Preparation method of cell-loaded silk fibroin hydrogel fiber and fiber

Abstract

The invention relates to a preparation method of cell-loaded silk fibroin hydrogel fibers and fibers, the preparation method of the cell-loaded silk fibroin hydrogel fibers comprises the following steps: mixing a silk fibroin solution with a riboflavin solution to obtain a mixed solution, adding a hydrogen peroxide solution into the mixed solution to obtain a precursor solution, and carrying out ultrasonic treatment on the precursor solution to obtain the cell-loaded silk fibroin hydrogel fibers. Adding stem cells into the precursor solution, and then carrying out photocuring reaction under the irradiation of an ultraviolet lamp to prepare the hydrogel fiber. The fiber is obtained through the preparation method. According to the preparation method of the cell-loaded silk fibroin hydrogel fiber and the fiber, the gel forming time is greatly shortened, efficient and uniform loading of cells is achieved, the survival rate of the cells is increased, no organic reagent residues exist in the preparation process, operation is easy, the technological process is short, and the method is suitable for industrial production. The prepared fiber is free of biotoxicity, excellent in mechanical property, good in light transmission and good in biocompatibility, and has certain potential and application prospects in the biomedical field.

Description

Technical Field

[0001] The invention relates to a preparation method of hydrogel fiber and the fiber, belonging to the technical field of polymer gel, and in particular to a preparation method of cell-loaded silk fibroin hydrogel fiber and the fiber. Background Art

[0002] Hydrogel refers to a material that can absorb a large amount of water without dissolving. There are many methods for preparing hydrogels, such as chemical crosslinking, physical crosslinking, and biocatalysis. Different preparation methods can produce hydrogels with different properties to meet the application needs of different fields, such as facial masks, fever-reducing patches, preservatives in the food industry, and drug carriers in the medical field. Silk fibroin is a natural protein extracted from silk. It has excellent mechanical properties, processability, biocompatibility, and biodegradability. It has received widespread attention in the fields of tissue engineering and drug sustained release.

[0003] The Chinese invention patent with application number 201811006463.3 and application date 2018.08.30 discloses a silk fibroin photocurable hydrogel and a preparation method thereof. The preparation method uses silk fibroin as raw material and prepares a silk fibroin hydrogel with an interpenetrating network structure by ultraviolet light curing. The monomer N-vinyl pyrrolidone is dissolved in the silk fibroin solution, and the N-vinyl pyrrolidone is polymerized by ultraviolet light and horseradish peroxidase to form a polyvinyl pyrrolidone macromolecule that is physically entangled with the silk fibroin molecular chain, thereby obtaining a new type of silk fibroin hydrogel with good light transmittance and excellent degradation performance. However, since this method adds more chemical reagents inside the gel, it will have a greater impact on the biocompatibility of the material, thereby causing damage to the loaded cells, and the survival rate of the loaded cells is too low.

[0004] The information disclosed in this background technology section is only intended to increase the understanding of the overall background of the application, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art already known to ordinary technicians in this field. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem of low cell survival rate of silk fibroin hydrogel loaded in the above-mentioned prior art, and to provide a preparation method and fiber of silk fibroin hydrogel fiber loaded with cells with high cell survival rate.

[0006] To achieve the above objectives, the technical solution of the present invention is: A method for preparing cell-loaded silk fibroin hydrogel fibers comprises the following steps: The first step is to mix the silk fibroin solution with the riboflavin aqueous solution to obtain a mixed solution, then add the hydrogen peroxide solution to the mixed solution to obtain a precursor solution, and then add the cells to the precursor solution to obtain a gel precursor solution; In the second step, the obtained gel precursor solution is first irradiated under ultraviolet light to perform a photocuring reaction to obtain a silk fibroin hydrogel, and then the obtained silk fibroin hydrogel is made into a filament to obtain a cell-loaded silk fibroin hydrogel fiber.

[0007] In the first step, the method for adding the cells to the precursor solution for mixing is pipetting.

[0008] The blowing refers to using a blowing tool to control the air flow and liquid flow to evenly disperse the cells in the precursor solution, and the number of blowing is 3-5 times.

[0009] In the first step, the concentration of the silk fibroin solution is 1.5-7wt%, the concentration of the riboflavin aqueous solution is 0.05-10mmol / L, and the volume ratio of the silk fibroin solution to the riboflavin aqueous solution is 5-20:1.

[0010] In the first step, the volume percentage concentration of the hydrogen peroxide solution is 5-30%, and the volume ratio of the hydrogen peroxide solution to the mixed solution is 1:5-30.

[0011] In the second step, the power of the ultraviolet lamp is 100-900W, and the time of the photocuring reaction is 2-30 minutes.

