WO2018107573A1 - 一种丝素蛋白纤维支架及其制备方法 - Google Patents

一种丝素蛋白纤维支架及其制备方法 Download PDF

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WO2018107573A1
WO2018107573A1 PCT/CN2017/073015 CN2017073015W WO2018107573A1 WO 2018107573 A1 WO2018107573 A1 WO 2018107573A1 CN 2017073015 W CN2017073015 W CN 2017073015W WO 2018107573 A1 WO2018107573 A1 WO 2018107573A1
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silk
silk fibroin
fiber
fibroin fiber
fiber scaffold
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PCT/CN2017/073015
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English (en)
French (fr)
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张锋
黄继伟
左保齐
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南通纺织丝绸产业技术研究院
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Publication of WO2018107573A1 publication Critical patent/WO2018107573A1/zh

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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
    • A61L27/14—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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents

Definitions

  • the present invention relates to a silk fibroin fiber scaffold and a preparation method thereof, which can be used in the field of regenerative medicine such as soft tissue, hard tissue repair and drug sustained release.
  • Silk fibroin is a natural polymer biomaterial derived from nature. It has excellent mechanical properties, controlled biodegradability, and easy processing. Especially its biocompatibility with collagen is an ideal regenerative medicine.
  • the raw material of the bracket. China is a major producer of silk, and silk production accounts for more than 70% of world production.
  • the research and application of silk has extended from the traditional textile field to high-tech fields, such as optoelectronics and biomedical materials, especially as biomedical materials have made important progress.
  • the fiber scaffold prepared by the electrospinning technique is considered to be an ideal scaffold structure for tissue engineering; however, the electrospinning technique is complicated in processing, low in yield, and compact in the structure of the electrospun fiber membrane, which is also disadvantageous for cell and tissue growth.
  • overcoming the above-mentioned problems of the existing processing technology and the structure of the silk protein scaffold, Preparation methods, and the construction of silk fibroin scaffolds that are conducive to cell and tissue growth are of great significance for the application of silk fibroin in the field of biomedical materials and the clinical application of regenerative medicine.
  • An object of the present invention is to provide a method for preparing a silk fibroin fiber scaffold, and a silk fibroin fiber scaffold prepared by the method, which has the advantages of a large pore size and a high void ratio of a porous scaffold, and has a fibrous interior.
  • Structural features that greatly promote cell growth, such as cell proliferation and migration, and tissue growth, are very beneficial for clinical application of tissue engineering techniques.
  • the present invention provides a method for preparing a silk fibroin fiber scaffold, comprising the following steps:
  • the silk fiber frozen body of the step (2) is immersed in water or an organic solvent to remove formic acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the silk is one or more of mulberry silk, tussah silk, ramie silk, and silk;
  • the organic solvent is one or more of methanol, ethanol, and propanol.
  • the invention swells the silk with the acid solution, has wide applicability, can directly swell the mulberry silk, and particularly can swell the wild silk, such as tussah silk, ramie silk, and silk, thereby obtaining a single or several blended silk fiber dispersion.
  • the silk part can be used as a binder, so that the fiber support has certain mechanical properties and is not easy to fall apart, so as to enrich the silk product.
  • the acid is one of formic acid, trifluoroacetic acid, and hydrofluoric acid; and the concentration of the acid solution is 50 to 99 wt%.
  • the invention adopts acid-dispersed silk fibroin fiber, which is not dissolved, and the natural silk fibroin fiber scaffold has an internal structure mainly composed of fibers, and has high porosity, high penetration rate and excellent mechanical properties.
  • the dispersion treatment of the daytime is 5 to 30 minutes, has good dispersibility to the silk, combined with the slightly soluble bonding, the internal structure of the prepared silk fibroin fiber stent is mainly fiber, and has high Porosity.
  • the silk fiber dispersion has a mass concentration of 0.5 to 10%, and the stent thus obtained has a more uniform porous structure and a suitable porosity, and is more suitable for cell and tissue growth.
  • the temperature of the freezing treatment is the freezing point temperature of the acid solution; in the step (4), the temperature of the cold treatment is -1 to -80 ° C, freeze-dried The temperature is -1 to -60 °C.
