Preparation method of an absorbable silk fibroin biomembrane with a bilayer structure

Abstract

The present invention discloses a preparation method of an absorbable silk fibroin biomembrane with a double-layer structure, comprising the steps of: (1) mixing an aqueous solution of silk fibroin I with a film-forming agent, coating the mixture on the surface of a substrate and drying it to form a dense layer; (2) mixing an aqueous solution of silk fibroin II with a cross-linking agent, coating the mixture on the surface of the dense layer and thermally cross-linking it to form a hydrogel film; (3) drying the hydrogel film to form a loose layer, removing the substrate, and obtaining an absorbable silk fibroin biomembrane with a double-layer structure. The absorbable silk fibroin biomembrane prepared by the method of the present invention has good mechanical properties and biocompatibility, can effectively prevent soft tissue cells from growing and migrating into the defect area, and promote the growth of osteoblasts in the defect area to heal the wound.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and more specifically, to a preparation method of an absorbable silk fibroin biomembrane with a bilayer structure. Background Art

[0002] Soft tissue repair is one of the key fields of medical research, and biomaterials play an indispensable role. Although traditional repair materials such as silicone and polyethylene have achieved certain results in clinical applications, they have problems such as being prone to infection, displacement, and tissue rejection. In view of this, scientific researchers have been constantly seeking innovative biomaterials to optimize the effect of soft tissue repair, aiming to overcome the limitations of existing materials and improve the safety and effectiveness of treatment.

[0003] Silk fibroin, as a natural polymer material, has good biocompatibility and biodegradability and has been widely used in the medical field. Silk fibroin can be made into materials with different morphologies such as films, scaffolds, and microspheres, and is used in fields such as tissue engineering, drug delivery, and hemostatic materials. However, traditional silk fibroin materials have disadvantages such as poor mechanical properties and being easily degraded by enzymes, which limit their application in soft tissue repair.

[0004] In recent years, some researchers have compounded silk fibroin with film-forming agents, cross-linking agents, etc. to prepare an absorbable silk fibroin biomembrane with a bilayer structure. This biomembrane has good mechanical properties and biocompatibility, and can effectively prevent the growth and migration of soft tissue cells into the defect area, and promote the growth of osteoblasts in the defect area to heal the wound. However, there are some problems in the preparation process of the existing absorbable silk fibroin biomembrane, such as uneven coating and insufficient cross-linking, which affect the performance and application effect. Therefore, although the absorbable silk fibroin biomembrane has certain application prospects in soft tissue repair, there are still some problems to be solved in the existing preparation methods. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a preparation method of an absorbable silk fibroin biomembrane with a bilayer structure. This absorbable silk fibroin biomembrane has good mechanical properties and biocompatibility, and can effectively prevent the growth and migration of soft tissue cells into the defect area, and promote the growth of osteoblasts in the defect area to heal the wound.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A preparation method of an absorbable silk fibroin biomembrane with a bilayer structure, comprising the following steps: (1) Mix an aqueous solution I of silk fibroin with a film-forming agent, coat the mixture on the surface of a substrate, and dry it to form a dense layer; (2) Mix an aqueous solution II of silk fibroin with a crosslinking agent, coat the mixture on the surface of the dense layer, and thermally crosslink it to form a hydrogel film; (3) Dry the hydrogel film to form a loose layer, remove the substrate, and obtain an absorbable silk fibroin biomembrane with a bilayer structure.

[0008] Further, in step (1), the concentration of the aqueous solution I of silk fibroin is 1-10%; the mass ratio of the aqueous solution I of silk fibroin to the film-forming agent is 1:1-1:0.1; the substrate is a PET release film.

[0009] Further, the film-forming agent is sodium hyaluronate, chitosan, gelatin or collagen.

[0010] Further, in step (1), the coating method is a doctor blade coating method, the coating speed is 20-40 m / min, the wet film thickness is 200-300 μm, and the coating temperature is 20-30 °C.

[0011] Further, in step (1), the drying temperature is 30-50 °C.

[0012] Further, the hydrogel film is prepared by a casting method. The specific steps are as follows: Prepare an aqueous solution II of silk fibroin, add a crosslinking agent to the aqueous solution II of silk fibroin and mix evenly to obtain a silk fibroin / crosslinking agent mixture; Fix a mold on the dense layer substrate, pour the silk fibroin / crosslinking agent mixture, scrape it flat, control the wet film thickness to be 0.2-2 mm, and then place it in an oven for thermal crosslinking to obtain a hydrogel film loaded on the surface of the dense layer.