[0012] In the second step, the gel precursor solution is first loaded into a light-transmitting syringe and then placed under an ultraviolet lamp for photocuring reaction. After the gel is formed, the piston core rod of the syringe is pressed to squeeze the gel out of the syringe to obtain a filament, which is the cell-loaded silk protein hydrogel fiber.

[0013] The syringe is a 5 mL syringe with a needle size of 27G.

[0014] In the first step, the cell density in the gel precursor solution is 100,000-1,000,000 cells / mL, the cells remain active, and the cells are any one of stem cells, osteoblasts or fibroblasts.

[0015] A fiber is prepared by the method for preparing a cell-loaded silk fibroin hydrogel fiber.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. In the preparation method of a cell-loaded silk fibroin hydrogel fiber of the present invention, a silk fibroin solution is used as a base, a riboflavin solution is used as a photocrosslinker and mixed with the silk fibroin solution to obtain a mixed solution, and then a hydrogen peroxide solution is added to the mixed solution as a catalyst to obtain a precursor solution, and then the cells are added to the precursor solution and mixed, and the silk fibroin hydrogel is solidified under ultraviolet light to obtain a filamentous fiber. In this preparation method, riboflavin can be excited to produce a triplet state under ultraviolet light, react with oxygen to form oxygen free radicals, and oxygen free radicals can promote the mutual entanglement between silk fibroin macromolecules to form a gel, and hydrogen peroxide can decompose and produce oxygen under ultraviolet light, which can be used as an oxygen source to provide more oxygen for the system, promote the reaction, and greatly shorten the time for hydrogel formation, so that cells can be quickly solidified in the hydrogel, avoiding cell deposition. Therefore, the present invention not only has a short gel time, but also can realize efficient and uniform loading of cells.

[0017] 2. In the preparation method of a cell-loaded silk fibroin hydrogel fiber of the present invention, a light-curing method is used to prepare the silk fibroin hydrogel, and cross-linking is formed by mutual entanglement between silk fibroin macromolecules. The internal crystalline structure is low, and the internal β-folding structure of the silk fibroin is very small, and the main structure is random coil and α-helix structure. Therefore, the present invention not only has good light transmittance, but also has extremely high mechanical compression resilience and excellent biocompatibility.

[0018] 3. In the preparation method of a cell-loaded silk fibroin hydrogel fiber of the present invention, a syringe is used to make a silk fibroin hydrogel block into a filamentous fiber. This is because compared with a hydrogel block, the hydrogel fiber is more uniform in loading cells, and the hydrogel fiber loaded with cells has a larger area in contact with the culture medium during cultivation, which is convenient for the exchange of cell nutrition and metabolic waste, thereby facilitating the survival of cells. In addition, the hydrogel fiber has good injectability and shape adaptability, which protects the loaded cells from damage such as external mechanical stress and chemical substances to a certain extent, and maintains the stability of the cells. Therefore, the present invention can not only efficiently and uniformly load cells, but also improve the survival rate of loaded cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a physical picture of the cell-loaded silk fibroin hydrogel fiber prepared in Example 1.

[0020] Figure 2 This is a fluorescent staining image of the cell-loaded silk fibroin hydrogel fiber prepared in Example 1.

[0021] Figure 3 : This is the XRD pattern of the cell-loaded silk fibroin hydrogel prepared in Example 1.

[0022] Figure 4 This is a graph showing the resilience test of the cell-loaded silk fibroin hydrogel prepared in Example 1.

[0023] Figure 5 This is a graph showing the compressive strength test of the cell-loaded silk fibroin hydrogel prepared in Example 1 and pure silk fibroin hydrogel. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0025] See also Figure 1 — Figure 5 , a method for preparing cell-loaded silk fibroin hydrogel fibers, comprising the following steps: The first step is to mix the silk fibroin solution with the riboflavin aqueous solution to obtain a mixed solution, then add the hydrogen peroxide solution to the mixed solution to obtain a precursor solution, and then add the cells to the precursor solution to obtain a gel precursor solution; In the second step, the obtained gel precursor solution is first irradiated under ultraviolet light to perform a photocuring reaction to obtain a silk fibroin hydrogel, and then the obtained silk fibroin hydrogel is made into a filament to obtain a cell-loaded silk fibroin hydrogel fiber.

[0026] In the first step, the method for adding the cells to the precursor solution for mixing is pipetting.

[0027] The blowing refers to using a blowing tool to control the air flow and liquid flow to evenly disperse the cells in the precursor solution, and the number of blowing is 3-5 times.

[0028] In the first step, the concentration of the silk fibroin solution is 1.5-7wt%, the concentration of the riboflavin aqueous solution is 0.05-10mmol / L, and the volume ratio of the silk fibroin solution to the riboflavin aqueous solution is 5-20:1.

