WO2018090341A1 - 人工复合膜、制备方法及其应用 - Google Patents

人工复合膜、制备方法及其应用 Download PDF

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WO2018090341A1
WO2018090341A1 PCT/CN2016/106455 CN2016106455W WO2018090341A1 WO 2018090341 A1 WO2018090341 A1 WO 2018090341A1 CN 2016106455 W CN2016106455 W CN 2016106455W WO 2018090341 A1 WO2018090341 A1 WO 2018090341A1
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Prior art keywords
collagen
type
purified
silk fibroin
preparation
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PCT/CN2016/106455
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English (en)
French (fr)
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邢孟秋
商海涛
魏泓
蒋坤
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深圳市成农生物材料有限公司
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Priority to CN201680012639.9A priority Critical patent/CN108778353A/zh
Priority to PCT/CN2016/106455 priority patent/WO2018090341A1/zh
Publication of WO2018090341A1 publication Critical patent/WO2018090341A1/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
    • 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
    • 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
    • A61L27/26—Mixtures of macromolecular compounds
    • 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

Definitions

  • the invention relates to biomedical materials, in particular to an artificial composite membrane, a preparation method and an application thereof.
  • dural transplantation is particularly important for replacing the defect's dura mater and promoting its regeneration.
  • Insufficient repair of the dura mater can lead to many complications, such as adhesions, cerebrospinal fluid leakage (CSF), infection, etc.
  • CSF cerebrospinal fluid leakage
  • some biological materials can be obtained from the same body or allogeneic, they can also cause the spread of diseases, cause rejection reactions, and scars and adhesions are still inevitable.
  • the traditional method of repairing the damaged dura mater is to suture directly, but it is not convenient to suture after some parts are damaged.
  • Some synthetic polymers such as expanded polytetrafluoroethylene (ePTFE), can be expanded by fibrin glue.
  • the ideal graft should meet the following conditions: (1) low (or no) immunogenicity, (2) good stretch strength, (3) appropriate pore size, (4) anti-cerebrospinal fluid leakage, (5) anti-adhesion, (6) Proper degradation rate, (7) Easy surgical operation.
  • Collagen membrane has obvious advantages in transplantation and has been successfully applied in clinical practice. Collagen is the main component of the extracellular matrix and is essential for cell survival and division. Because of its good biocompatibility and biodegradability, it promotes cell adhesion. Clinical application of collagen to dural transplantation has been nearly a century. However, two major drawbacks limit its widespread use. The poor tensile strength and shear resistance limit the application of collagen membrane in surgery. In addition, the purity of collagen also affects the therapeutic effect.
  • the main object of the present invention is to overcome the above deficiencies of the prior art and to provide a high strength artificial composite film, a preparation method and application thereof.
  • the present invention adopts the following technical solutions:
  • a method for preparing an artificial composite membrane comprising mixing silk fibroin with collagen and preparing a composite membrane.
  • collagen is type I collagen.
  • the silk fibroin is extracted from silkworm pupa.
  • type I collagen is extracted from an animal's knotted tissue such as bovine tendon.
  • the method includes a type I collagen preparation step, and the type I collagen preparation step comprises:
  • Type I collagen was purified by filtration through a filter.
  • step of preparing the type I collagen further comprises:
  • the purified type I collagen is added to glacial acetic acid to dissolve it;
  • the precipitate is added to glacial acetic acid, purified by filtration through a membrane, and the filtrate is added to distilled water, and then purified by filtration through a membrane;
  • the type I collagen is purified by freeze-drying to obtain a powder type I collagen.
  • the precipitate is successively added to glacial acetic acid at a concentration of 2 to 4%, 0.8 to 1.5%, and 0.2 to 0.7%, and purified by a membrane, and then purified by adding a filter to distilled water.
  • the method comprises a silk fibroin preparation step, the silk fibroin preparation step comprising:
  • the silkworm cocoons are boiled in a Na 2 CO 3 solution to remove the gum;
  • the filtrate is added to distilled water and purified by filtration through a membrane;
  • the silk fibroin is purified by freeze-drying to obtain a powdered silk fibroin.
  • silk fibroin and type I collagen are mixed in an acetic acid solution, homogenized, and then the resulting slurry is used to prepare the composite film.
  • the acetic acid solution has a pH of 2.5 to 3.5.
  • the mixture is homogenized at 2 to 6 ° C for 1 to 3 hours.
  • the obtained slurry was poured into a mold to form a film having a uniform pore diameter under freeze-drying conditions; the film was baked in a vacuum oven to be crosslinked.
  • the weight ratio of the type I collagen to the silk fibroin is (3:1) to (5:1).
  • An artificial composite film is an artificial composite film produced by the above preparation method.
  • An artificial composite membrane produced by the preparation method described above is used for repairing a dura mater after craniocerebral surgery or repairing a spinal capsule after spinal surgery.
  • the present invention introduces silk fibroin in collagen slurry for the first time to overcome the shortcomings of simple collagen membrane tensile and shear resistance. Studies have shown that silk fibroin has good biocompatibility and biodegradability and is much stronger than collagen.
  • the composite membrane prepared by mixing the two natural biopolymers can greatly increase the strength of the membrane, thereby overcoming the disadvantages of the collagen membrane in transplantation applications.
  • the process of the invention is capable of extracting high-purity collagen, and the prepared composite membrane has good biocompatibility. In addition, it has a uniform microporous structure and good toughness, and is convenient for surgical operation and suturing.
  • the artificial composite membrane prepared by the method of the present invention can be used in neurosurgery to replace the resected dura mater, for repairing and regenerating the dura mater after craniocerebral surgery, and promoting dura mater regeneration.