[0013] Further, the concentration of the aqueous solution II of silk fibroin is 9-40%; the addition amount of the crosslinking agent is 0.01-0.05% of the mass of the aqueous solution II of silk fibroin.

[0014] Further, the crosslinking agent is horseradish peroxidase, glutaraldehyde, genipin.

[0015] Further, the thermal crosslinking temperature is 35-60 °C.

[0016] Further, the hydrogel film is dried by a thermal drying method. The drying temperature is 35-40 °C, the humidity is 75-90%, and the drying time is 24-48 h.

[0017] The beneficial effects of the present invention are:

[0018] The present invention uses silk fibroin and a film-forming agent as raw materials to prepare a dense-layer base film on the surface of a substrate, uses silk fibroin and a cross-linking agent as raw materials to prepare a hydrogel film on the surface of the dense layer, and dries the hydrogel film to form a loose layer, finally obtaining an absorbable silk biomembrane with a bilayer structure, which has good mechanical properties and biocompatibility, can effectively prevent soft tissue cells from growing and migrating into the defect area, and promote the growth of osteoblasts in the defect area to heal the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 SEM image of the absorbable silk biomembrane prepared in Example 1;

[0020] Figure 2 CCK-8 cell experiment results of the absorbable silk biomembrane prepared in Example 1 at 1 day, 3 days, and 5 days. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] A preparation method of an absorbable silk biomembrane with a bilayer structure according to the present invention includes the steps of: (1) mixing an aqueous solution of silk fibroin I with a film-forming agent and then coating the mixture on the surface of a substrate and drying to form a dense layer; (2) mixing an aqueous solution of silk fibroin II with a cross-linking agent and then coating the mixture on the surface of the dense layer and thermally cross-linking to form a hydrogel film; (3) drying the hydrogel film to form a loose layer, and removing the substrate to obtain an absorbable silk biomembrane with a bilayer structure. Specifically:

[0023] Step (1): Weigh the regenerated silk fibroin, add purified water, stir and dissolve it at room temperature to prepare an aqueous solution of silk fibroin I with a concentration of 1-10%; add a film-forming agent (the film-forming agent is sodium hyaluronate, chitosan, gelatin or collagen; the mass ratio of the aqueous solution of silk fibroin I to the film-forming agent is 1:1-1:0.1), stir evenly at room temperature to form a silk fibroin / film-forming agent mixed solution; uniformly coat the silk fibroin / film-forming agent mixed solution on the substrate by a doctor blade method (the doctor blade speed is 20-40 m / min, the wet film thickness is 200-300 μm, the doctor blade temperature is 20-30 °C, and the substrate is a PET release film), and then place it in an oven at 30-50 °C to dry to obtain a dense-layer base film.

[0024] Step (2): Weigh the regenerated silk fibroin, add purified water and stir to dissolve at room temperature to prepare an aqueous silk fibroin solution II with a concentration of 9-40%; add a cross-linking agent with an addition amount of 0.01-0.05% of the mass of the aqueous silk fibroin solution II (the cross-linking agent is horseradish peroxidase, glutaraldehyde, genipin or a mixture of genipin and thermosensitive hydrogel microcapsules of pink lotus essential oil, and the mass ratio of genipin to thermosensitive hydrogel microcapsules of pink lotus essential oil is 1:0.1-0.5, and the thermosensitive hydrogel microcapsules of pink lotus essential oil are prepared according to the method in the paper (Analysis of Chemical Components of Pink Lotus Essential Oil and Its Hydrogel Promoting Wound Healing [D]. Sun Runzhou. Qingdao University of Science and Technology, No. 01, 2023)), stir evenly at room temperature; add hydrogen peroxide with an addition amount of 0.1-0.5% of the mass of the aqueous silk fibroin solution II to obtain a silk fibroin / cross-linking agent mixture; fix a mold on the dense layer substrate, pour in the silk fibroin / cross-linking agent mixture, scrape it flat, control the wet film thickness to be 0.2-2 mm, and then place it in an oven for thermal cross-linking (the thermal cross-linking temperature is 35-60 °C) to obtain a hydrogel film loaded on the surface of the dense layer.