[0029] In the first step, the volume percentage concentration of the hydrogen peroxide solution is 5-30%, and the volume ratio of the hydrogen peroxide solution to the mixed solution is 1:5-30.

[0030] In the second step, the power of the ultraviolet lamp is 100-900W, and the time of the photocuring reaction is 2-30 minutes.

[0031] In the second step, the gel precursor solution is first loaded into a light-transmitting syringe and then placed under an ultraviolet lamp for photocuring reaction. After the gel is formed, the piston core rod of the syringe is pressed to squeeze the gel out of the syringe to obtain a filament, which is the cell-loaded silk protein hydrogel fiber.

[0032] The syringe is a 5 mL syringe with a needle size of 27G.

[0033] In the first step, the cell density in the gel precursor solution is 100,000-1,000,000 cells / mL, the cells remain active, and the cells are any one of stem cells, osteoblasts or fibroblasts.

[0034] A fiber is prepared by the method for preparing a cell-loaded silk fibroin hydrogel fiber.

[0035] Embodiment 1: A 3wt% silk fibroin solution and a 0.5mmol / L riboflavin aqueous solution were mixed at a volume ratio of 20:1 to obtain a mixed solution, and then a 30vol% hydrogen peroxide solution was added to the mixed solution at a volume ratio of 1:30 to the mixed solution, and the precursor solution was obtained after sufficient mixing.

[0036] The cells are added to the obtained precursor solution, and the gel precursor solution is obtained after repeated blowing and mixing, and the cell density in the solution is 1 million / mL. The gel precursor solution is loaded into a 5mL light-transmitting syringe, and then placed under ultraviolet light for light curing. After the gel is formed, it is squeezed out of the syringe to obtain a filament, which is the silk protein hydrogel fiber loaded with cells. The power of the above-mentioned ultraviolet lamp is 400W, the light curing reaction time is 2min, and the syringe needle size is 27G.

[0037] The obtained cell-loaded silk fibroin hydrogel fibers were immersed in a culture dish filled with deionized water solution and observed under ultraviolet light. Figure 1 It can be observed that the hydrogel fibers can be suspended in the aqueous solution and are in the form of uniform and continuous filaments with good molding.

[0038] The cell-loaded silk fibroin hydrogel fibers were stained with CAM / PI counterstaining solution and observed under a fluorescence microscope. Figure 2 As shown, green cells are live cells and red cells are dead cells. It can be seen that the cells are evenly distributed, and green live cells are in the majority, with only a small number of dead cells. The reason for cell death is that ultraviolet light is introduced during the photocuring process. A certain amount of ultraviolet radiation can cause cell apoptosis, resulting in reduced cell activity or even death. However, only a small number of cells in the cell-loaded silk fibroin hydrogel fiber are apoptotic, indicating that the photocuring method causes less damage to cells and will not cause large-scale death. The cells in the hydrogel fiber all maintain a high level of activity, confirming that the photocured silk fibroin hydrogel fiber has excellent biocompatibility, which can not only efficiently and evenly load cells, but also improve cell survival rate.

[0039] In order to test the light transmittance of a cell-loaded silk fibroin hydrogel fiber of the present invention, the silk fibroin hydrogel prepared in Example 1 was characterized by XRD. The results are as follows: Figure 3 As shown, it can be seen that the prepared silk fibroin hydrogel has a diffraction peak at 19.7°, which mainly shows the silkI structure, indicating that the silk fibroin mainly reacts in the non-crystalline region during the formation of the hydrogel. This shows that the silk fibroin hydrogel has good light transmittance, and thus it is concluded that the cell-loaded silk fibroin hydrogel fiber of the present invention has good light transmittance.

[0040] In order to test the resilience of a cell-loaded silk fibroin hydrogel fiber of the present invention, the silk fibroin hydrogel prepared in Example 1 was tested. The silk fibroin hydrogel was prepared into a cylinder with a diameter of 2 mm and a height of 5 mm. A compression mechanical test was performed on a TMS-PRO texture analyzer. The test mainly performed three compressions, the first compression deformation was 20%, the second compression deformation was 40%, and the third compression deformation was 60%. The stress-strain curves of the silk fibroin hydrogel during compression and rebound are shown in FIG. Figure 4 As shown, it can be seen that the area enclosed by the closed curve formed by the loading curve and the unloading curve is very small, that is, the elastic loss during the cyclic compression process is very small, indicating that the light-cured silk fibroin hydrogel prepared by the present invention has high elastic properties, thereby concluding that the cell-loaded silk fibroin hydrogel fiber of the present invention has good resilience.