  • a method of making an artificial composite membrane comprising mixing silk fibroin with collagen, preferably type I collagen, and making a high strength composite membrane.
  • the artificial composite membrane is a uniform porous matrix, and the thickness can be adjusted according to clinical needs, for example, 6 mm.
  • High-intensity artificial composite membranes include type I collagen and silk fibroin, and natural polymeric collagen and silk fibroin can be extracted from animal tendons and silkworm cocoons, respectively.
  • Collagen and silk fibroin are natural proteins with good biocompatibility and degradation, and are non-antigenic and immunogenic.
  • the preparation process is as follows:
  • the bovine tendon is sterilized by ultraviolet disinfection and concentration of 70% alcohol.
  • bovine tendon After the cattle are sacrificed, 10 grams of bovine tendon is taken and sent to the cleaning room as soon as possible, UV disinfection or 70% alcohol spray for the next step.
  • the bovine tendon was placed in a homogenizer to prepare a tissue homogenate.
  • the bovine tendon was made into a tissue homogenate using a homogenizer, and dry ice was added to prevent collagen denaturation.
  • Polysaccharides can increase the viscosity of collagen and cause an immune response in the body.
  • 100 ml of a concentration of 0.3% NaH 2 PO 4 was added to the adipose tissue-removing bovine tendon, soaked for 3 days, and NaH 2 PO 4 was replaced every 12 hours.
  • the collagen was first crudely extracted with a 10% NaCl solution.
  • the NaCl solution was added to the bovine tendon, and the white fiber precipitated was collagen at 4 ° C overnight.
  • Collagen was filtered through a filter (cutoff molecular mass 20,000 D) in distilled water and purified for 24 hours.
  • the collagen precipitate was placed in a filter tube with a molecular weight of 20,000 D, placed in a beaker containing distilled water, and placed at 4 ° C for 24 hours, and distilled water was changed every 8 hours.
  • collagen was added to a concentration of 3% (v / v) glacial acetic acid, soaked for 3 days, fully dissolved.
  • the collagen solution was transferred to a 50 ml high-speed centrifuge tube, centrifuged at 14,000 g for 45 minutes at 4 ° C, and the supernatant was collected.
  • the supernatant was transferred to a test tube, and a 10% NaCl solution was added thereto, and the mixture was ice-cooled overnight, and the precipitated colloidal white fiber was collagen.
  • the precipitated colloidal white fiber was added to the concentration of 3%, 1%, 0.5% glacial acetic acid, filtered through a filter and purified for 24 hours, then placed in distilled water and filtered through a filter for 3 days to obtain purified.
  • Collagen Collagen
  • the purified collagen was transferred to a test tube, placed at -80 ° C for 48 hours, and then transferred to a lyophilizer to obtain a collagen lyophilized powder in about 4 days, and stored under dry conditions at 4 ° C.
  • a boiled 0.5% (w/v) Na 2 CO 3 solution was added to the silkworm cocoons, washed three times with distilled water, and the sericin was removed and dried in a vacuum oven at 65 ° C for 24 hours.
  • the degummed silk was dissolved in a mixed solution containing alcohol and CaCl 2 to obtain a silk fibroin solution.
  • the molar ratio of 100 ml of the mixed solution CaCl 2 , distilled water, and alcohol was 1:8:2.
  • the silk fibroin solution was filtered through a filter paper under a vacuum pump suction negative pressure condition.
  • the filtrate was transferred to a filtration tube, placed in distilled water for 3 days to remove CaCl 2 and alcohol, and distilled water was changed every 12 hours.
  • the purified collagen was transferred to a test tube, placed at -80 ° C for 48 hours, and then transferred to a lyophilizer for about 3 to 5 days to obtain a collagen lyophilized powder, which was stored under dry conditions at 4 ° C.
  • Collagen and silk fibroin were added to a homogenizer, and stirred at 15,000 rpm for 2 hours at 4 ° C, and then the homogenate was transferred into a bottle to which a vacuum pump was attached to remove air bubbles.
  • the collagen silk fibroin homogenate is injected into a metal mold.
  • the mold was placed in a lyophilizer at room temperature (20 ° C), cooled to a freezing temperature (-40 ° C) at a constant rate, and placed at -40 ° C for 60 minutes; then moved into a vacuum environment ( ⁇ 200 mT) or 0 ° C It was allowed to stand for 17 hours until a homogeneous, porous solid composite membrane was formed.
  • the film was crosslinked in a vacuum oven at 105 °C.
  • the aluminum foil packaging composite film is sealed on three sides, one side is opened to evaporate water, and the film is crosslinked in a vacuum oven at 105 ° C.
  • the final film has a thickness of about 6 mm.
  • the preparation process is as follows:
  • the burdock is sterilized by ultraviolet disinfection and 70% alcohol.
  • the cattle are sacrificed to obtain bovine tendon. Take 50 grams of bovine tendon and send it to the clean room as soon as possible. UV disinfection or 70% alcohol spray for the next step.
  • the bovine tendon was made into a tissue homogenate using a homogenizer (this process was added to dry ice to prevent collagen denaturation).
  • bovine tendon 50 grams was added to 500 ml of acetone (concentration > 98%), soaked for 3 days, and acetone was changed every 24 hours to remove adipose tissue from the bovine tendon. The entire operation was carried out at 4 °C.
  • Polysaccharides can increase the viscosity of collagen and cause an immune response in the body.
  • 1000 g of 0.3% NaH 2 PO 4 was added to the bovine tendon from which adipose tissue was removed, and soaked for 3 days, and NaH 2 PO 4 was replaced every 12 hours.
