CN118718109B - Preparation method of absorbable silk fibroin biomembrane with double-layer structure - Google Patents

Preparation method of absorbable silk fibroin biomembrane with double-layer structure

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CN118718109B
CN118718109B CN202410783130.0A CN202410783130A CN118718109B CN 118718109 B CN118718109 B CN 118718109B CN 202410783130 A CN202410783130 A CN 202410783130A CN 118718109 B CN118718109 B CN 118718109B
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silk fibroin
double
absorbable
film
layer
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CN118718109A (en
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金天成
成佳琪
周泽平
陈枫
金伟伟
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Shaoxing Microsource Biomedical Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Materials For Medical Uses (AREA)

Abstract

The invention discloses a preparation method of an absorbable silk fibroin biomembrane with a double-layer structure, which comprises the steps of (1) mixing a silk fibroin aqueous solution I with a film forming agent, coating the mixture on the surface of a substrate, drying to form a compact layer, (2) mixing a silk fibroin aqueous solution II with a cross-linking agent, coating the mixture on the surface of the compact layer, and thermally crosslinking to form a hydrogel film, (3) drying the hydrogel film to form a loose layer, and removing the substrate to obtain the absorbable silk fibroin biomembrane with the double-layer structure. The absorbable silk fibroin biomembrane prepared by the method has good mechanical property and biocompatibility, can effectively prevent soft tissue cells from growing and migrating into a defect area, and promotes the growth of osteoblasts in the defect area so as to heal wounds.

Description

Preparation method of absorbable silk fibroin biomembrane with double-layer structure
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of an absorbable silk fibroin biological film with a double-layer structure.
Background
Soft tissue repair is one of the key areas of medical research, where the role of biological materials is indispensable. Although traditional repair materials such as silica gel and polyethylene have certain effects in clinical application, the problems of easy infection, displacement, tissue rejection and the like exist. In view of this, researchers are continually seeking to create biomaterials to optimize the effect of soft tissue repair, aiming at overcoming the limitations of existing materials and improving the safety and effectiveness of treatment.
The silk fibroin is used as a natural polymer material, has good biocompatibility and degradability, and is widely applied in the medical field. The silk fibroin can be prepared into materials with different morphologies such as films, stents, microspheres and the like, and is used in the fields of tissue engineering, drug delivery, hemostatic materials and the like. However, the traditional silk fibroin material has the defects of poor mechanical property, easy enzymatic degradation and the like, and limits the application of the silk fibroin material in soft tissue repair.
In recent years, some researchers have compounded silk fibroin with a film forming agent, a crosslinking agent, and the like to prepare an absorbable silk fibroin biofilm having a bilayer structure. The biological film has good mechanical property and biocompatibility, can effectively prevent the growth of soft tissue cells from migrating into a defect area, and promote the growth of osteoblasts in the defect area to heal wounds. However, the existing absorbable silk fibroin biomembrane has some problems in the preparation process, such as uneven coating, insufficient crosslinking and the like, and influences the performance and application effect. Therefore, although the absorbable silk fibroin biomembrane has a certain application prospect in soft tissue repair, the existing preparation method still has some problems to be solved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a preparation method of an absorbable silk fibroin biomembrane with a double-layer structure, which has good mechanical property and biocompatibility, can effectively prevent soft tissue cells from growing and migrating into a defect area, and promote the growth of osteoblasts in the defect area so as to heal wounds.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a process for preparing the absorbable silk fibroin film with dual-layer structure includes such steps as mixing aqueous silk fibroin solution I with filming agent, coating it on the surface of substrate, drying to obtain compact layer, mixing aqueous silk fibroin solution II with cross-linking agent, coating it on the surface of compact layer, thermally cross-linking to obtain hydrogel film, drying to obtain loose layer, and removing substrate.
Further, in the step (1), the concentration of the silk fibroin aqueous solution I is 1-10%, the mass ratio of the silk fibroin aqueous solution I to the film forming agent is 1:1-1:0.1, and the base material is a PET release film.
