.

[0013] As a preferred embodiment of the preparation method of the present invention, in step S1, the 28wt% gelatin solution in the water-in-oil emulsion method is obtained by dissolving gelatin in water at 70°C.

[0014] In a preferred embodiment of the preparation method described in this invention, in step S1, the temperature of the liquid paraffin in the water-in-oil emulsion method is 70-80°C, and the amount used is ≥400mL. In the water-in-oil emulsion method, the liquid paraffin serves as the oil phase. To prevent the gelatin from immediately turning into a solid upon contact with the oil phase, the oil phase needs to be heated to 70-80°C. Simultaneously, to ensure the gelatin microspheres are more intact and to prevent aggregation, the amount of oil phase used is at least 400mL to avoid causing shape defects in the gelatin microspheres.

[0015] As a preferred embodiment of the preparation method of the present invention, in step S1, the temperature of the liquid paraffin in the water-in-oil emulsion method is 70°C, and the amount used is 400-500 mL.

[0016] In a preferred embodiment of the preparation method of the present invention, in step S1, the stirring speed is 400-700 rpm.

[0017] In a preferred embodiment of the preparation method described in this invention, in step S1, the sedimentation in the water-in-oil emulsion method involves standing at 23-27°C for 12-16 hours. Standing at 23-27°C for 12-16 hours effectively solidifies and settles the gelatin microspheres, facilitating collection.

[0018] In a preferred embodiment of the preparation method described in this invention, in step S1, the cleaning in the water-in-oil emulsion method involves cleaning the gelatin microspheres with cyclohexane and / or acetone. Cyclohexane and acetone can effectively remove liquid paraffin from the surface of the gelatin microspheres, preventing the liquid paraffin from affecting the subsequent preparation of the hydrogel scaffold.

[0019] In a preferred embodiment of the preparation method of the present invention, in step S2, the volume percentage of acetic acid in the acetic acid solution is 3-5%.

[0020] In a preferred embodiment of the preparation method described in this invention, in step S2, the mass ratio of chitosan to proanthocyanidins in the chitosan-proanthocyanidin solution is (50-65):(1-3). Within a specific ratio range, chitosan and proanthocyanidins can enhance the in-situ crosslinking effect of gelatin microspheres, making it easier to prepare high-porosity hydrogel scaffolds.

[0021] As a preferred embodiment of the preparation method of the present invention, in step S2, the mass ratio of chitosan to proanthocyanidins in the chitosan-proanthocyanidin solution is (55-60):(1.5-2).

[0022] As a preferred embodiment of the preparation method of the present invention, in step S3, the ratio of gelatin microspheres to chitosan-proanthocyanidin solution is gelatin microspheres: chitosan-proanthocyanidin solution = (12-15) g: 10 mL.

[0023] In a preferred embodiment of the preparation method described in this invention, in step S3, the freeze-drying process yields a freeze-dried scaffold, and the alkali treatment involves immersing the freeze-dried scaffold in a 1.5-3M sodium hydroxide solution for 8-12 minutes.

[0024] In a preferred embodiment of the preparation method of the present invention, in step S3, the freeze-drying process yields a freeze-dried scaffold, and the alkali treatment involves immersing the freeze-dried scaffold in a 2M sodium hydroxide solution for 10 minutes.

[0025] In a preferred embodiment of the preparation method of the present invention, in step S3, the alkaline treatment is followed by cleaning with deionized water.

[0026] In a preferred embodiment of the preparation method described in this invention, in step S3, the heating is performed in a water bath at 45-55°C for at least 30 minutes. In this invention, the heat treatment here is to completely dissolve the gelatin microspheres to achieve a macroporous structure.

[0027] In a preferred embodiment of the preparation method of the present invention, in step S3, the heating is performed by water bath heating at 45-55°C for 30-45 minutes.

[0028] In a preferred embodiment of the preparation method described in this invention, in step S3, the template includes, but is not limited to, at least one of a square template, a circular template, a cylindrical template, and a rhomboid template. The template selected in this invention is a commonly used template in any template leaching method, and the template size can be adjusted according to actual needs.

[0029] Secondly, the present invention provides a cell super-infiltrating hydrogel scaffold, which is mainly prepared by the above-mentioned preparation method.