  • the 10% NaCl solution was added to the bovine tendon, and the collagen was crudely extracted. The concentration was adjusted with distilled water. At 4 ° C overnight, the white floc precipitated in the solution was collagen.
  • Collagen particles were filtered through a filter (cutoff molecular mass 20,000 D) in distilled water and purified for 24 hours.
  • the collagen precipitate was transferred to a filter tube having a molecular weight cut-off of 20,000 D, placed in a beaker containing distilled water, and placed at 4 ° C for 24 hours, and distilled water was changed every 8 hours.
  • collagen was added to a concentration of 3% (v / v) glacial acetic acid, soaked for 3 days, fully dissolved.
  • the collagen solution was transferred to a 50 ml high-speed centrifuge tube, centrifuged at 14,000 g for 45 minutes at 4 ° C, and the supernatant was collected.
  • the supernatant was transferred to a test tube, and a 10% NaCl solution was added thereto, and the mixture was ice-cooled overnight to precipitate a colloidal white fiber which was both collagen.
  • the purified collagen was transferred to a test tube, placed at -80 ° C for 48 hours, and then transferred to a lyophilizer to obtain 45 g of collagen lyophilized powder in about 4 days, and stored under dry conditions at 4 ° C.
  • a boiled 0.5% (w/v) Na 2 CO 3 solution was added to the silkworm cocoons, washed several times with distilled water, the sericin was removed, and dried in a vacuum oven at 65 ° C for 24 hours.
  • the degummed silkworm cocoons (10 g) were dissolved in a mixed solution containing alcohol and CaCl 2 .
  • the silk fibroin solution was obtained, and the molar ratio of 100 mL of the mixed solution CaCl 2 , distilled water, and alcohol was 1:8:2.
  • the silk fibroin solution was filtered through a filter paper under a vacuum pump suction negative pressure condition.
  • the silk fibroin solution was filtered through a filter in distilled water and purified for 72 hours.
  • the filtrate was transferred to a filtration tube and placed in distilled water for 3 days to remove CaCl 2 and alcohol, and the distilled water was changed every 12 hours.
  • the purified collagen was transferred to a test tube, placed at -80 ° C for 48 hours, and then transferred to a lyophilizer for about 3 to 5 days to obtain a collagen lyophilized powder, which was stored under dry conditions at 4 ° C.
  • Collagen and silk fibroin were added to a homogenizer, and stirred at 15,000 rpm for 2 hours at 4 ° C, and then the homogenate was transferred into a bottle to which a vacuum pump was attached to remove air bubbles.
  • Collagen silk fibroin homogenate is injected into a metal mold.
  • the mold was placed in a lyophilizer at room temperature (20 ° C), cooled to a freezing temperature (-40 ° C) at a constant rate, and placed at -40 ° C for 60 minutes; then moved into a vacuum environment ( ⁇ 200 mT) or 0 ° C It was allowed to stand for 17 hours until a homogeneous, porous solid composite membrane was formed.
  • the film was crosslinked in a vacuum oven at 105 °C.
  • the aluminum foil packaging composite film is sealed on three sides, one side is opened to evaporate water, and the film is crosslinked in a vacuum oven at 105 ° C.
  • the final film has a thickness of about 6 mm.
  • the preparation process is as follows:
  • the bovine tendon was placed in a homogenizer to prepare a tissue homogenate.
  • the bovine tendon was made into a tissue homogenate using a homogenizer (this process was added to dry ice to prevent collagen denaturation).
  • Polysaccharides can increase the viscosity of collagen and cause immune response.
  • 10 liters of 0.3% NaH 2 PO 4 was added to the adipose tissue-removing tendon, soaked for 3 days, and NaH 2 PO 4 was replaced every 12 hours.
  • the 10% NaCl solution was added to the bovine tendon, the collagen was crudely extracted, and the concentration was adjusted with distilled water. At 4 ° C overnight, the white floc precipitated was collagen.
  • the collagen precipitate was transferred to a filter tube having a molecular weight cut-off of 20,000 D, placed in a beaker containing distilled water, and allowed to stand at 4 ° C for 24 hours, and distilled water was changed every 8 hours.
  • collagen was added to a concentration of 3% (v / v) glacial acetic acid, soaked for 3 days, fully dissolved.
  • the collagen solution was transferred to a 50 ml high-speed centrifuge tube, centrifuged at 14,000 g for 45 minutes at 4 ° C, and the supernatant was collected.
  • the supernatant was transferred to a test tube, and a 10% NaCl solution was added thereto, and the mixture was ice-cooled overnight to precipitate a colloidal white fiber which was both collagen.
  • the purified collagen was placed in a test tube, placed at -80 ° C for 48 hours, and then transferred to a lyophilizer to obtain 45 g of collagen lyophilized powder in about 4 days, and stored under dry conditions at 4 ° C.
  • a boiled 0.5% (w/v) Na 2 CO 3 solution was added to the silkworm cocoons, washed several times with distilled water, the sericin was removed, and dried in a vacuum oven at 65 ° C for 24 hours.
  • the degummed silk (100 g) was dissolved in a solution containing alcohol and CaCl 2 to obtain a silk fibroin solution.
  • the molar ratio of the 1 L mixed solution CaCl 2 , distilled water, and alcohol was 1:8:2.
  • the silk fibroin solution was filtered through a filter paper under a vacuum pump suction negative pressure condition.
  • the filtrate was transferred to a filtration tube and placed in distilled water for 3 days to remove CaCl 2 and alcohol, and the distilled water was changed every 12 hours.
  • the purified collagen was transferred into a test tube, placed at -80 ° C for 48 hours and then transferred to a lyophilizer, about 3 ⁇
  • the collagen lyophilized powder was obtained in 5 days and stored under dry conditions at 4 °C.