[0041] In order to test the compressive strength of a cell-loaded silk fibroin hydrogel fiber of the present invention, the silk fibroin hydrogel prepared in Example 1 was prepared into a cylinder with a diameter of 2 mm and a height of 5 mm, and a compression mechanical test was performed on a TMS-PRO texture analyzer, and its deformation was set to 90%. Pure silk fibroin hydrogel was used as a control sample. The pure silk fibroin hydrogel was obtained by ultrasonic treatment of a 5wt% silk fibroin solution under 300W power for 1 min. Figure 5 It can be seen that the pure silk hydrogel ruptured when the compression deformation reached 40%, and its compression strength was about 20 kPa. The light-cured silk protein hydrogel in this embodiment began to rupture when the deformation reached 82%, and its compression strength reached about 80 kPa. Therefore, it is concluded that the cell-loaded silk protein hydrogel fiber of the present invention has extremely high compression resistance.

[0042] Embodiment 2: The basic content is the same as that of Example 1, except that: 30 g of raw silk was placed in 900 mL of 0.1 wt% sodium carbonate aqueous solution and boiled for 1.5 h to obtain degumming treatment to obtain degummed silk fibroin fibers. The degummed silk fibroin fibers were immersed in 300 mL of 10 mol / L LiBr aqueous solution and heated to 80°C to dissolve for 3 h to obtain a crude solution. The crude solution was placed in a dialysis bag, and the dialysis bag was dialyzed in 0°C deionized water for 5 days. The dialyzed solution was filtered to remove impurities to obtain a silk fibroin solution. The silk fibroin solution was put into an aluminum box and frozen at -80°C for 4 h, then put into a vacuum dryer. After three days, it was taken out to obtain the re-dissolved silk fibroin, which was put into a sealed bag for later use.

[0043] The silk fibroin and riboflavin powder were sterilized by ultraviolet light for 20 minutes.

[0044] The obtained re-dissolved silk fibroin was re-dissolved to obtain a silk fibroin solution with a mass fraction of 3 wt%.

[0045] The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiment. Any equivalent modifications or changes made by ordinary technicians in this field based on the contents disclosed by the present invention should be included in the protection scope recorded in the claims.

Claims

1. A method for preparing cell-loaded silk fibroin hydrogel fibers, characterized in that: The preparation method comprises the following steps: The first step is to mix the silk fibroin solution with the riboflavin aqueous solution to obtain a mixed solution, then add the hydrogen peroxide solution to the mixed solution to obtain a precursor solution, and then add the cells to the precursor solution to obtain a gel precursor solution; In the second step, the obtained gel precursor solution is first irradiated under ultraviolet light to perform a photocuring reaction to obtain a silk fibroin hydrogel, and then the obtained silk fibroin hydrogel is made into a filament to obtain a cell-loaded silk fibroin hydrogel fiber.

2. The method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 1, characterized in that: In the first step, the method for adding the cells to the precursor solution for mixing is pipetting.

3. The method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 2, characterized in that: The blowing refers to using a blowing tool to control the air flow and liquid flow to evenly disperse the cells in the precursor solution, and the number of blowing is 3-5 times.

4. The method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 1, 2 or 3, characterized in that: In the first step, the concentration of the silk fibroin solution is 1.5-7wt%, the concentration of the riboflavin aqueous solution is 0.05-10mmol / L, and the volume ratio of the silk fibroin solution to the riboflavin aqueous solution is 5-20:

1.

5. The method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 1, 2 or 3, characterized in that: In the first step, the volume percentage concentration of the hydrogen peroxide solution is 5-30%, and the volume ratio of the hydrogen peroxide solution to the mixed solution is 1:5-30.

6. The method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 1, 2 or 3, characterized in that: In the second step, the power of the ultraviolet lamp is 100-900W, and the time of the photocuring reaction is 2-30 minutes.

7. The method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 1, 2 or 3, characterized in that: In the second step, the gel precursor solution is first loaded into a light-transmitting syringe and then placed under an ultraviolet lamp for photocuring reaction. After the gel is formed, the piston core rod of the syringe is pressed to squeeze the gel out of the syringe to obtain a filament, which is the cell-loaded silk protein hydrogel fiber.

8. The method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 7, characterized in that: The syringe is a 5 mL syringe with a needle size of 27G.

9. The method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 1, 2 or 3, characterized in that: In the first step, the cell density in the gel precursor solution is 100,000-1,000,000 cells / mL, the cells remain active, and the cells are any one of stem cells, osteoblasts or fibroblasts.

10. A fiber, characterized in that: The fiber is prepared by the method for preparing a cell-loaded silk fibroin hydrogel fiber according to claim 1, 2 or 3.

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