  • the advantage of this treatment is that both energy conservation and stable acquisition of the porous structure are achieved, and the resulting porous structure is more conducive to cell adhesion and growth, and tissue growth.
  • the present invention also discloses a silk fibroin fiber scaffold prepared according to the above preparation method; the silk fibroin fibrous porous scaffold is composed of fibers having a diameter of 5 ⁇ m to 20 ⁇ m; and the porosity of the silk fibroin porous scaffold More than 60%, the pore size is 5 (Vm ⁇ 2mm.
  • the natural silk fibroin porous scaffold of the present invention has a three-dimensional porous structure, can provide a three-dimensional space for cell adhesion proliferation and tissue regeneration, and facilitates the transport of nutrients, and thus the present invention is further disclosed.
  • the present invention directly constructs a fibrous porous material by using silk fibroin fibers, and the preparation method utilizes the good dispersibility and slightly soluble adhesion of the silk to the silk, and the silk fibroin prepared by the combined treatment process.
  • the internal structure of the fiber scaffold is mainly composed of fibers, has high porosity, high penetration rate, excellent mechanical properties, is very beneficial for nutrient transport, cell migration, tissue growth, and is an ideal tissue engineering scaffold, and the present invention has The advantages of simple process, low cost and easy batch processing.
  • the method for preparing a porous, large-diameter silk fibroin fiber scaffold is simple, and only acid-dispersed silk is used, and no other chemical reagents are needed, and on the one hand, the reagent residue is not conducive to the in vivo application of the biological material.
  • the problem reduces the purification step and the daytime, which can greatly maintain the natural properties of the silk fiber and facilitate industrial production.
  • FIG. 1 is a photograph and a scanning electron micrograph (right) of a wet (left), dry (medium) silk fibroin scaffold obtained in Example 1.
  • Example 2 is a photograph (left) and a scanning electron microscope (right) of a silk fibroin scaffold obtained in Example 2;
  • 3 is a scanning electron micrograph of a cross section of a silk fibroin support obtained in Example 3;
  • Example 4 is a scanning electron micrograph of a section of a natural silk fibroin support obtained in Example 4.
  • Example 5 is a scanning electron micrograph of a section of a natural silk fibroin support obtained in Example 5;
  • FIG. 6 is a fluorescence diagram of the sixth embodiment after inoculation of bone marrow mesenchymal stem cells
  • FIG. 7 is a fluorescence diagram of a sixth stent after inoculation of bone marrow mesenchymal stem cells in one embodiment
  • FIG. 8 is a fluorescence diagram of the sixth stent after inoculation of bone marrow mesenchymal stem cells for five days.
  • the natural mulberry silk is boiled for 30 minutes with a sodium carbonate solution having a mass fraction of 0.05% by weight, and the silk fibroin fiber is obtained after repeating three times; the silk fibroin fiber dispersion is obtained by dispersing the silk fibroin fiber in a solvent of 88% formic acid. Concentration 2%;
  • the silk fiber frozen body of the step (2) is immersed in methanol to remove formic acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the wet silk fibroin fiber scaffold obtained in the step (3) was frozen at -20 ° C, and then freeze-dried at -40 ° C to obtain a silk fibroin scaffold.
  • the inside of the stent of the present invention is mainly composed of fibers, the fiber
  • the stent has a compression modulus of about 15 kPa.
  • the natural mulberry silk is boiled for 30 minutes with a sodium carbonate solution having a mass fraction of 0.5 wt%, and the silk fibroin fiber is obtained after repeating three times; the silk fibroin fiber dispersion is obtained by dispersing the silk fibroin fiber in a solvent of 98% formic acid. Concentration 3%;
  • the wet silk fibroin fiber scaffold was frozen at -10 ° C, and then freeze-dried at -20 ° C to obtain a silk fibroin scaffold.
  • the fiber holder has a compression modulus of about 10 kPa.
  • FIG. 3 is a scanning electron micrograph of the cross section of the silk fibroin scaffold obtained above.
  • the inside of the scaffold is mainly composed of fibers, and the fibers have no obvious fibrillation and macroscopic structure damage, and the compression modulus of the fiber scaffold is about 1 00kPa.