Further, the film forming agent is sodium hyaluronate, chitosan, gelatin or collagen.
Further, in the step (1), the coating modes are knife coating methods, the knife coating speeds are 20-40 m/min, the wet film thicknesses are 200-300 mu m, and the knife coating temperatures are 20-30 ℃.
Further, in the step (1), the drying temperature is 30-50 ℃.
Further, the hydrogel film is prepared by a casting method, and the specific steps are that a silk fibroin aqueous solution II is prepared, a cross-linking agent is added into the silk fibroin aqueous solution II and uniformly mixed to obtain a silk fibroin/cross-linking agent mixed solution, a die is fixed on a compact layer substrate, the silk fibroin/cross-linking agent mixed solution is poured into the die, the die is scraped, the thickness of a wet film is controlled to be 0.2-2 mm, and then the die is placed in an oven for thermal cross-linking, so that the hydrogel film loaded on the surface of the compact layer is obtained.
Further, the concentration of the silk fibroin aqueous solution II is 9-40%, and the addition amount of the cross-linking agent is 0.01-0.05% of the mass of the silk fibroin aqueous solution II.
Further, the cross-linking agent is horseradish peroxidase, glutaraldehyde and genipin.
Further, the thermal crosslinking temperature is 35-60 ℃.
Further, the hydrogel film is dried by a thermal drying method, the drying temperature is 35-40 ℃, the humidity is 75-90%, and the drying time is 24-48 h.
The beneficial effects of the invention are as follows:
The invention prepares a compact layer basal membrane on the surface of a substrate by taking silk fibroin and a film forming agent as raw materials, prepares a hydrogel film on the surface of the compact layer by taking silk fibroin and a crosslinking agent as raw materials, dries the hydrogel film into a loose layer, finally obtains the absorbable silk fibroin biomembrane with a double-layer structure, has good mechanical property and biocompatibility, can effectively prevent soft tissue cells from growing and migrating into a defect area, and promotes the growth of osteoblasts in the defect area so as to heal wounds.
Drawings
FIG. 1 is an SEM image of an absorbable silk fibroin biofilm prepared in example 1;
FIG. 2 shows the results of CCK-8 cell experiments on absorbable silk fibroin biofilms prepared in example 1 for 1 day, 3 days and 5 days.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention relates to a preparation method of an absorbable silk fibroin biomembrane with a double-layer structure, which comprises the steps of (1) mixing a silk fibroin aqueous solution I with a film forming agent, coating the mixture on the surface of a substrate, drying to form a compact layer, (2) mixing a silk fibroin aqueous solution II with a cross-linking agent, coating the mixture on the surface of the compact layer, and thermally crosslinking to form a hydrogel film, (3) drying the hydrogel film to form a loose layer, and removing the substrate to obtain the absorbable silk fibroin biomembrane with the double-layer structure. The method comprises the following steps:
The preparation method comprises the steps of (1) weighing regenerated silk fibroin, adding purified water, stirring and dissolving at room temperature to prepare a silk fibroin aqueous solution I with the concentration of 1-10%, adding a film forming agent (the film forming agent is sodium hyaluronate, chitosan, gelatin or collagen, the mass ratio of the silk fibroin aqueous solution I to the film forming agent is 1:1-1:0.1), stirring uniformly at room temperature to form a silk fibroin/film forming agent mixed solution, uniformly coating the silk fibroin/film forming agent mixed solution on a substrate by a doctor blade coating method (the doctor blade coating speed is 20-40 m/min, the wet film thickness is 200-300 mu m, the doctor blade coating temperature is 20-30 ℃, the substrate is a PET release film), and then drying in a 30-50 ℃ oven to obtain a compact layer base film.