  • Collagen and silk fibroin were added to a homogenizer, and stirred at 15,000 rpm for 2 hours at 4 ° C, and then the homogenate was transferred into a bottle to which a vacuum pump was attached to remove air bubbles.
  • the collagen silk fibroin homogenate is injected into a metal mold.
  • the mold was placed in a lyophilizer at room temperature (20 ° C), cooled to a freezing temperature (-40 ° C) at a constant rate, and placed at -40 ° C for 60 minutes; then moved into a vacuum environment ( ⁇ 200 mT) or 0 ° C It was allowed to stand for 17 hours until a homogeneous, porous solid composite membrane was formed.
  • the film was crosslinked in a vacuum oven at 105 °C.
  • the aluminum foil packaging composite film is sealed on three sides, one side is opened to evaporate water, and the film is crosslinked in a vacuum oven at 105 ° C.
  • the final film has a thickness of about 6 mm.
  • the collagen composite membrane of the foregoing examples was used for the repair of a dural defect of a human or animal. During the operation, the membrane can be trimmed to a certain size, soaked and attached to the surface of the animal's brain. Experimental animals using the composite membrane were compared to intraoperative dura mater or tightly sutured animals. The upper midline curved incision, the right top microscopic craniotomy, to avoid damage to the cerebral cortex and to remove the dura mater of about 15 ⁇ 15mm 2 size, place a suitable size of dura mater on the brain surface and tightly suture with the surrounding dura mater.
  • the collagen composite membrane of the foregoing examples was used for the repair of the meninges after human or animal spinal surgery.

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  • Life Sciences & Earth Sciences (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Transplantation (AREA)
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  • Animal Behavior & Ethology (AREA)
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Abstract

一种人工复合膜的制备方法，包括将丝素蛋白与胶原蛋白混合，并制作成复合膜。一种人工复合膜，是由所述的制备方法制作的人工复合膜。一种将由所述的制备方法制作的人工复合膜用于颅脑手术后硬膜的修复或脊椎手术后脊膜的修复的应用。该复合膜可大大提高膜的强度，从而克服现有的胶原蛋白膜在移植应用中的缺点。

Description

人工复合膜、制备方法及其应用 技术领域
