WO2022028105A1 - 一种仿生复合骨支架及其制备方法 - Google Patents

一种仿生复合骨支架及其制备方法 Download PDF

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WO2022028105A1
WO2022028105A1 PCT/CN2021/099612 CN2021099612W WO2022028105A1 WO 2022028105 A1 WO2022028105 A1 WO 2022028105A1 CN 2021099612 W CN2021099612 W CN 2021099612W WO 2022028105 A1 WO2022028105 A1 WO 2022028105A1
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silk
solution
sericin
silk fibroin
preparation
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PCT/CN2021/099612
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French (fr)
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王建南
裔洪根
殷音
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苏州大学
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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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the invention relates to the technical field of biomedical materials, in particular to a bionic composite bone support and a preparation method thereof.
  • Bone or teeth are the hardest connective tissue in the human body. Taking bone as an example, with the aging of the world population and the increasing number of accidents, there are countless patients with bone diseases, and clinical medical treatment related to bone is facing more and more problems. stress, especially for bone repair and bone grafting. As early as the 19th century, bone graft surgery began to be used to repair extensive bone defects. At present, the common bone repair methods include traditional autologous bone transplantation, allogeneic bone transplantation, and artificial bone transplantation. Although autologous bone graft repair has the best therapeutic effect, there are serious constraints on many factors such as damage to the donor site, insufficient amount and complications. Allogeneic bone transplantation faces problems such as immune rejection and disease transmission.
  • Bioceramic materials have hydroxyapatite with the same important components as biological bones and teeth, and have excellent biological activity and biomechanical strength, but their high brittleness and poor toughness are not conducive to the remodeling of new bone.
  • Synthetic organic polymer materials have the advantages of wide sources, adjustable physicochemical properties, and excellent processability, such as polyester, polyanhydride, polyorthoester, polyphosphazene, polylactic acid, polyglycolic acid and their copolymers. It is a polymer, but the material lacks biological activity, has poor cell compatibility, and does not degrade or the degradation products affect the tissue microenvironment.
  • Natural organic polymers are widely favored in the research of tissue engineering materials due to their excellent biological activities, such as collagen, gelatin, alginate, hyaluronic acid, chitosan, silk protein, etc.
  • silk is composed of silk fibroin (70-80% by weight) and sericin (20-30% by weight) covered with a unique composition and protein.
  • the purity is very high, and the preparation method can be relatively simple and mild, that is, it can control the small damage to the fibroin macromolecules, unlike natural macromolecules such as collagen, which are complex in purification, small in molecular weight, scattered in distribution, and volatile.
  • the silk fibroin macromolecule is connected by disulfide bonds with 6 heavy chains of about 390kDa and 6 light chains of about 26kDa, and then combined with another glycoprotein/P25 of about 25kDa hydrophobic interaction, wherein the heavy chain has the ability to form a compact structure, high performance Sequential structural basis of materials.
  • the molecular weight of sericin is also relatively large. There are as many as 9 kinds of sericin. Shibukawa Akira pointed out from histological point of view that sericin has three layers, the middle and the inner layer.
  • Komatsu Keichi proposed that the outer sericin exists from the outside to the inside 4 kinds, namely sericin I, sericin II, sericin III and sericin IV, and the solubility from the outside to the inside is getting lower and lower, and the ⁇ conformation increases.
  • Silk fibroin and sericin have excellent cell, blood and tissue compatibility and controllable forming.
  • tissue engineering scaffold materials they have been widely studied in tissue engineering applications such as skin, blood vessels, and bone.
  • the regenerated silk fibroin solutions are all obtained by dissolving strong alkali weak acid salts or enzyme degumming salts. These methods cause the destruction of silk fibroin macromolecules; the regenerated sericin solutions are mainly derived from silkworm cocoons, and the two solutions are generally prepared independently.
  • CN02138129.1 discloses the preparation of a silk fibroin and sericin composite tissue engineering scaffold.