The preparation method comprises the steps of weighing regenerated silk fibroin, adding purified water, stirring and dissolving at room temperature to prepare a silk fibroin aqueous solution II with the concentration of 9-40%, adding a cross-linking agent (the cross-linking agent is horseradish peroxidase, glutaraldehyde, genipin or a genipin and lotus flower essential oil microcapsule temperature-sensitive hydrogel mixture with the mass ratio of genipin to lotus flower essential oil microcapsule temperature-sensitive hydrogel of 1:0.1-0.5), stirring uniformly at room temperature, adding a silk fibroin/cross-linking agent mixture with the additive amount of 0.1-0.5%, fixing a mould on a compact layer substrate, scraping the silk fibroin/cross-linking agent mixture, controlling the film thickness to be 0.2m, and then placing the compact layer substrate in a hot water gel film at a temperature of 2m to a compact layer (the temperature of a thermal load of 60 ℃), wherein the silk fibroin/cross-linking agent mixture is obtained by a method in a paper (analysis of chemical components of lotus flower essential oil and hydrogel promotion wound is carried out by the technology [ D ]. Sun Runzhou, iceland university, 2023, phase 01), and placing the compact layer on a compact layer substrate at a thermal load of 2m to obtain a thermal hydrogel film at the temperature of 60 DEG.
And (3) placing the hydrogel film loaded on the surface of the compact layer film into a constant temperature and humidity box for drying, wherein the constant temperature and humidity box is set to be at 35-40 ℃, the humidity is 75-90%, and the drying time is 24-48 h. And after the drying is finished, taking out the film sample from the box, and separating the absorbable silk fibroin biomembrane from the surface of the PET release film.
The preferred embodiments are:
example 1
The preparation method comprises the steps of (1) weighing regenerated silk fibroin, adding purified water, stirring and dissolving at room temperature to prepare a silk fibroin aqueous solution I with the concentration of 1%, adding sodium hyaluronate (the mass ratio of the silk fibroin aqueous solution I to the sodium hyaluronate is 1:1), stirring uniformly at room temperature to form a silk fibroin/film forming agent mixed solution, uniformly coating the silk fibroin/film forming agent mixed solution on the surface of a PET release film by a knife coating method (the knife coating speed is 20m/min, the wet film thickness is 200 mu m, and the knife coating temperature is 25 ℃), and then drying in a 40 ℃ oven to obtain a compact layer base film.
The method comprises the steps of (2) weighing regenerated silk fibroin, adding purified water, stirring and dissolving at room temperature to prepare silk fibroin aqueous solution II with the concentration of 10%, adding horseradish peroxidase with the addition amount of 0.01% of the silk fibroin aqueous solution II, stirring uniformly at room temperature, adding hydrogen peroxide with the addition amount of 0.1% of the silk fibroin aqueous solution II to obtain silk fibroin/cross-linking agent mixed solution, fixing a mould on a compact layer substrate, pouring the silk fibroin/cross-linking agent mixed solution, strickling (controlling the thickness of a wet film to be 1 mm), and then placing in an oven for thermal cross-linking at 40 ℃ to obtain the hydrogel film loaded on the surface of the compact layer.
And (3) placing the hydrogel film loaded on the surface of the compact layer film into a constant temperature and humidity box for drying, wherein the constant temperature and humidity box is set to be 37 ℃, the humidity is 85%, and the drying time is 24 hours. And after the drying is finished, taking out the film sample from the box, and separating the absorbable silk fibroin biomembrane from the surface of the PET release film.
Test results:
The sectional structure of the absorbable silk fibroin biomembrane obtained in example 1 is shown in fig. 1, and it is known that the absorbable silk fibroin biomembrane prepared in this example has an obvious bilayer structure.
The absorbable silk fibroin biomembrane obtained in example 1 is soaked in purified water at 37 ℃, drained after water absorption saturation, and the mechanical properties of the absorbable silk fibroin biomembrane in a wet state are tested, so that the tensile strength of the absorbable silk fibroin biomembrane prepared in example 1 is 8.3MPa, and the elongation at break is 149%.
The experiment of CCK-8 cells shows that the absorbable silk fibroin biomembrane with a double-layer structure obtained in the example 1 has good biocompatibility, and the result is shown in figure 2, wherein the OD value is increased along with the increase of the culture time compared with a blank control, namely the number of osteoblasts is increased along with the culture time.