本发明涉及生物医学材料，特别是一种人工复合膜、制备方法及其应用。
背景技术
在脑膜瘤切除术、颅脑损伤以及其他一些涉及硬脑膜损伤的情况下，硬膜的移植对于替代缺损的硬膜，促进其再生尤为重要。硬膜修复不全可导致诸多的并发症，如粘连、脑脊液漏(CSF)、感染等。一些生物材料虽可从同体或异体中获得，但同样可导致疾病的传播，引起排异反应，且疤痕、粘连仍不可避免。传统修复损伤硬膜的方法是直接缝合，但某些部位损伤后不便于缝合。有的合成聚合物，如膨体聚四氟乙烯(ePTFE)又可因纤维蛋白胶而发生膨胀。
理想的移植物应满足以下条件：(1)低(或无)免疫原性，(2)良好的伸展强度，(3)孔径大小合适，(4)可防脑脊液漏，(5)抗粘连，(6)合适的降解速率，(7)易于手术操作。
胶原蛋白膜在移植方面具有明显的优势，并已成功应用于临床。胶原蛋白是细胞外基质的主要成分，为细胞存活和分裂的必需物质。由于其具有良好的生物相容性和生物降解能力，故能促进细胞粘附。临床上将胶原蛋白应用于硬膜移植已将近一个世纪。然而两大缺点限制了它的广泛应用。抗拉能力、抗剪能力较差限制了胶原蛋白膜在手术中的应用。此外，胶原蛋白的纯度也会影响治疗效果。
发明内容
本发明的主要目的在于克服现有技术的上述不足，提供一种高强度的人工复合膜、制备方法及其应用。
为实现上述目的，本发明采用以下技术方案：
一种人工复合膜的制备方法，包括将丝素蛋白与胶原蛋白混合，并制作成复合膜。
进一步地，所述胶原蛋白为I型胶原蛋白。
进一步地，所述丝素蛋白从蚕茧中提取。
进一步地，所述I型胶原蛋白从动物的结蹄组织例如牛肌腱中提取。
进一步地，所述方法包括I型胶原蛋白制备步骤，所述I型胶原蛋白制备步骤包括：
对牛肌腱进行灭菌处理；
将牛肌腱制成组织匀浆；
去除牛肌腱中的脂肪组织；
去除牛肌腱中的多糖；
加入NaCl溶液，取沉淀物获得I型胶原蛋白；
经滤膜过滤纯化I型胶原蛋白。
进一步地，所述I型胶原蛋白制备步骤还包括：
将经纯化I型胶原蛋白加入冰醋酸中使其溶解；
对溶液进行离心处理，取上清液；
向上清液中加入NaCl溶液，再进行离心处理，取沉淀物；
将沉淀物加入冰醋酸中，经滤膜过滤纯化，滤液再加入蒸馏水中，再经滤膜过滤纯化；
冷冻干燥经纯化I型胶原蛋白，得到粉末I型胶原蛋白。
进一步地，取所述沉淀物后，将沉淀物先后加入浓度为2～4％、0.8～1.5％、0.2～0.7％的冰醋酸中经滤膜纯化，再加入蒸馏水中经滤膜纯化。
进一步地，所述方法包括丝素蛋白制备步骤，所述丝素蛋白制备步骤包括：
将蚕茧在Na2CO3溶液中煮沸以去除胶质；
将脱胶的蚕茧溶于含有酒精和CaCl2的混合溶液中；
对蚕茧溶液用无菌滤纸过滤；
滤液加入蒸馏水中，再经滤膜过滤纯化；
冷冻干燥经纯化丝素蛋白，得到粉末丝素蛋白。
进一步地，将丝素蛋白与I型胶原蛋白置于醋酸溶液中混合，再进行匀浆处理，然后用所得的浆料制作所述复合膜。
进一步地，所述醋酸溶液的PH值为2.5～3.5。
进一步地，在2～6℃下匀浆处理1～3小时。
进一步地，将所得的浆料注入模具中，在冷冻干燥条件下，制成孔径均一的膜；将膜置于真空烤箱烘烤，使其交联。
进一步地，所述I型胶原蛋白与丝素蛋白的重量比为(3:1)～(5:1)。
一种人工复合膜，是由所述的制备方法制作的人工复合膜。
一种将由所述的制备方法制作的人工复合膜用于颅脑手术后硬膜的修复或脊椎手术后脊膜的修复的应用。
本发明的有益效果：
本发明首次在胶原浆中引入了丝素蛋白以克服单纯胶原蛋白膜抗拉、抗剪能力差的缺点。研究显示丝素蛋白具有良好的生物相容性和生物降解性，且强度远高于胶原蛋白。将这两种天然的生物高聚物混合制备的复合膜可大大提高膜的强度，从而克服胶原蛋白膜在移植应用中的缺点。本发明的工艺能够提取高纯度胶原蛋白，所制备的复合膜具有良好的生物相容性，此外，它还具有均一的微孔结构和良好的韧性，便于手术操作和缝合。由本发明的方法制备的人工复合膜在神经外科手术中可用以替代切除的硬膜，用于颅脑手术后硬膜的修复和再生，促进硬膜再生。
具体实施方式
以下对本发明的实施方式作详细说明。应该强调的是，下述说明仅仅是示例性的，而不是为了限制本发明的范围及其应用。
在一种实施例中，一种人工复合膜的制备方法，包括将丝素蛋白与胶原蛋白优选I型胶原蛋白混合，并制作成高强度的复合膜。该人工复合膜是一种均一的多孔状基质，厚度可根据临床需要进行调整，例如6mm。高强度的人工复合膜包括I型胶原蛋白和蚕丝蛋白，天然聚合胶原蛋白和丝素蛋白可分别从动物肌腱和蚕茧中提取。胶原蛋白和蚕丝蛋白是天然的蛋白质，具有良好的生物相容性和降解能力，对人体具有无抗原性和低免疫原性的特点。
实施例1
制备流程如下：
一、提取高纯度的I型胶原蛋白
A、用紫外消毒法和浓度70％酒精对牛肌腱进行灭菌处理。
牛处死取得10克牛肌腱后尽快送往清洁操作间，紫外线消毒或浓度70％酒精喷洒以备下一步使用。
B、4℃条件下将牛肌腱放入匀浆搅拌器中制成组织匀浆。
使用匀浆搅拌器将牛肌腱制成组织匀浆，此过程加入干冰防止胶原变性。
C、加入丙酮(浓度>98％)，充分浸泡3天，以去除肌腱中的脂肪组织。
10克牛肌腱浸泡入50ml丙酮(浓度>98％)中，每24小时更换一次丙酮。整个操作在4℃条件下进行。