  • the composite material is prepared by mixing pre-prepared regenerated silk fibroin and regenerated sericin in proportion.
  • the degumming method uses a strong base and weak acid salt solvent, and sericin is extracted from the outer part of the silkworm cocoon silk covered with sericin.
  • Another example is CN201510353363.8, which discloses a bone tissue engineering scaffold material containing silk fibroin and sericin and a preparation method thereof. The regenerated silk fibroin and regenerated sericin are also obtained in steps and then mixed to prepare the scaffold material. The preparation of silk fibroin does not disclose specific process conditions.
  • the sericin is obtained from silk cocoons and treated with organic solvents such as alcohol.
  • the regenerated silk (silk fibroin, sericin) protein materials studied above also have poor mechanical properties and are difficult to meet the requirements for replacement of hard tissues such as bones and teeth.
  • the macromolecular structure of protein and sericin is greatly damaged, and the degree of polymerization (molecular weight) of the polymer is an extremely important intrinsic factor that determines the performance of the polymer.
  • the technical problem solved by the present invention is to provide a preparation method of a biomimetic composite skeleton, and the biomimetic composite skeleton has high strength and modulus.
  • the present application provides a preparation method of a bionic composite bone scaffold, comprising the following steps:
  • A) silk is placed in warm water for pretreatment, then placed in 65 ⁇ 95 °C of water for treatment, and dried to obtain the silk of initial treatment;
  • the temperature of the warm water is 35 ⁇ 60° C., and the time of the pretreatment is 1 ⁇ 10 min.
  • the treatment time in water at 65-95° C. is 1-8 h.
  • the ratio of the silk to warm water is 1 g: (40-60) ml
  • the ratio of the pretreated silk to water is 1 g: (40-60) ml.
  • the method further comprises: taking out the pretreated silk, washing and dehydrating in water at 25-30°C;
  • the method further includes: washing the treated silk in water at 25-30° C. and then dehydrating; the drying adopts hot air at 40-60° C. for drying.
  • the neutral salt solution is an 8-10 M lithium bromide aqueous solution, and the ratio of the initially treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml.
  • the temperature of the treatment is 50-80°C.
  • the purifying method is specifically:
  • the obtained silk fibroin sericin composite solution is poured into a dialysis bag with a molecular weight cut-off of 10-50 kDa, and placed in deionized water for continuous dialysis for 1-3 days.
  • the small molecule monohydric alcohol is an ethanol solution with a concentration of 70-80%, the treatment time is 10-60 min; the freezing temperature of the freeze-drying is -10--80°C, The time is 12-36h, and the drying time is 24-72h.
  • the application also provides a biomimetic composite bone scaffold, which is prepared from a silk fibroin sericin composite solution, and the preparation method of the silk fibroin sericin composite solution includes the following steps:
  • A) silk is placed in warm water for pretreatment, then placed in 65 ⁇ 95 °C of water for treatment, and dried to obtain the silk of initial treatment;
  • the present application provides a method for preparing a biomimetic composite skeleton.
  • the silk fibroin sericin composite solution is prepared by mild degumming and one-step dissolving. Partial or complete removal of the glue protein, the existence of sericin increases the strong hydrogen bonding and network structure between the molecular chains of the composite membrane, and improves the strength and modulus of the composite skeleton, or degumming in water at a specific temperature, silk During the subsequent degumming process of fibroin, the macromolecular structure of silk fibroin is protected, so that the molecular weight of regenerated silk fibroin is high to improve the strength of the composite skeleton; The types, structures and properties of sericin are also different.
  • high-strength and high-tenacity silk fibroin composite three-dimensional scaffolds with different compositions and controllable mechanical properties are obtained, which solves the problem of natural biopolymer tissue engineering three-dimensional scaffolds for bone regeneration and repair.