Example 2
An absorbable silk fibroin biofilm having a bilayer structure was prepared as in example 1, except that the crosslinking agent was replaced with glutaraldehyde in equal amounts.
Example 3
An absorbable silk fibroin biofilm having a bilayer structure was prepared as in example 1, except that the crosslinking agent was replaced with genipin in equal amounts.
Example 4
An absorbable silk fibroin biomembrane with a double-layer structure is prepared by the method of the embodiment 1, but the cross-linking agent is replaced by a mixture of genipin and the temperature-sensitive hydrogel of the trollius chinensis essential oil microcapsule in an equivalent quantity, and the mass ratio of the genipin to the temperature-sensitive hydrogel of the trollius chinensis essential oil microcapsule is 1:0.2.
Example 5
An absorbable silk fibroin biofilm with a bilayer structure was prepared as in example 4, but genipin in the original crosslinker was replaced with an equivalent amount of horseradish peroxidase.
Example 6
An absorbable silk fibroin biofilm having a bilayer structure was prepared as in example 4, but genipin in the original crosslinker was replaced with an equivalent amount of glutaraldehyde.
The mechanical properties of the absorbable silk fibroin biomembranes prepared in examples 2-6 in a wet state are measured, the tensile strength of the absorbable silk fibroin biomembranes prepared in examples 2-6 is 8.21, 8.42, 9.37, 8.33 and 8.26MPa in sequence, the elongation at break is 137, 142.8, 173.6, 149.2 and 145% in sequence, and the absorbable silk fibroin biomembrane is prepared by taking a genipin and lotus flower essential oil microcapsule temperature-sensitive hydrogel mixture as a cross-linking agent, so that the mechanical properties of the absorbable silk fibroin biomembranes are optimal. The experiment result of CCK-8 cell biocompatibility shows that the OD value is increased along with the increase of the culture time, the absorbable silk fibroin biological membranes prepared in the examples 2-6 have good biocompatibility, and the OD values are 1.2, 1.3, 2.2, 1.6 and 1.5 respectively when the absorbable silk fibroin biological membranes prepared in the examples 2-6 are cultured until the 5 th day. Therefore, the genipin and the trollius chinensis essential oil microcapsule temperature-sensitive hydrogel exert a synergistic effect.
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 examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims (8)

1.一种具有双层结构的可吸收丝素生物膜的制备方法，其特征在于，包括以下步骤：（1）将丝素蛋白水溶液Ⅰ与成膜剂混合后涂覆于基材表面并干燥，形成致密层；（2）将丝素蛋白水溶液Ⅱ与交联剂混合后涂覆于致密层表面并热交联，形成水凝胶膜；（3）水凝胶膜经干燥形成疏松层，去除掉基材，得到具有双层结构的可吸收丝素生物膜；1. A method for preparing an absorbable silk biofilm having a double-layer structure, characterized in that it comprises the following steps: (1) mixing a silk fibroin aqueous solution I with a film-forming agent, coating the mixture on the surface of a substrate, and drying the mixture to form a dense layer; (2) mixing a silk fibroin aqueous solution II with a cross-linking agent, coating the mixture on the surface of the dense layer, and thermally cross-linking the mixture to form a hydrogel film; (3) drying the hydrogel film to form a loose layer, and removing the substrate to obtain an absorbable silk biofilm having a double-layer structure; 所述成膜剂为透明质酸钠、壳聚糖、明胶或胶原蛋白；所述交联剂为京尼平与粉莲花精油微胶囊温敏水凝胶混合物，京尼平与粉莲花精油微胶囊温敏水凝胶的质量比为1:0.1-0.5。