[0030] The cell super-wetting hydrogel scaffold of the present invention contains macroporous and microporous structures, with the two pore structures hierarchically arranged, possessing high porosity and multi-level pore synergistic characteristics, which can achieve deep and effective cell adhesion and growth.

[0031] Thirdly, the present invention provides the application of the above-mentioned cell-super-infiltrating hydrogel scaffold in the preparation of tissue engineering scaffolds and / or wound dressings.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention mixes gelatin microspheres with chitosan-proanthocyanidin solution and constructs the structure based on template leaching and freeze-drying. Freeze-drying can form ice crystal templates in the hydrogel. After freeze-drying, the ice crystals sublimate to form a microporous structure. Further heating treatment can dissolve the gelatin microspheres and crosslink them in situ with proanthocyanidins, so that the gelatin molecules are fixed on the inner wall of the pores. This results in the formation of a macroporous structure in the hydrogel scaffold while enhancing the mechanical properties of the scaffold.

[0033] (2) The hydrogel scaffold of the present invention is obtained by mixing gelatin microspheres and chitosan-proanthocyanidin solution, placing them in a template, freeze-drying, alkali treatment, and heating, to obtain a hydrogel scaffold with high porosity and hierarchical pores. Proanthocyanidins, as polyphenol compounds, have the property of crosslinking gelatin molecules, so in-situ crosslinking of gelatin can be achieved without the addition of additional crosslinking agents. At the same time, the hydrogen bonds between chitosan molecules and the crosslinking of chitosan molecules and gelatin molecules by proanthocyanidins enable the hydrogel scaffold to maintain a large pore structure and have good mechanical stability, thus balancing the contradiction between large pore structure and mechanical stability.

[0034] (3) The cell super-wetting hydrogel scaffold of the present invention contains macroporous structure and microporous structure. The two pore structures are hierarchically arranged, which has high porosity, high cell spreading area and multi-level pore synergy characteristics, and can realize deep and effective cell adhesion and growth. Attached Figure Description

[0035] Figure 1 The preparation process of the cell super-infiltrating hydrogel scaffold described in this invention; Figure 2 The images shown are scanning electron microscope images of different cell-infiltrating hydrogel scaffolds in Example 1 of the present invention, where a is Example 1, b is Example 3, c is Example 4, and d is Example 5. Figure 3 The image shows a scanning electron microscope (SEM) image of the inner wall of the macroporous structure of the cell super-infiltrating hydrogel scaffold in Example 1 of the present invention. Figure 4 This is a scanning electron microscope image of the cell-super-infiltrating hydrogel scaffold obtained in Comparative Example 2 of Example 1 of the present invention; Figure 5The porosity statistics of different cell-super-infiltrating hydrogel scaffolds in Example 1 of the present invention are shown. Figure 6 This is a statistical result of the cell spreading area of ​​different cell-super-infiltrating hydrogel scaffolds in Example 1 of the present invention; Figure 7 The mechanical stability test results of the cell-super-infiltrating hydrogel scaffold obtained in Example 1 of the present invention are shown in Example 1. Figure 8 The images shown are laser confocal microscope images of different cell super-infiltrating hydrogel scaffolds and primary cardiomyocytes after co-culturing in Example 2 of the present invention, where a is Example 1, b is Example 3, c is Example 4, and d is Example 5. Figure 9 The images shown are laser confocal microscopy images of different cell super-infiltrating hydrogel scaffolds and primary cardiomyocytes after co-culturing in Example 2 of the present invention, where a is Comparative Example 1, b is Comparative Example 2, and c is Comparative Example 3. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0037] Unless otherwise specified, all other materials and reagents used in the examples, comparative examples, and effect examples are commercially available.

[0038] The method for verifying the average particle size of gelatin microspheres is as follows: Images of the cell-super-infiltrating hydrogel scaffold are captured by scanning electron microscopy (SEM). Using image software, diameter lines are drawn along the longest axis of the macroporous structures in the cell-super-infiltrating hydrogel scaffold. Particles with overlap, edge cuts, and roundness ≤0.25 are excluded. The diameters of at least 5 macroporous structures are measured, and the average value is calculated, which is the average particle size of the gelatin microspheres.