  • the natural mulberry silk is boiled for 30 minutes with a sodium carbonate solution having a mass fraction of 0.5 wt%, and the silk fibroin fiber is obtained after repeating three times; the silk fibroin fiber is dispersed in a solvent of 90% trifluoroacetic acid to obtain a silk fiber.
  • the concentration of the dispersion is 4%;
  • step (3) The silk fiber frozen body of step (2) is immersed in ethanol to remove trifluoroacetic acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the inside of the stent is mainly composed of fibers, and a network of a plurality of microfibers is formed in the same layer. This structure will facilitate cell adhesion and growth, and the fiber stent has a compression modulus of about 60 kPa.
  • the natural mulberry silk is boiled for 30 minutes with a sodium carbonate solution of a mass fraction of 0.05 wt%, and the silk fibroin fiber is obtained after repeating three times; the silk fibroin fiber is dispersed in a 60% hydrofluoric acid solvent to obtain a silk fiber.
  • the concentration of the dispersion is 3%;
  • the silk fiber frozen body of the step (2) is immersed in ethanol to remove hydrofluoric acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the wet silk fibroin fiber scaffold was frozen at -100 ° C, and then freeze-dried at -50 ° C to obtain a natural silk fibroin scaffold.
  • the inside of the stent is mainly composed of fibers, and a network of a plurality of fine fibers is formed in the same layer. This structure will facilitate cell adhesion and growth, and the fiber stent has a compression modulus of about 50 kPa.
  • the natural mulberry silk is boiled for 30 minutes with a sodium carbonate solution of a mass fraction of 0.05 wt%, and the silk fibroin fiber is obtained after repeating three times; the silk fibroin fiber dispersion is obtained by dispersing the silk fibroin fiber in a solvent of 98% formic acid. Concentration 8%;
  • the silk fiber frozen body of the step (2) is immersed in ethanol to remove formic acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the wet silk fibroin fiber scaffold was frozen at -10 ° C, and then freeze-dried at -20 ° C to obtain a silk fibroin scaffold, and the inside of the scaffold was mainly composed of fibers.
  • the fiber holder has a compression modulus of about 150 kPa.
  • FIG. 6-8 is a fluorescent picture of the above-mentioned scaffold after inoculation of bone marrow mesenchymal stem cells; The cytoplasmic sputum adhered to the scaffold; one day later (Fig. 7), the cells began to spread and arranged in the direction of the fibers; after 5 days of inoculation (Fig. 8), the cells proliferated in large amounts.
  • the scaffold On the surface of the cell experiment, the scaffold has good cell biocompatibility, supports cell biological behavior such as cell adhesion, spreading and proliferation, and is a good biological scaffold.
  • the silk fiber frozen body of the step (2) is immersed in ethanol to remove formic acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the wet silk fibroin fiber scaffold was frozen at -20 ° C, and then freeze-dried at -20 ° C to obtain a silk fibroin scaffold, and the inside of the scaffold was mainly composed of fibers.
  • the fiber stent has a compression modulus of about 50 kPa.
  • the natural silk is boiled for 30 minutes with a sodium carbonate solution having a mass fraction of 0.5 wt%, and the silk fibroin fiber is obtained after repeating 3 times; the silk fibroin dispersion is obtained by dispersing the silk fibroin fiber in a solvent of 78% formic acid. Concentration 10%;
  • the silk fiber frozen body of the step (2) is immersed in ethanol to remove formic acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the wet silk fibroin fiber scaffold was frozen at -10 ° C, and then freeze-dried at -60 ° C to obtain a natural silk fibroin fiber scaffold, and the inside of the scaffold was mainly composed of fibers.
  • the fiber holder has a compression modulus of about 70 kPa.
  • the silk fiber frozen body of the step (2) is immersed in methanol to remove formic acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the wet silk fibroin fiber scaffold was frozen at -80 ° C, and then freeze-dried at -20 ° C to obtain a natural silk fibroin fiber scaffold, and the inside of the scaffold was mainly composed of fibers.