D、加入浓度0.3％NaH2PO4，浸泡3天，去除肌腱中的多糖。
多糖可增加胶原蛋白的粘性，引起机体免疫反应。4℃，将100ml的浓度0.3％NaH2PO4加入去除脂肪组织的牛肌腱中，浸泡3天，每12小时更换一次NaH2PO4。
E、加入浓度10％NaCl溶液，获得I型胶原蛋白。
首先用浓度10％NaCl溶液粗提胶原蛋白。将NaCl溶液加入牛肌腱中，4℃过夜，析出的白色纤维即为胶原蛋白。
F、在蒸馏水中将胶原蛋白经滤膜(截留分子质量20，000D)过滤并纯化24小时。
胶原蛋白沉淀置入截留分子质量为20，000D的滤管中，放入盛有蒸馏水的烧杯中，4℃下，放置24小时，每8小时更换一次蒸馏水。
G、胶原蛋白中加入浓度3％(v/v)冰醋酸，浸泡3天，充分溶解。
4℃条件下，胶原蛋白中加入浓度3％(v/v)冰醋酸，浸泡3天以充分溶解，每12小时更换一次冰醋酸溶液。3ml冰醋酸加入997ml蒸馏水中即为所用冰醋酸溶液，4℃下储存以备使用。
H、进行14，000g的离心，取上清液。
胶原蛋白溶液移入50ml高速离心管中，4℃下，14，000g离心45分钟，收集上清液。
I、加入浓度10％NaCl溶液，14，000g离心，取胶原蛋白沉淀物。
将上清液移入试管中，加入浓度10％NaCl溶液，冰浴过夜，析出的胶状白色纤维即为胶原蛋白。
J、析出的胶状白色纤维先后加入浓度3％、1％、0.5％冰醋酸中，经滤膜过滤并各纯化24小时后，放入蒸馏水中经滤膜过滤并纯化3天，获得纯化的胶原蛋白。
K、冷冻干燥得到固体粉末。
纯化的胶原蛋白移入试管，-80℃下放置48小时后移入冻干机，约4天即可获得胶原蛋白冻干粉末，4℃干燥条件下保存。
二、从蚕茧中提取丝素蛋白
A、于浓度0.5％Na2CO3中煮沸蚕茧以去除丝胶。
蚕茧中加入煮沸的浓度0.5％(w/v)Na2CO3溶液，蒸馏水洗涤3次，去除丝胶蛋白，真空烤箱中65℃条件下干燥24小时，备用。
B、90℃条件下，将脱胶的蚕丝溶解于含有酒精和CaCl2的混合溶液中得到丝素液。
100ml混合溶液CaCl2、蒸馏水、酒精的摩尔比为1:8:2。
C、无菌滤纸过滤
真空泵抽气负压条件下，丝素蛋白溶液经滤纸过滤。
D、在蒸馏水中将丝素蛋白溶液经滤膜过滤纯化72小时
滤液移入滤过管，在蒸馏水中放置3天以去除CaCl2和酒精，每12小时更换一次蒸馏水。
E、冷冻干燥得到固体粉末
将纯化的胶原蛋白移入试管，-80℃下放置48小时后移入冻干机，约3～5天即可获得胶原蛋白冻干粉末，4℃干燥条件下保存。
三、取0.8克胶原蛋白和0.25克丝素蛋白溶于100ml醋酸(PH＝3)溶液中
四、4℃下，匀浆处理2小时。
将胶原蛋白和丝素蛋白加入匀浆搅拌器中，4℃下，15，000rpm搅拌2小时，随后将匀浆移入接有真空泵的瓶子中以去除气泡。
五、制膜
A、将胶原蛋白丝素蛋白匀浆注入金属模具中。
75ml匀浆倒入12cm×12cm的金属模具中。
B、冷冻(-40℃)干燥条件下，利用可控程序制成多孔状膜。
室温条件下(20℃)将模具放入冻干机中，以恒定的速率降温至冷冻温度(-40℃)，并在-40℃放置60分钟；随后移入真空环境(<200mT)或0℃放置17个小时，直至形成质地均一、多孔状的固体复合膜。
C、105℃真空烤箱中使膜交联。
铝箔包装复合膜，三面密封，一面敞开以蒸发水分，105℃真空烤箱中使膜交联，最终膜的厚度约为6mm。
六、将膜切割成一定大小以备临床应用。
七、环氧乙烷灭菌。
八、包装至成品。
实施例2
制备流程如下：
一、提取高纯度的I型胶原蛋白
A、用紫外线消毒法和浓度70％酒精对牛腱进行灭菌处理。
牛处死获取牛肌腱，取50克牛肌腱尽快送往清洁的操作间，紫外线消毒或浓度70％酒精喷洒以备下一步使用。
B、4℃条件下将新鲜牛肌腱放入匀浆搅拌器中制成组织匀浆。
使用匀浆搅拌器将牛肌腱制成组织匀浆(此过程加入干冰防止胶原变性)。
C、加入丙酮(浓度>98％)，充分浸泡3天，以去除肌腱中的脂肪组织。
50克牛肌腱加入500ml丙酮(浓度>98％)，浸泡3天，每24小时更换一次丙酮，以去除牛肌腱中的脂肪组织。整个操作在4℃条件下进行。
D、加入浓度0.3％NaH2PO4，浸泡3天，去除肌腱中的多糖。
多糖可增加胶原蛋白的粘性，引起机体免疫反应。4℃条件下，去除脂肪组织的牛肌腱中加入1000ml的浓度0.3％NaH2PO4，浸泡3天，每12小时更换一次NaH2PO4。
E、加入浓度10％NaCl溶液，获得I型胶原蛋白。
浓度10％NaCl溶液加入牛肌腱中，粗提胶原蛋白，蒸馏水调节浓度，4℃下过夜，溶液中析出的白色絮状物即为胶原蛋白。
F、在蒸馏水中将胶原蛋白颗粒经滤膜(截留分子质量20，000D)过滤并纯化24小时。
胶原蛋白沉淀移入截留分子质量为20，000D的滤管中，放入盛有蒸馏水的烧杯中，4℃下，放置24小时，每8小时更换一次蒸馏水。
G、胶原蛋白中加入浓度3％(v/v)冰醋酸，浸泡3天，充分溶解。
4℃条件下，胶原蛋白中加入浓度3％(v/v)冰醋酸，浸泡3天以充分溶解，每12小时更换一次冰醋酸溶液。9ml冰醋酸加入2991ml蒸馏水中即为所用冰醋酸溶液，4℃储存以备使用。
H、进行14，000g离心，取上清液。