  • the technical problem of neck-stuck is insufficient mechanical properties of the scaffold, and the composite scaffold has the osteogenic activity of in situ to rapidly induce hydroxyapatite deposition.
  • the present application provides a biomimetic composite skeleton.
  • the composite skeleton is prepared from a silk fibroin sericin composite solution.
  • a large molecular weight and high viscosity silk fibroin sericin composite solution is prepared by deionized water, mild degumming and one-step dissolution.
  • the method can avoid the damage of the macromolecules of silk fibroin fibers, and finally make the silk fibroin in the prepared silk fibroin sericin composite solution have high viscosity and large molecular weight, and then the silk fibroin sericin biomimetic composite skeleton prepared from the silk fibroin sericin composite skeleton has Good compressive modulus and strength.
  • the embodiment of the present invention discloses a preparation method of a bionic composite bone scaffold, which includes the following steps:
  • A) silk is placed in warm water for pretreatment, then placed in 65 ⁇ 95 °C of water for treatment, and dried to obtain the silk of initial treatment;
  • the present application first places the silk in warm water for pretreatment, and then places it in water at 65-95°C for treatment and drying to obtain the pre-treated silk; during this process In the process, the sericin of silk is completely or partially dissolved, while the silk fibroin is still in the fibrous state. Exist in water, in solution state.
  • the temperature of the warm water is 35-60°C, that is, the silk can be pretreated in warm water at 35°C, 40°C, 45°C, 50°C, 55°C or 60°C, and the ratio of the silk to the warm water is 1 g : (40-60) ml, more specifically, the ratio of the silk to the warm water is 1g: 42ml, 1g: 45ml, 1g: 48ml, 1g: 52ml, 1g: 55ml, 1g: 58ml.
  • the pretreatment is carried out in the above-mentioned warm water at a constant temperature, and the time of the pretreatment is 1-10 minutes, and the above-mentioned pretreatment is always carried out under stirring, so as to ensure that the silk can be initially and completely immersed in the water.
  • the pretreated silk is taken out and washed and dehydrated in deionized water at 25-30°C.
  • the dehydrated silk is treated in water at 65 to 95°C. , 90 °C or 93 °C water treatment.
  • the silk fibroin macromolecular structure of the present application is pretreated in water and then treated in water, which can dissolve sericin while protecting the macromolecular structure of silk fibroin.
  • the ratio of the pretreated silk to water is 1g:(40 ⁇ 60)ml, more specifically, the ratio of the pretreated silk to water is 1g:42ml, 1g:45ml, 1g:48ml, 1g : 50ml, 1g: 52ml, 1g: 55ml, 1g: 58ml or 1g: 60ml.
  • the degumming rate of silk sericin can be adjusted according to the different temperature and time of the water, so as to realize the preparation of the composite solution of silk fibroin sericin with different viscosities and different molecular weights; in this application, the water treatment time is preferably 1-8h , so that the silk is partially or completely degummed; more specifically, the water treatment time is 1.5h, 2h, 2.5h, 3h, 3.2h, 3.6h, 4.2h, 4.5h, 5.0h, 5.5h, 6.0h , 6.5h, 6.8h, 7.0h or 7.5h. After the treatment, the treated silk is washed in water at 25-30°C, then dehydrated and dried; the drying adopts hot air drying at 40-60°C.
  • the silk undergoes the above-mentioned treatment to obtain the initially treated silk, which is still in a fibrous state, and realizes the protection of the silk fibroin macromolecules, so as to ensure the viscosity of the silk fibroin sericin.
  • the initially treated silk is then treated in a neutral salt solution and purified to obtain a silk fibroin sericin composite solution.
  • the fibrous silk that is initially treated is dissolved to obtain a composite solution of silk fibroin sericin.
  • the inner core of the silk is silk fibroin, and the periphery is wrapped with different contents of sericin.