  • the fiber stent has a compression modulus of about 300 kPa.
  • the silk fiber frozen body of the step (2) is immersed in ethanol to remove formic acid, and then washed with deionized water to obtain a wet silk fibroin fiber scaffold;
  • the wet silk fibroin fiber scaffold is frozen at -20 ° C, and then freeze-dried at -20 ° C to obtain natural silk fibroin.

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Abstract

一种丝素蛋白纤维支架及其制备方法，该制备方法包括：蚕丝脱胶后，浸泡在酸溶液中进行分散处理，得到蚕丝纤维分散液；将蚕丝纤维分散液注入模具中进行冷冻处理得到蚕丝纤维冷冻体；将蚕丝纤维冷冻体浸入水或有机溶剂中去除酸，然后用去离子水浸泡洗涤得到湿态丝素蛋白纤维支架；将湿态丝素蛋白纤维支架进行冷冻处理得到冷冻体，然后对冷冻体进行冷冻干燥得到丝素蛋白纤维支架。该丝素蛋白纤维支架内部结构以纤维为主，有利于营养物质的输送、细胞的迁移、组织的生长。

Description

一种丝素蛋白纤维支架及其制备方法 技术领域
[0001] 本发明涉及一种丝素蛋白纤维支架及其制备方法， 可用于软组织、 硬组织修复 及药物缓释等再生医学领域。
背景技术
[0002] 由于疾病和事故导致的器官或组织损伤和功能缺失的病人每年都有数百万之多 ， 仅美国每年需要 800多万次手术对这类病人进行救治， 其经济花费在 4000亿美 元以上。 随着现代医学和外科手术技术的发展， 通过组织或器官移植来修复功 能损失已经被广泛接受， 然而却面临着巨大的供体缺口。 通过再生医学手段体 在体内或体外形成组织或器官为受损功能的修复提供了新的治疗方案。 其中， 组织工程支架材料的选择及构建成为该治疗方法的关键之一。
[0003] 蚕丝蛋白是来源于自然界的天然高分子生物材料， 具有优异的力学性质、 可控 的生物降解性、 易加工性， 特别是其与胶原同等的生物相容性而成为理想的再 生医学支架的原材料。 我国是蚕丝的主要生产国， 蚕丝产量占世界产量的 70%以 上。 近年来， 蚕丝的研究与应用从传统的纺织领域延伸到高新技术领域， 如光 电子与生物医用材料， 特别是作为生物医用材料已经取得了重要进展。
[0004] 目前， 制备丝素蛋白多孔支架的方法有很多， 包括冷冻干燥、 盐析法、 气体发 泡法、 三维打印等， 然而这些方法都依然存在一些难以克服的不足。 例如， 冷 冻干燥法易形成片层结构， 不利于细胞与组织生长， 尽管现有技术报道了一种 反复进行膜溶解控制丝素蛋白自组装形成纳米纤维结构， 进而形成多孔支架， 但效率低， 重复性差， 并且孔壁结构的存在直接限制细胞的迁移与相互作用， 组织生长也因此受限。
技术问题
[0005] 利用静电纺丝技术制备的纤维支架被认为是组织工程理想的支架结构； 然而静 电纺丝技术加工复杂、 产量低、 且静电纺纤维膜结构致密， 也不利于细胞与组 织生长。 为此， 克服现有加工技术与丝蛋白支架结构的上述问题， 幵发一种制 备方法， 并构建出有利于细胞与组织生长的丝素蛋白支架对丝素蛋白在生物医 用材料领域的应用及再生医学的临床应用都具有非常重大的意义。
问题的解决方案
技术解决方案
[0006] 本发明的目的是提供一种丝素蛋白纤维支架的制备方法， 及由该方法制备的丝 素蛋白纤维支架， 具有多孔支架的大孔径高空隙率优点， 同吋具有纤维状的内 部结构特征， 极大促进细胞生长， 如细胞增殖与迁移、 和组织生长， 对组织工 程技术临床应用非常有利。
[0007] 为达到上述目的， 本发明提供一种丝素蛋白纤维支架的制备方法， 包括以下步 骤：
[0008] (1) 蚕丝脱胶后， 浸泡在酸溶液中进行分散， 得到蚕丝纤维分散液；