[0039] Rat primary cardiomyocytes were prepared according to the following literature: He Y, Li Q, Chen P, Duan Q, Zhan J, Cai X, Wang L, Hou H, Qiu X. Asmart adhesive Janus hydrogel for non-invasive cardiac repair and tissueadhesion prevention. Nat Commun. 2022 Dec 12;13(1):7666.

[0040] Example 1 This embodiment provides a cell superinfiltrating hydrogel scaffold and its preparation method. The operation flow of the preparation method is as follows: Figure 1 As shown, the specific steps include: (1) Add gelatin to water and heat to 70°C to completely dissolve the gelatin, thus preparing a 28wt% gelatin solution; (2) Slowly drop the gelatin solution obtained in step (1) into 400 mL of liquid paraffin at 70 °C, and mechanically stir at 700 rpm to obtain gelatin droplets. Continue stirring and gradually cool down. When it drops to room temperature (25 ± 2 °C), let it stand for 12 h, collect the gelatin microspheres through a sieve and wash them with cyclohexane. The washed gelatin microspheres are the gelatin microspheres obtained. At this time, the average particle size of the gelatin microspheres is 200-300 μm. (3) Chitosan and proanthocyanidins were added to a solution containing 3% v / v acetic acid at a mass ratio of 55:2 and stirred for 12 hours to obtain a chitosan-proanthocyanidin solution, wherein the final concentration of proanthocyanidins in the chitosan-proanthocyanidin solution was 2 mg / mL. (4) Mix 12g of gelatin microspheres obtained in step (2) with 10mL of chitosan-proanthocyanidin solution obtained in step (3), place them in a cylindrical template with a diameter of 11mm, freeze-dry them, and obtain a hydrogel scaffold loaded with gelatin microspheres. (5) Add the hydrogel scaffold obtained in step (4) to a 2M sodium hydroxide solution and treat for 10 minutes. After the sodium hydroxide solution fully wets the hydrogel scaffold, wash it thoroughly with deionized water and heat it in a 50°C water bath for 30 minutes to fully dissolve the gelatin microspheres and obtain a cell super-infiltrated hydrogel scaffold.

[0041] Example 2 This embodiment provides a cell superinfiltrating hydrogel scaffold and its preparation method, the preparation method including the following steps: (1) Gelatin microspheres were prepared according to steps (1) to (2) of Example 1; (2) Chitosan and proanthocyanidins were added to a solution containing 3% v / v acetic acid at a mass ratio of 60:1.5 and stirred for 12 hours to obtain a chitosan-proanthocyanidin solution, wherein the final concentration of proanthocyanidins in the chitosan-proanthocyanidin solution was 1.5 mg / mL. (3) Mix 15g of gelatin microspheres obtained in step (1) with 10mL of chitosan-proanthocyanidin solution obtained in step (2), place them in a cylindrical template with a diameter of 11mm, freeze dry, and obtain a hydrogel scaffold loaded with gelatin microspheres. (4) Add the hydrogel scaffold obtained in step (3) to a 2M sodium hydroxide solution and treat for 10 minutes to allow the sodium hydroxide solution to fully wet the hydrogel scaffold. Then rinse thoroughly with deionized water and heat in a 70°C water bath for 45 minutes to fully dissolve the gelatin microspheres and obtain a cell super-infiltrated hydrogel scaffold.

[0042] Example 3 This embodiment provides a cell superinfiltrating hydrogel scaffold and its preparation method, the preparation method including the following steps: (1) Add gelatin to water and heat to 70°C to completely dissolve the gelatin, thus preparing a 30wt% gelatin solution; (2) Slowly drop the gelatin solution obtained in step (1) into 500 mL of liquid paraffin at 70 °C, and mechanically stir at 500 rpm to obtain gelatin droplets. Continue stirring and gradually cool down. When it drops to room temperature (25 ± 2 °C), let it stand for 12 h, collect the gelatin microspheres through a sieve and wash them with cyclohexane. The washed gelatin microspheres are obtained. At this time, the average particle size of the gelatin microspheres is 300-400 μm. (3) Cell super-infiltrating hydrogel scaffolds were prepared according to steps (3) to (5) in Example 1.