  • the neutral salt solution is specifically selected from the lithium bromide aqueous solution in this application, and its concentration is 8-10M, and the ratio of the initially treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml, more specifically, The ratio of the lithium bromide aqueous solution of the initially treated silk rain is 1g:22ml, 1g:24ml, 1g:25ml, 1g:27ml or 1g:29ml.
  • the temperature of the treatment is specifically 50-80°C, and more specifically, the temperature of the treatment is specifically 52°C, 57°C, 62°C, 65°C, 67°C, 68°C, 72°C, 75°C or 78°C.
  • the obtained silk fibroin sericin dissolving solution is purified to remove lithium bromide and low molecular weight silk fibroin sericin; the purification method is specifically pouring the obtained silk fibroin sericin complex dissolving solution into Place in a dialysis bag with a molecular weight cut-off of 10-50kDa and place it in deionized water for continuous dialysis for 1-3 days.
  • This application finally uses the prepared silk fibroin sericin composite solution to form a skeleton. Specifically, the silk fibroin sericin composite solution is concentrated to a certain extent, and then pre-frozen at -20 ⁇ -80°C for 12 minutes after removing air bubbles.
  • the obtained initial composite skeleton is placed in a small molecule monohydric alcohol solution for 10-60min, so that the obtained composite skeleton is insoluble in water and tissue fluid, and finally soaked in water, air-dried at constant temperature and humidity , and then freeze-dried to obtain the silk fibroin sericin biomimetic composite framework; more specifically, the treatment time is 15min, 20min, 22min, 26min, 32min, 36min, 47min, 46min, 52min, 54min, 56min or 58min.
  • the invention adopts the mild degumming and one-step dissolving method to prepare the silk fibroin and sericin composite solution, which breaks through the traditional method of separately preparing silk fibroin and sericin, dissolving them separately and then mixing them to prepare composite materials.
  • the present invention provides a preparation technology of high-performance regenerated silk fibroin sericin composite material based on the molecular weight of the polymer, which is the fundamental factor affecting the performance of the polymer.
  • Mild degumming One-step dissolving method In the process of preparing silk protein solution, hot water method is used for degumming, so that the silk fibroin macromolecular chain (especially the heavy chain) will not be destroyed during the degumming process.
  • the molecular weight of silk protein in this composite material is concentrated at 150kDa
  • the molecular weight of the silk fibroin prepared by the reported conventional method or the material prepared by mixing silk fibroin with sericin presents a continuous distribution, mainly below 85kDa to 15kDa or even smaller, so the silk fibroin provided by the present invention
  • Vegetarian protein has a large molecular weight and a small degree of molecular weight dispersion, which improves the strength and modulus of the composite three-dimensional framework.
  • the presence of sericin increases the strong hydrogen bonding between the molecular chains of the composite backbone, and also improves the strength and modulus of the composite backbone.
  • the sericin in the outer layer of silk fibroin is different from the outside to the inside of the coating layer, and the type, structure and properties (especially mechanical properties) of sericin are also different.
  • the invention obtains high-strength and high-tenacity silk fibroin sericin composite three-dimensional scaffolds with different compositions and controllable mechanical properties, and solves the neck-stuck technology of insufficient mechanical properties of natural biopolymer tissue engineering three-dimensional scaffolds for bone regeneration and repair difficult, and the composite scaffold has osteogenic activity to rapidly induce hydroxyapatite deposition in situ.
  • the present application also provides a biomimetic composite bone scaffold prepared from a silk fibroin sericin composite solution.
  • the preparation method of the silk fibroin sericin composite solution includes the following steps:
  • A) silk is placed in warm water for pretreatment, then placed in 65 ⁇ 95 °C of water for treatment, and dried to obtain the silk of initial treatment;
  • steps A) and B) are the process of preparing the silk fibroin sericin composite solution, which will not be repeated here.
  • the silk fibroin sericin biomimetic composite bone scaffold described in the present application is prepared from a silk fibroin sericin composite solution.