[0009] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理得到蚕丝纤维冷冻体；
[0010] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入水或有机溶剂中去除甲酸， 然后用去 离子水浸泡洗涤得到湿态丝素蛋白纤维支架；
[0011] (4) 将步骤 （3) 得到的湿态丝素蛋白纤维支架进行冷处理得到冷冻体， 然后 对冷冻体进行冷冻干燥即得到丝素蛋白纤维支架。
[0012] 上述技术方案中， 所述蚕丝为桑蚕丝、 柞蚕丝、 蓖麻蚕丝、 天蚕丝中的一种或 者几种； 所述有机溶剂为甲醇、 乙醇、 丙醇中的一种或几种。 本发明以酸溶液 溶胀蚕丝， 具有广泛适用性， 可直接溶胀桑蚕丝， 特别还可以溶胀柞蚕丝、 蓖 麻蚕丝、 天蚕丝等野蚕丝， 从而获得单种或几种共混蚕丝纤维分散液， 其中蚕 丝部分可以作为粘合剂， 使得纤维支架具有一定力学性能且不易散架， 为丰富 蚕丝制品夯实基础。
[0013] 上述技术方案中， 所述酸为甲酸、 三氟乙酸、 氢氟酸中的一种； 所述酸溶液的 浓度 50〜99 wt%。 本发明以酸分散丝素蛋白纤维， 而不溶解， 制备的天然丝素 纤维支架内部结构以纤维为主， 具有高孔隙率、 高贯通率、 优良的力学性能。
[0014] 上述技术方案中， 所述分散处理的吋间为 5〜30分钟， 对蚕丝具有良好分散性 ， 结合微溶黏结作用， 制备的丝素蛋白纤维支架内部结构以纤维为主， 具有高 孔隙率。 [0015] 上述技术方案中， 所述蚕丝纤维分散液的质量浓度为 0.5〜10%， 由此得到的支 架具有更为均匀的多孔结构、 适宜的孔隙率， 更适合细胞与组织生长。
[0016] 上述技术方案中， 步骤 （2) 中， 所述冷冻处理的温度为酸溶液的冰点温度； 步骤 （4) 中， 所述冷处理的温度为 -1〜- 80°C， 冷冻干燥的温度为 -1〜- 60°C。 这 样处理的优点是兼顾能源节约与多孔结构的稳定获取， 由此得到的多孔结构更 有利于细胞的黏附与生长， 及组织长入。
[0017] 本发明还公幵了根据上述制备方法制备的丝素蛋白纤维支架； 所述丝素蛋白纤 维多孔支架由直径为 5μηι〜20μιη的纤维组成； 所述丝素蛋白纤维多孔支架的孔 隙率大于 60%， 孔径为 5(Vm〜2mm。
[0018] 本发明的天然丝素丝蛋白多孔支架因具有三维多孔结构， 可以为细胞的粘附增 殖、 及组织的再生提供三维空间， 并且有利于营养物质的传输， 因此本发明进 一步公幵了上述丝素蛋白纤维支架在制备细胞与组织生长材料中的应用。
发明的有益效果
有益效果
[0019] 与现有技术相比， 本发明直接以蚕丝丝素纤维构建纤维多孔材料， 该制备方法 利用酸对蚕丝的良好分散性及微溶黏结作用， 结合处理工艺， 所制备的丝素蛋 白纤维支架内部结构以纤维为主， 具有高孔隙率、 高贯通率、 优良的力学性能 ， 非常有利于营养物质的输送、 细胞的迁移、 组织的生长， 是理想的组织工程 支架， 并且本发明具有工艺简单、 成本低、 易于批量化加工的优点。
[0020] 特别的， 本发明公幵的制备多孔、 大孔径丝素蛋白纤维支架的方法简单， 仅采 用酸分散蚕丝， 无需其他化学试剂的使用， 一方面避免试剂残留不利于生物材 料体内应用的问题， 另一方面减少纯化步骤与吋间， 可以极大地保持蚕丝纤维 的天然性能并利于工业生产。
对附图的简要说明
附图说明
[0021] 图 1为实施例一获得的湿态 （左） 、 干态 （中） 丝素纤维支架的照片和扫描电 镜图 （右） ；
[0022] 图 2为实施例二获得的丝素纤维支架的照片 （左） 和扫描电镜图 （右） ； [0023] 图 3为实施例三获得的丝素纤维支架断面的扫描电镜图；
[0024] 图 4为实施例四获得的天然丝素纤维支架断面的扫描电镜图；
[0025] 图 5为实施例五获得的天然丝素纤维支架断面的扫描电镜图；
[0026] 图 6为实施例六支架接种骨髓间充质干细胞后的荧光图；
[0027] 图 7为实施例六支架接种骨髓间充质干细胞一天后的荧光图；
[0028] 图 8为实施例六支架接种骨髓间充质干细胞五天后的荧光图。
本发明的实施方式
[0029] 实施例一