The film-forming agent is sodium hyaluronate, chitosan, gelatin or collagen; the cross-linking agent is a mixture of genipin and pink lotus essential oil microcapsule thermosensitive hydrogel, and the mass ratio of genipin to pink lotus essential oil microcapsule thermosensitive hydrogel is 1:0.1-0.5. 2.根据权利要求1所述一种具有双层结构的可吸收丝素生物膜的制备方法，其特征在于，步骤（1）中，所述丝素蛋白水溶液Ⅰ浓度为1~10%；所述丝素蛋白水溶液Ⅰ与成膜剂的质量比为1:1~1:0.1；所述基材为PET离型膜。2. The method for preparing an absorbable silk fibroin biofilm with a double-layer structure according to claim 1, characterized in that, in step (1), the concentration of the silk fibroin aqueous solution I is 1-10%; the mass ratio of the silk fibroin aqueous solution I to the film-forming agent is 1:1-1:0.1; and the substrate is a PET release film. 3.根据权利要求1所述一种具有双层结构的可吸收丝素生物膜的制备方法，其特征在于，步骤（1）中，涂覆方式为刮涂法，刮涂速度为20~40m/min，湿膜厚度为200~300 μm，刮涂温度为20~30℃。3. The method for preparing a double-layer absorbable silk biofilm according to claim 1, characterized in that in step (1), the coating method is a scraping method, the scraping speed is 20-40 m/min, the wet film thickness is 200-300 μm, and the scraping temperature is 20-30°C. 4.根据权利要求1所述一种具有双层结构的可吸收丝素生物膜的制备方法，其特征在于，步骤（1）中，干燥温度为30-50℃。4. The method for preparing a double-layer absorbable silk biofilm according to claim 1, wherein in step (1), the drying temperature is 30-50°C. 5.根据权利要求1所述一种具有双层结构的可吸收丝素生物膜的制备方法，其特征在于，水凝胶膜通过浇铸法制备，具体步骤为：配制丝素蛋白水溶液Ⅱ，向丝素蛋白水溶液Ⅱ中加入交联剂并混合均匀，得到丝素蛋白/交联剂混合液；在致密层基底上固定模具，倒入丝素蛋白/交联剂混合液，刮平，控制湿膜厚度为0.2~2 mm，然后置于烘箱中热交联，得到负载在致密层表面的水凝胶膜。5. The method for preparing an absorbable silk fibroin biofilm with a double-layer structure according to claim 1 is characterized in that the hydrogel film is prepared by a casting method, and the specific steps are: preparing a silk fibroin aqueous solution II, adding a crosslinker to the silk fibroin aqueous solution II and mixing them evenly to obtain a silk fibroin/crosslinker mixed solution; fixing a mold on the base of the dense layer, pouring the silk fibroin/crosslinker mixed solution, scraping it flat, controlling the wet film thickness to 0.2~2 mm, and then placing it in an oven for thermal crosslinking to obtain a hydrogel film loaded on the surface of the dense layer. 6.根据权利要求1所述一种具有双层结构的可吸收丝素生物膜的制备方法，其特征在于，所述丝素蛋白水溶液Ⅱ浓度为9~40%；交联剂添加量为丝素蛋白水溶液Ⅱ质量的0.01~0.05%。6. The method for preparing an absorbable silk fibroin biofilm with a double-layer structure according to claim 1, characterized in that the concentration of the silk fibroin aqueous solution II is 9-40%; and the amount of the cross-linking agent added is 0.01-0.05% by mass of the silk fibroin aqueous solution II. 7.根据权利要求1所述一种具有双层结构的可吸收丝素生物膜的制备方法，其特征在于，热交联温度为35~60℃。7 . The method for preparing a double-layer absorbable silk biofilm according to claim 1 , wherein the thermal cross-linking temperature is 35-60° C. 8.根据权利要求1-7中任意一项所述一种具有双层结构的可吸收丝素生物膜的制备方法，水凝胶膜通过热干燥法进行干燥，干燥温度为35~40℃，湿度为75~90%，干燥时间为24~48h。8. The method for preparing a double-layer absorbable silk biofilm according to any one of claims 1 to 7, wherein the hydrogel film is dried by a thermal drying method at a drying temperature of 35-40°C, a humidity of 75-90%, and a drying time of 24-48 hours.
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