[0043] Example 4 This embodiment provides a cell super-infiltrating hydrogel scaffold and its preparation method. The preparation method is similar to that in Embodiment 2, except that in step (1), the average particle size of the gelatin microspheres is 400-500 μm, and the other steps and parameters remain unchanged.

[0044] Example 5 This embodiment provides a cell super-infiltrating hydrogel scaffold and its preparation method. The preparation method is similar to that in Embodiment 2, except that in step (1), the average particle size of the gelatin microspheres is 500-600 μm, and the other steps and parameters remain unchanged.

[0045] Comparative Example 1 This comparative example provides a cell super-infiltrating hydrogel scaffold and its preparation method. The preparation method is similar to that of Example 4, except that: in step (2), proanthocyanidins are not added, and the prepared product is a chitosan solution. The remaining steps and parameters remain unchanged.

[0046] Comparative Example 2 This comparative example provides a cell super-infiltrating hydrogel scaffold and its preparation method. The preparation method is similar to that of Example 4, except that in step (3), the gelatin microspheres obtained in step (1) are not added, while the remaining steps and parameters remain unchanged.

[0047] Comparative Example 3 This comparative example provides a cell super-infiltrating hydrogel scaffold and its preparation method. The preparation method is similar to that of Example 4, except that in step (3), the amount of gelatin microspheres used is 8g, and the other steps and parameters remain unchanged.

[0048] Example 1 1. The cell-super-infiltrating hydrogel scaffolds of Examples 1-5 and Comparative Examples 1-2 were observed by scanning electron microscopy. The results are shown in the figure below. Figure 2-4 .

[0049] like Figure 2 As shown, hydrogel scaffolds prepared from gelatin microspheres of different sizes can exhibit macroporous structures with different pore sizes. Therefore, hydrogel scaffolds with macroporous structures of different pore sizes can be prepared according to requirements.

[0050] like Figure 3 As shown in the magnified view of the inner wall of the cell super-infiltrating hydrogel scaffold in Example 1, there are some gelatin fiber residues on the inner wall of the pores. The gelatin molecules that are cross-linked and retained by proanthocyanidins and the proanthocyanidins can synergistically promote cell adhesion on the inner wall of the pores.

[0051] like Figure 4 As shown, the cell super-infiltrating hydrogel scaffold in Comparative Example 2 does not have a significant macroporous structure; it only has a pore structure constructed from ice crystals, and cannot achieve the characteristics of having both macroporous and microporous structures.

[0052] 2. The porosity of the cell-super-infiltrating hydrogel scaffolds obtained in Example 1 and Comparative Example 2 was measured according to the method described in Section 2.3, Part 2 of Reference 1. The results are shown in [Figure 1]. Figure 5 .

[0053] Document 1: Li Z, et al. Fabrication of regular macro-mesoporous reduced graphene aerogel beads with ultra-high mechanical property for efficient bilirubin adsorption. Mater Sci Eng C Mater Biol Appl. 2020Jan;106:110282. like Figure 5 As shown, the porosity of the cell super-infiltrating hydrogel scaffold obtained in Example 1 is 94.4%, while the porosity of the cell super-infiltrating hydrogel scaffold obtained in Comparative Example 2 is 81.3%. Moreover, the porosity of Example 1 is significantly higher than that of Comparative Example 2 (p < 0.05), indicating that the cell super-infiltrating hydrogel scaffold of this application has more porous structures and can further improve the cell loading capacity.

[0054] 3. The cell superinfiltrating hydrogels obtained in Example 1 and Comparative Example 2 were compared with 5×10 6 Primary mouse cardiomyocytes were co-cultured for 5 days, and the cell spreading area was calculated using ImageJ software. The results are shown below. Figure 6 .

[0055] like Figure 6 As shown, the cell spreading area of ​​the cell-super-infiltrating hydrogel scaffold obtained in Example 1 was 27.64%, while that of the cell-super-infiltrating hydrogel scaffold obtained in Comparative Example 2 was 5.60%. The cell spreading area of ​​Example 1 was significantly higher than that of Comparative Example 2 (p < 0.05), indicating that the cell-super-infiltrating hydrogel scaffold of this application can accommodate more cells, which helps the hydrogel scaffold release more target cells to the affected area in vivo.