胶原蛋白溶液移入50ml高速离心管中，4℃下，14，000g离心45分钟，收集上清液。
I、加入浓度10％NaCl溶液，14，000g离心，取沉淀物获得胶原蛋白。
上清液移入试管中，加入浓度10％NaCl溶液，冰浴过夜，析出的胶状白色纤维既为胶原蛋白。
J、先后加入浓度3％、1％、0.5％冰醋酸中，经滤膜过滤并各纯化24小时后，加入蒸馏水中经滤膜过滤并纯化3天，获得胶原蛋白。
K、冷冻干燥得到固体粉末。
将纯化的胶原蛋白移入试管，-80℃下放置48小时后移入冻干机，约4天即可获得胶原蛋白冻干粉末45克，4℃下干燥条件下保存。
二、从蚕茧中提取丝素蛋白
A、于浓度0.5％Na2CO3中煮沸蚕茧以去除丝胶。
蚕茧中加入煮沸的浓度0.5％(w/v)Na2CO3溶液，蒸馏水洗涤若干次，去除丝胶蛋白，真空烤箱中65℃条件下干燥24小时，备用。
B、90℃条件下，将脱胶的蚕茧(10g)溶解于含有酒精和CaCl2的混合溶液中。
得到丝素液，100mL混合溶液CaCl2、蒸馏水、酒精的摩尔比为1:8:2。
C、无菌滤纸过滤。
真空泵抽气负压条件下，丝素蛋白溶液经滤纸过滤。
D、在蒸馏水中将丝素蛋白溶液经滤膜过滤后纯化72小时。
将滤液移入滤过管，在蒸馏水中放置3天以去除CaCl2和酒精，蒸馏水每12小时更换一次。
E、冷冻干燥得到固体粉末。
将纯化的胶原蛋白移入试管，-80℃下放置48小时后移入冻干机，约3～5天即可获得胶原蛋白冻干粉末，4℃干燥条件下保存。
三、取0.8克胶原蛋白和0.25克丝素蛋白溶于100ml醋酸(PH＝3)溶液中。
四、4℃下，匀浆2小时。
将胶原蛋白和丝素蛋白加入匀浆搅拌器中，4℃下，15，000rpm搅拌2小时，随后将匀浆移入接有真空泵的瓶子中以去除气泡。
五、制膜
A、胶原蛋白丝素蛋白匀浆注入金属模具中。
300ml匀浆倒入24cm×24cm的金属模具中，共18个模具。
B、冷冻(-40℃)干燥条件下，利用可控程序制成多孔状膜。
室温条件下(20℃)将模具放入冻干机中，以恒定的速率降温至冷冻温度(-40℃)，并在-40℃放置60分钟；随后移入真空环境(<200mT)或0℃放置17个小时，直至形成质地均一、多孔状的固体复合膜。
C、105℃真空烤箱中使膜交联。
铝箔包装复合膜，三面密封，一面敞开以蒸发水分，105℃真空烤箱中使膜交联，最终膜的厚度约为6mm。
六、将膜切割成一定大小以备临床应用。
七、环氧乙烷灭菌。
八、包装至成品。
实施例3
制备过程如下：
一、提取高纯度的I型胶原蛋白
A、用紫外线消毒法和浓度70％酒精对牛肌腱进行灭菌处理
牛处死后取500克牛肌腱尽快送往清洁的操作间，紫外线消毒或浓度70％酒精喷洒以备下一步使用。
B、4℃条件下将牛肌腱放入匀浆搅拌器中制成组织匀浆。
使用匀浆搅拌器将牛肌腱制成组织匀浆(此过程加入干冰防止胶原变性)。
C、加入丙酮(浓度>98％)，充分浸泡3天，以去除肌腱中的脂肪组织。
500克牛肌腱加入5000ml丙酮(浓度>98％)，浸泡3天，每24小时更换一次丙酮，以去除牛肌腱中的脂肪组织。整个操作在4℃条件下进行。
D、加入浓度0.3％NaH2PO4，浸泡3天，去除肌腱中的多糖。
多糖可增加胶原蛋白粘性，引起机体免疫反应。4℃条件下，将10升浓度0.3％NaH2PO4加入去除脂肪组织的肌腱中，浸泡3天，每12小时更换一次NaH2PO4。
E、加入浓度10％NaCl溶液，获得I型胶原蛋白。
浓度10％NaCl溶液加入牛肌腱中，粗提胶原蛋白，蒸馏水调节浓度，4℃下过夜，析出的白色絮状物即为胶原蛋白。
F、在蒸馏水中将胶原蛋白经滤膜(截留分子质量20，000D)过滤纯 化24小时。
胶原蛋白沉淀移入截留分子质量为20，000D的滤管中，放入盛有蒸馏水的烧杯中，4℃下放置24小时，每8小时更换一次蒸馏水。
G、胶原蛋白中加入浓度3％(v/v)冰醋酸，浸泡3天，充分溶解。
4℃下，胶原蛋白中加入浓度3％(v/v)冰醋酸，浸泡3天以充分溶解，每12小时更换一次冰醋酸溶液。90ml冰醋酸加入29910ml蒸馏水中即为所用冰醋酸溶液，4℃下储存以备使用。
H、14，000g离心，取上清液。
胶原蛋白溶液移入50ml高速离心管中，4℃下，14，000g离心45分钟，收集上清液。
I、加入浓度10％NaCl溶液，14，000g离心，取胶原蛋白沉淀物。
上清液移入试管中，加入浓度10％NaCl溶液，冰浴过夜，析出的胶状白色纤维既为胶原蛋白。
J、先后在浓度3％、1％、0.5％冰醋酸中，经滤膜过滤并各纯化24小时后，于蒸馏水中经滤膜过滤并纯化3天，获得胶原蛋白。
K、冷冻干燥得到固体粉末。
纯化的胶原蛋白放入试管，-80℃下放置48小时后移入冻干机，约4天即可获得胶原蛋白冻干粉末45克，4℃下干燥条件下保存。
二、从蚕茧中提取丝素蛋白
A、于浓度0.5％Na2CO3中煮沸蚕茧以去除丝胶。
蚕茧中加入煮沸的浓度0.5％(w/v)Na2CO3溶液，蒸馏水洗涤若干次，去除丝胶蛋白，真空烤箱中65℃条件下干燥24小时，备用。
B、90℃下，将脱胶的蚕丝(100g)溶解于含有酒精和CaCl2的溶液中得到丝素液。
1L混合溶液CaCl2、蒸馏水、酒精的摩尔比为1:8:2。
C、无菌滤纸过滤。
真空泵抽气负压条件下，丝素蛋白溶液经滤纸过滤。