[0030] ( 1) 天然桑蚕丝用质量分数 0.05wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次后 获得丝素纤维； 将丝素纤维分散于 88%甲酸溶剂中， 得到的蚕丝纤维分散液浓度 2%；
[0031] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -20°C) 得到蚕丝纤维 冷冻体；
[0032] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入甲醇中去除甲酸， 然后用去离子水浸 泡洗涤得到湿态丝素蛋白纤维支架；
[0033] (4) 将步骤 （3) 得到的湿态丝素蛋白纤维支架在 -20°C冷冻， 然后 -40°C冷冻 干燥， 得到丝素纤维支架。
[0034] 附图 1为上述获得的湿态 （左） 、 干态 （中） 丝素纤维支架的照片和扫描电镜 图片 （右） ， 由图可见， 本发明支架内部主要由纤维构成， 该纤维支架的压缩 模量约 15kPa。
[0035] 实施例二
[0036] ( 1) 天然桑蚕丝用质量分数 0.5wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次后 获得丝素纤维； 将丝素纤维分散于 98%甲酸溶剂中， 得到的蚕丝纤维分散液浓度 3%；
[0037] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -10°C) 得到蚕丝纤维 冷冻体；
[0038] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入乙醇中去除甲酸， 然后用去离子水浸 泡洗涤得到湿态丝素蛋白纤维支架；
[0039] (4) 湿态丝素蛋白纤维支架在 -10°C冷冻， 然后 -20°C冷冻干燥， 得到丝素纤维 支架。
[0040] 附图 2为上述获得的丝素纤维支架的照片 （左） 和扫描电镜图 （右） ； 由图可 见， 支架内部主要由纤维构成， 且有部分细小纤维分散于蚕丝纤维表面与纤维 间， 该纤维支架的压缩模量约 10kPa。
[0041] 实施例三
[0042] (1) 天然柞蚕丝用质量分数 0.05wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次后 获得丝素纤维； 将丝素纤维分散于 98%甲酸溶剂中， 得到的蚕丝纤维分散液浓度 5%；
[0043] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -30°C) 得到蚕丝纤维 冷冻体；
[0044] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入丙醇中去除甲酸， 然后用去离子水浸 泡洗涤得到湿态丝素蛋白纤维支架；
[0045] (4) 湿态丝素蛋白纤维支架在 -20°C冷冻， 然后 -80°C冷冻干燥， 得到天然丝素 纤维支架。
[0046] 附图 3为上述获得的丝素纤维支架断面的扫描电镜图， 由图可见， 支架内部主 要由纤维构成， 纤维未有明显分纤与宏观结构破坏， 该纤维支架的压缩模量约 1 00kPa。
[0047] 实施例四
[0048] (1) 天然桑蚕丝用质量分数 0.5wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次后 获得丝素纤维； 将丝素纤维分散于 90%三氟乙酸溶剂中， 得到的蚕丝纤维分散液 浓度 4%;
[0049] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -80°C) 得到蚕丝纤维 冷冻体；
[0050] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入乙醇中去除三氟乙酸， 然后用去离子 水浸泡洗涤得到湿态丝素蛋白纤维支架；
[0051] (4) 湿态丝素蛋白纤维支架在 -196°C冷冻， 然后 -10°C冷冻干燥， 得到天然丝 素纤维支架。