[0056] 4. The cell-super-infiltrating hydrogel scaffold obtained in Example 1 was placed in a universal testing machine for cyclic compression experiments. The parameters were set to 60% compressive strain, and the compression rate was 100 mm / min. The number of cyclic compressions was 5000. The results are as follows: Figure 7 As shown, after 5000 cycles, its mechanical stability is good, indicating that the cell super-infiltrating hydrogel scaffold of the present invention has good mechanical properties.

[0057] Example 2 The cell infiltration was observed after co-culturing the cell-superinfiltrating hydrogel scaffolds of Example 4 and Comparative Examples 1-3 with primary cardiomyocytes, as follows: 5×10 6 Primary cardiomyocytes were cultured in a superinfiltrated hydrogel scaffold for 5 days and observed using a laser confocal microscope. Results are shown below. Figure 8-9 .

[0058] like Figure 8 As shown, the cell penetration depth increases with the increase of the macropore size of the cell superinfiltrated hydrogel scaffold, indicating that primary cardiomyocytes can not only effectively adhere and permeate in the cell superinfiltrated hydrogel scaffold, but also penetrate deep into the hydrogel scaffold to achieve cell superinfiltration, and the cells are in good condition, indicating that the cell superinfiltrated hydrogel scaffold has good safety.

[0059] like Figure 9 As shown, in the hydrogel scaffold without proanthocyanidins (Comparative Example 1), although primary cardiomyocytes could penetrate well into the macroporous structure, the cardiomyocytes' extension state was poor. Figure 9 a); In the non-macroporous hydrogel scaffold (Comparative Example 2), most of the primary cardiomyocytes remained on the surface of the hydrogel scaffold and did not penetrate into the interior of the hydrogel. Figure 9 (b) indicates that macroporous structure is one of the key factors for cell hyperinfiltration; in Comparative Example 3, most of the primary cardiomyocytes remained on the surface of the hydrogel scaffold, and although some cells infiltrated inside the hydrogel scaffold, the number was lower than that of the hydrogel scaffold in the examples (b). Figure 9c), which indicates that only when the solid-liquid ratio of the gelatin microsphere mass to the chitosan-proanthocyanidin solution is greater than 1 can a macroporous structure and cell superinfiltration be achieved.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a cell-super-infiltrating hydrogel scaffold, characterized in that, Includes the following steps: S1. Gelatin microspheres were prepared from gelatin using a water-in-oil emulsion method. S2. Mix chitosan, proanthocyanidins and acetic acid solution to obtain chitosan-proanthocyanidin solution; S3. The gelatin microspheres described in step S1 are mixed with the chitosan-proanthocyanidin solution described in step S2 and placed in a template. The mixture is then freeze-dried, treated with alkali, and heated to obtain a cell-super-infiltrating hydrogel scaffold.

2. The preparation method according to claim 1, characterized in that, In step S1, the average particle size of the gelatin microspheres is 200-600 μm.

3. The preparation method according to claim 1, characterized in that, In step S1, the water-in-oil emulsion method mainly involves adding a 26-30 wt% gelatin solution to liquid paraffin, stirring, allowing it to settle, sieving, and washing to obtain gelatin microspheres.

4. The preparation method according to claim 1, characterized in that, In step S2, the volume percentage of acetic acid in the acetic acid solution is 3-5%.

5. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of chitosan to proanthocyanidins in the chitosan-proanthocyanidin solution is (50-65):(1-3).

6. The preparation method according to claim 1, characterized in that, In step S3, the ratio of gelatin microspheres to chitosan-proanthocyanidin solution is gelatin microspheres: chitosan-proanthocyanidin solution = (12-15) g: 10 mL.

7. The preparation method according to claim 1, characterized in that, In step S3, the freeze-drying process yields a freeze-dried scaffold, and the alkali treatment involves immersing the freeze-dried scaffold in a 1.5-3M sodium hydroxide solution for 8-12 minutes.

8. The preparation method according to claim 1, characterized in that, In step S3, the heating is performed by water bath heating at 45-55°C for at least 30 minutes.

9. A cell-super-infiltrating hydrogel scaffold, characterized in that, It is mainly prepared by the preparation method described in any one of claims 1-8.

10. The use of the cell-super-infiltrating hydrogel scaffold as described in claim 9 in the preparation of tissue engineering scaffolds and / or wound dressings.

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