[0025] Step (3): Put the hydrogel film loaded on the surface of the dense layer film into a constant temperature and humidity box for drying. The constant temperature and humidity box is set with a drying temperature of 35-40 °C, a humidity of 75-90%, and a drying time of 24-48 h. After drying, take out the film sample from the box, and detach the absorbable silk fibroin biomembrane from the surface of the PET release film.

[0026] The preferred embodiment is:

[0027] Example 1

[0028] Step (1): Weigh the regenerated silk fibroin, add purified water, and stir to dissolve at room temperature to prepare an aqueous silk fibroin solution I with a concentration of 1%; add sodium hyaluronate (the mass ratio of the aqueous silk fibroin solution I to sodium hyaluronate is 1:1), stir evenly at room temperature to form a silk fibroin / film-forming agent mixed solution; evenly coat the silk fibroin / film-forming agent mixed solution on the surface of the PET release film by the doctor blade method (the doctor blade speed is 20 m / min, the wet film thickness is 200 μm, and the doctor blade temperature is 25 °C), and then place it in an oven at 40 °C for drying to obtain a dense layer base film.

[0029] Step (2): Weigh the regenerated silk fibroin, add purified water and stir to dissolve at room temperature to prepare an aqueous silk fibroin solution II with a concentration of 10%; add horseradish peroxidase with an addition amount of 0.01% of the mass of the aqueous silk fibroin solution II, and stir evenly at room temperature; add hydrogen peroxide with an addition amount of 0.1% of the mass of the aqueous silk fibroin solution II to obtain a silk fibroin / crosslinking agent mixture; fix a mold on the dense layer substrate, pour in the silk fibroin / crosslinking agent mixture, scrape flat (control the wet film thickness to be 1 mm), and then place it in an oven for thermal crosslinking at 40 °C to obtain a hydrogel film loaded on the surface of the dense layer.

[0030] Step (3): Place the hydrogel film loaded on the surface of the dense layer film into a constant temperature and humidity chamber for drying. The constant temperature and humidity chamber is set with a drying temperature of 37 °C, a humidity of 85%, and a drying time of 24 h. After drying, take out the film sample from the chamber, and detach the absorbable silk fibroin biomembrane from the surface of the PET release film.

[0031] Test results:

[0032] The cross-sectional structure of the absorbable silk fibroin biomembrane obtained in Example 1 is as Figure 1 shown, and it can be seen that the absorbable silk fibroin biomembrane prepared in this example has an obvious bilayer structure.

[0033] Immerse the absorbable silk fibroin biomembrane obtained in Example 1 in purified water at 37 °C, drain the water after water absorption saturation, and test the mechanical properties of the absorbable silk fibroin biomembrane in the wet state. The tensile strength of the absorbable silk fibroin biomembrane prepared in Example 1 is measured to be 8.3 MPa, and the elongation at break is 149%.

[0034] Test the biocompatibility of the absorbable silk fibroin biomembrane with a bilayer structure obtained in Example 1 through a CCK-8 cell experiment. The results are as Figure 2 shown. Compared with the blank control, as the culture time increases, the OD value also increases, that is, the number of osteoblasts increases with the culture time, indicating that the absorbable silk fibroin biomembrane obtained in Example 1 has good biocompatibility.

[0035] Example 2

[0036] Prepare an absorbable silk fibroin biomembrane with a bilayer structure according to the method of Example 1, but replace the crosslinking agent with glutaraldehyde in equal amount.

[0037] Example 3

[0038] Prepare an absorbable silk fibroin biomembrane with a bilayer structure according to the method of Example 1, but replace the crosslinking agent with genipin in equal amount.

[0039] Example 4

[0040] Prepare an absorbable silk fibroin biofilm with a bilayer structure according to the method of Example 1, but replace the crosslinking agent with an equal amount of a mixture of genipin and thermosensitive hydrogel microcapsules of pink lotus essential oil, and the mass ratio of genipin to thermosensitive hydrogel microcapsules of pink lotus essential oil is 1:0.2.

[0041] Example 5

[0042] Prepare an absorbable silk fibroin biofilm with a bilayer structure according to the method of Example 4, but replace the genipin in the original crosslinking agent with an equal amount of horseradish peroxidase.

[0043] Example 6

[0044] Prepare an absorbable silk fibroin biofilm with a bilayer structure according to the method of Example 4, but replace the genipin in the original crosslinking agent with an equal amount of glutaraldehyde.