[0052] 附图 4为上述获得的天然丝素纤维支架断面的扫描电镜图， 右图为放大图； 由 图可见， 支架内部主要由纤维构成， 同吋出现大量微细纤维组成的网状结构， 该结构将有利于细胞的黏附与生长， 该纤维支架的压缩模量约 60kPa。
[0053] 实施例五
[0054] (1) 天然桑蚕丝用质量分数 0.05wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次后 获得丝素纤维； 将丝素纤维分散于 60%氢氟酸溶剂中， 得到的蚕丝纤维分散液浓 度 3%;
[0055] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -80°C) 得到蚕丝纤维 冷冻体；
[0056] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入乙醇中去除氢氟酸， 然后用去离子水 浸泡洗涤得到湿态丝素蛋白纤维支架；
[0057] (4) 湿态丝素蛋白纤维支架在 -100°C冷冻， 然后 -50°C冷冻干燥， 得到天然丝 素纤维支架。
[0058] 附图 5为上述获得的天然丝素纤维支架断面的扫描电镜图， 右图为放大图； 由 图可见， 支架内部主要由纤维构成， 同吋出现大量微细纤维组成的网状结构， 该结构将有利于细胞的黏附与生长， 该纤维支架的压缩模量约 50kPa。
[0059] 实施例六
[0060] (1) 天然桑蚕丝用质量分数 0.05wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次后 获得丝素纤维； 将丝素纤维分散于 98%甲酸溶剂中， 得到的蚕丝纤维分散液浓度 8%；
[0061] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -50°C) 得到蚕丝纤维 冷冻体；
[0062] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入乙醇中去除甲酸， 然后用去离子水浸 泡洗涤得到湿态丝素蛋白纤维支架；
[0063] (4) 湿态丝素蛋白纤维支架在 -10°C冷冻， 然后 -20°C冷冻干燥， 得到丝素纤维 支架， 支架内部主要由纤维构成。 该纤维支架的压缩模量约 150kPa。
[0064] 附图 6-8为上述支架接种骨髓间充质干细胞后的荧光图片； 由图 6可见接种后细 胞幵始在支架黏附； 1天后 （图 7) 细胞幵始铺展， 并沿纤维方向排列； 接种 5天 后 （图 8) ， 细胞大量增殖。 细胞实验表面， 该支架具有良好的细胞生物相容性 ， 支持细胞的黏附、 铺展与增殖等细胞生物学行为， 是一种良好的生物支架。
[0065] 实施例七
[0066] ( 1) 天然蓖麻蚕丝用质量分数 0.5wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次 后获得丝素纤维； 将丝素纤维分散于 98%甲酸溶剂中， 得到的蚕丝纤维分散液浓 度 5% ;
[0067] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -70°C) 得到蚕丝纤维 冷冻体；
[0068] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入乙醇中去除甲酸， 然后用去离子水浸 泡洗涤得到湿态丝素蛋白纤维支架；
[0069] (4) 湿态丝素蛋白纤维支架在 -20°C冷冻， 然后 -20°C冷冻干燥， 得到丝素纤维 支架， 支架内部主要由纤维构成。 该纤维支架的压缩模量约 50kPa。
[0070] 实施例八
[0071] ( 1) 天然天蚕丝用质量分数 0.5wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次后 获得丝素纤维； 将丝素纤维分散于 78%甲酸溶剂中， 得到的蚕丝纤维分散液浓度 10%；
[0072] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -40°C) 得到蚕丝纤维 冷冻体；
[0073] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入乙醇中去除甲酸， 然后用去离子水浸 泡洗涤得到湿态丝素蛋白纤维支架；