D、在蒸馏水中将丝素蛋白溶液经滤膜过滤并纯化72小时
滤液移入滤过管，蒸馏水中放置3天以去除CaCl2和酒精，蒸馏水每12小时更换一次。
E、冷冻干燥得到固体粉末。
纯化的胶原蛋白移入试管，-80℃下放置48小时后移入冻干机，约3～ 5天即可获得胶原蛋白冻干粉末，4℃下干燥条件下保存。
三、取0.8克胶原蛋白和0.25克丝素蛋白溶于100ml醋酸(PH＝3)溶液中。
四、4℃下，匀浆2小时。
胶原蛋白和丝素蛋白加入匀浆搅拌器中，4℃下，15，000rpm搅拌2小时，随后将匀浆移入接有真空泵的瓶子中以去除气泡。
五、制膜
A、将胶原蛋白丝素蛋白匀浆注入金属模具中。
300ml匀浆倒入24cm×24cm的金属模具中，共180个模具。
B、冷冻(-40℃)干燥条件下，利用可控程序制成多孔状膜。
室温条件下(20℃)将模具放入冻干机中，以恒定的速率降温至冷冻温度(-40℃)，并在-40℃放置60分钟；随后移入真空环境(<200mT)或0℃放置17个小时，直至形成质地均一、多孔状的固体复合膜。
C、105℃真空烤箱中使膜交联。
铝箔包装复合膜，三面密封，一面敞开以蒸发水分，105℃真空烤箱中使膜交联，最终膜的厚度约为6mm。
六、将膜切割成一定大小以备临床应用。
七、环氧乙烷灭菌。
八、包装至成品。
实施例4
将前述实施例的胶原蛋白复合膜用于人或动物的硬膜缺损的修复。手术过程中，膜可修剪成一定大小，浸湿，贴附于动物脑表面。将应用复合膜的实验动物与术中硬膜敞开或是严密缝合的动物进行比较。头顶中线弧形切口，右顶部显微开颅，避免损伤脑皮质并切除大小约15×15mm2的硬脑膜，放置合适大小的硬脑膜替代物于脑表面并与周围硬膜严密缝合。
实施例5
将前述实施例的胶原蛋白复合膜用于人或动物脊椎手术后脊膜的修复。
以上内容是结合具体/优选的实施方式对本发明所作的进一步详细说 明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，其还可以对这些已描述的实施方式做出若干替代或变型，而这些替代或变型方式都应当视为属于本发明的保护范围。

Claims (15)

  1. 一种人工复合膜的制备方法，其特征在于，包括将丝素蛋白与胶原蛋白混合，并制作成复合膜。
  2. 如权利要求1所述的制备方法，其特征在于，所述胶原蛋白为I型胶原蛋白。
  3. 如权利要求1所述的制备方法，其特征在于，所述丝素蛋白从蚕茧中提取。
  4. 如权利要求2所述的制备方法，其特征在于，所述I型胶原蛋白从动物的结蹄组织提取，优选从牛肌腱提取。
  5. 如权利要求2所述的制备方法，其特征在于，包括I型胶原蛋白制备步骤，所述I型胶原蛋白制备步骤包括：
    对牛肌腱进行灭菌处理；
    将牛肌腱制成组织匀浆；
    去除牛肌腱中的脂肪组织；
    去除牛肌腱中的多糖；
    加入NaCl溶液，取沉淀物获得I型胶原蛋白；
    经滤膜过滤纯化I型胶原蛋白。
  6. 如权利要求5所述的制备方法，其特征在于，所述I型胶原蛋白制备步骤还包括：
    将经纯化I型胶原蛋白加入冰醋酸中使其溶解；
    对溶液进行离心处理，取上清液；
    向上清液中加入NaCl溶液，再进行离心处理，取沉淀物；
    将沉淀物加入冰醋酸中，经滤膜过滤纯化，滤液再加入蒸馏水中，再经滤膜过滤纯化；
    冷冻干燥经纯化I型胶原蛋白，得到粉末I型胶原蛋白。
  7. 如权利要求6所述的制备方法，其特征在于，取所述沉淀物后，将沉淀物先后加入浓度为2～4％、0.8～1.5％、0.2～0.7％的冰醋酸中经滤膜纯化，再加入蒸馏水中经滤膜纯化。
  8. 如权利要求2至7任一项所述的制备方法，其特征在于，包括丝素蛋白制备步骤，所述丝素蛋白制备步骤包括：
    将蚕茧在Na2CO3溶液中煮沸以去除胶质；
    将脱胶的蚕茧溶于含有酒精和CaCl2的混合溶液中；
    对蚕茧溶液用无菌滤纸过滤；
    滤液加入蒸馏水中，再经滤膜过滤纯化；
    冷冻干燥经纯化丝素蛋白，得到粉末丝素蛋白。
  9. 如权利要求2至8任一项至所述的制备方法，其特征在于，将丝素蛋白与I型胶原蛋白置于醋酸溶液中混合，再进行匀浆处理，然后用所得的浆料制作所述复合膜。
  10. 如权利要求9至所述的制备方法，其特征在于，所述醋酸溶液的PH值为2.5～3.5。
  11. 如权利要求9至所述的制备方法，其特征在于，在2～6℃下匀浆处理1～3小时。
  12. 如权利要求9所述的制备方法，其特征在于，将所得的浆料注入模具中，在冷冻干燥条件下，制成孔径均一的膜；将膜置于真空烤箱烘烤，使其交联。
  13. 如权利要求2至12任一项至所述的制备方法，其特征在于，所述I型胶原蛋白与丝素蛋白的重量比为(3:1)～(5:1)。
  14. 一种人工复合膜，其特征在于，是由如权利要求1至13任一项所述的制备方法制作的人工复合膜。
  15. 一种将由如权利要求1至13任一项所述的制备方法制作的人工复合膜用于颅脑手术后硬膜的修复或脊椎手术后脊膜的修复的应用。
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