[0045] Measure the mechanical properties of the absorbable silk fibroin biofilms prepared in Examples 2 - 6 under wet conditions. The tensile strengths of the absorbable silk fibroin biofilms prepared in Examples 2 - 6 are 8.21, 8.42, 9.37, 8.33, and 8.26 MPa in sequence, and the elongation at break are 137, 142.8, 173.6, 149.2, and 145% in sequence. It can be seen that when prepared with a mixture of genipin and thermosensitive hydrogel microcapsules of pink lotus essential oil as the crosslinking agent, the absorbable silk fibroin biofilm has the best mechanical properties. The results of the CCK-8 cell biocompatibility experiment show that as the culture time increases, the OD value also increases. The absorbable silk fibroin biofilms prepared in Examples 2 - 6 all have good biocompatibility; and when the absorbable silk fibroin biofilms prepared in Examples 2 - 6 are cultured to the 5th day, the OD values are 1.2, 1.3, 2.2, 1.6, and 1.5 respectively. It can be seen that genipin and thermosensitive hydrogel microcapsules of pink lotus essential oil play a synergistic effect.

[0046] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should also be regarded as within the protection scope of the present invention.

Claims

1. A preparation method of an absorbable silk fibroin biomembrane with a bilayer structure, characterized in that, It includes the following steps: (1) Mix the aqueous solution Ⅰ of silk fibroin with a film-forming agent, coat the mixture on the surface of a substrate and dry it to form a dense layer; (2) Mix the aqueous solution Ⅱ of silk fibroin with a cross-linking agent, coat the mixture on the surface of the dense layer and perform thermal cross-linking to form a hydrogel film; (3) Dry the hydrogel film to form a porous layer, remove the substrate, and obtain an absorbable silk fibroin biofilm with a bilayer structure; The film-forming agent is sodium hyaluronate, chitosan, gelatin or collagen; the cross-linking agent is a mixture of genipin and a thermosensitive hydrogel microcapsule of pink lotus essential oil, and the mass ratio of genipin to the thermosensitive hydrogel microcapsule of pink lotus essential oil is 1:0.1 - 0.

5.

2. The preparation method of an absorbable silk fibroin biomembrane with a bilayer structure according to claim 1, wherein, In step (1), the concentration of the aqueous solution Ⅰ of silk fibroin is 1 - 10%; the mass ratio of the aqueous solution Ⅰ of silk fibroin to the film-forming agent is 1:1 - 1:0.1; the substrate is a PET release film.

3. The preparation method of an absorbable silk fibroin biofilm with a double-layer structure according to claim 1, characterized in that, In step (1), the coating method is the doctor blade method, the coating speed is 20 - 40 m / min, the wet film thickness is 200 - 300 μm, and the coating temperature is 20 - 30°C.

4. The preparation method of an absorbable silk fibroin biomembrane with a bilayer structure according to claim 1, characterized in that, In step (1), the drying temperature is 30 - 50°C.

5. The preparation method of an absorbable silk fibroin biomembrane with a bilayer structure according to claim 1, characterized in that, The hydrogel film is prepared by the casting method. The specific steps are as follows: Prepare the aqueous solution Ⅱ of silk fibroin, add a cross-linking agent to the aqueous solution Ⅱ of silk fibroin and mix evenly to obtain a silk fibroin / cross-linking agent mixture; fix a mold on the dense layer substrate, pour the silk fibroin / cross-linking agent mixture, scrape it flat, control the wet film thickness to be 0.2 - 2 mm, and then place it in an oven for thermal cross-linking to obtain a hydrogel film loaded on the surface of the dense layer.

6. The preparation method of an absorbable silk fibroin biomembrane with a double-layer structure according to claim 1, characterized in that, The concentration of the aqueous solution Ⅱ of silk fibroin is 9 - 40%; the addition amount of the cross-linking agent is 0.01 - 0.05% of the mass of the aqueous solution Ⅱ of silk fibroin.

7. The preparation method of an absorbable silk fibroin biomembrane with a double-layer structure according to claim 1, characterized in that, The thermal cross-linking temperature is 35 - 60°C.

8. According to the preparation method of an absorbable silk fibroin biofilm with a bilayer structure described in any one of claims 1 - 7, the hydrogel film is dried by the thermal drying method, the drying temperature is 35 - 40°C, the humidity is 75 - 90%, and the drying time is 24 - 48 h.

Images