[0074] (4) 湿态丝素蛋白纤维支架在 -10°C冷冻， 然后 -60°C冷冻干燥， 得到天然丝素 纤维支架， 支架内部主要由纤维构成。 该纤维支架的压缩模量约 70kPa。
[0075] 实施例九
[0076] ( 1) 天然蓖麻蚕丝用质量分数 0.5wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次 后获得丝素纤维； 将丝素纤维分散于 98%甲酸溶剂中， 得到的蚕丝纤维分散液浓 度 10% ;
[0077] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -50°C) 得到蚕丝纤维 冷冻体；
[0078] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入甲醇中去除甲酸， 然后用去离子水浸 泡洗涤得到湿态丝素蛋白纤维支架；
[0079] (4) 湿态丝素蛋白纤维支架在 -80°C冷冻， 然后 -20°C冷冻干燥， 得到天然丝素 纤维支架， 支架内部主要由纤维构成。 该纤维支架的压缩模量约 300kPa。
[0080] 实施例十
[0081] (1) 天然天蚕丝用质量分数 0.5wt%的碳酸钠溶液煮沸 30min脱胶， 重复 3次后 获得丝素纤维； 将丝素纤维分散于 98%甲酸溶剂中， 得到的蚕丝纤维分散液浓度 6%；
[0082] (2) 将蚕丝纤维分散液注入模具中进行冷冻处理 （温度 -30°C) 得到蚕丝纤维 冷冻体；
[0083] (3) 将步骤 （2) 的蚕丝纤维冷冻体浸入乙醇中去除甲酸， 然后用去离子水浸 泡洗涤得到湿态丝素蛋白纤维支架；
[0084] (4) 湿态丝素蛋白纤维支架在 -20°C冷冻， 然后 -20°C冷冻干燥， 得到天然丝素

Claims

权利要求书 [权利要求 1] 一种丝素蛋白纤维支架的制备方法， 其特征在于， 包括如下步骤：
(1) 蚕丝脱胶后， 浸泡在酸溶液中进行分散处理， 得到蚕丝纤维分 散液；
(2) 将蚕丝纤维分散液注入模具中进行冷冻处理得到蚕丝纤维冷冻 体；
(3) 将步骤 （2) 的蚕丝纤维冷冻体浸入水或有机溶剂中去除甲酸， 然后用去离子水充分洗涤得到湿态丝素蛋白纤维支架；
(4) 将步骤 （3) 得到的湿态丝素蛋白纤维支架进行冷冻处理得到冷 冻体， 然后对冷冻体进行冷冻干燥即得到丝素蛋白纤维支架。
[权利要求 2] 根据权利要求 1所述丝素蛋白纤维支架的制备方法， 其特征在于： 所 述蚕丝为桑蚕丝、 柞蚕丝、 蓖麻蚕丝、 天蚕丝中的一种或者几种； 所 述有机溶剂为甲醇、 乙醇、 丙醇中的一种或几种。
[权利要求 3] 根据权利要求 1所述丝素蛋白纤维支架的制备方法， 其特征在于： 所 述酸为甲酸、 三氟乙酸、 氢氟酸中的一种； 所述酸溶液的浓度 50〜99 wt<¾。
[权利要求 4] 根据权利要求 1所述丝素蛋白纤维支架的制备方法， 其特征在于： 所 述分散处理的吋间为 5〜30分钟。
[权利要求 5] 根据权利要求 1所述丝素蛋白纤维支架的制备方法， 其特征在于： 所 述蚕丝纤维分散液的质量浓度为 0.5〜10%。
[权利要求 6] 根据权利要求 1所述丝素蛋白纤维支架的制备方法， 其特征在于： 步 骤 （2) 中， 所述冷冻处理的温度为酸溶液的冰点温度。
[权利要求 7] 根据权利要求 1所述丝素蛋白纤维支架的制备方法， 其特征在于： 步 骤 （4) 中， 所述冷处理的温度为 -1〜- 80°C; 冷冻干燥的温度为 -20〜
-60。C。
[权利要求 8] 根据权利要求 1所述丝素蛋白纤维支架的制备方法制备的丝素蛋白纤 维支架。
[权利要求 9] 根据权利要求 8所述丝素蛋白纤维支架， 其特征在于： 所述丝素蛋白 纤维支架由直径为 5μηι〜20μιη的纤维组成； 所述丝素蛋白纤维支架 的孔隙率大于 60%， 孔径为 5(Vm〜2mm。
[权利要求 10] 权利要求 8所述丝素蛋白纤维支架在制备细胞与组织生长材料中的应 用。
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