  • the dried silk was placed in preheated deionized water at 90°C and cooked at a constant temperature for 3 hours according to the liquor ratio of 1:50 (g/mL), and the silk containing sericin was taken out and thoroughly washed with deionized water at 25-30°C. After dehydration, dry it with hot air at 40-60 °C;
  • the degumming rate of silk was measured to be 14%;
  • the compressive strength and compressive modulus of silk fibroin and sericin composite scaffolds were measured by a texture analyzer up to 3 MPa and 101 MPa, respectively.
  • the dried silk was placed in deionized water preheated at 90°C and cooked at a constant temperature for 6 hours according to the liquor ratio of 1:50 (g/mL), and the silk containing sericin was taken out and thoroughly washed with deionized water at 25-30°C. After dehydration, dry it with hot air at 40-60 °C;
  • the compressive strength and compressive modulus of the silk fibroin sericin composite scaffolds were measured by a texture analyzer up to 4 MPa and 116 MPa, respectively.
  • the dried silk was placed in deionized water preheated at 90°C and cooked at a constant temperature for 6 hours according to the liquor ratio of 1:50 (g/mL), and the silk containing sericin was taken out and thoroughly washed with deionized water at 25-30°C. After dehydration, dry it with hot air at 40-60 °C;
  • the compressive strength and compressive modulus of silk fibroin and sericin composite scaffolds were measured by a texture analyzer up to 5MPa and 134MPa, respectively.
  • the degumming rate of silk was measured to be 23%
  • the anti-compression properties of silk fibroin scaffolds were measured using a texture analyzer: the compressive strength could reach 1.65MPa, and the compressive modulus could reach 40.5MPa.
  • the degumming rate of silk was measured to be 23%
  • the dehydrated silk was placed in preheated deionized water at 90°C and cooked at a constant temperature for 7 hours according to the liquor ratio of 1:50 (g/mL), the fibers were discarded, and the sericin aqueous solution was collected and freeze-dried to obtain solid sericin;
  • step 7 use a rotary evaporator to concentrate the silk fibroin aqueous solution obtained in step 4, add the sericin prepared in step 6 with the same content as in Example 3, dissolve and mix evenly, prepare the silk fibroin sericin composite solution with a concentration of 80 mg/mL, and remove the solution Add the bubbles in the air to a cylindrical or square vessel, freeze the sample at -10 ⁇ -80°C for 12 ⁇ 36h, then immediately put it into a freeze dryer to dry for 24 ⁇ 72h, take out and soak the sample with 80% ethanol solution About 20min, and then air-dried in a constant temperature and humidity room to obtain a silk fibroin scaffold.
  • the anti-compression properties of silk fibroin scaffolds measured by texture analyzer are as follows: the compressive strength can reach 1.4MPa, and the initial compressive modulus can reach 33MPa.

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Abstract

一种仿生复合骨支架的制备方法，包括以下步骤：A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液；C)将所述丝素丝胶蛋白复合溶液浓缩后冷冻干燥，将得到的初始骨架置于小分子一元醇中处理后冻干，得到仿生复合骨支架。

Description

一种仿生复合骨支架及其制备方法
本申请要求于2020年08月06日提交中国专利局、申请号为202010782576.3、发明名称为“一种仿生复合骨支架及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医用材料技术领域，尤其涉及一种仿生复合骨支架及其制备方法。
背景技术
骨或牙齿是人体中最坚硬的结缔组织，以骨为例，随着世界人口的老龄化及意外事故不断增加，骨疾病患者不计其数，与骨有关的临床医学治疗面临着越来越大的压力，尤其是骨修补、骨移植。早在19世纪，骨移植手术开始用来修复大范围的骨缺损。目前，常见的骨修复方法有传统的自体骨移植，同种异体骨移植，人工骨移植。自体骨移植修复虽然治疗效果最佳，但存在对供区破坏、取量不足及并发症等诸多因素的严重制约。异体骨移植面临着免疫排斥反应、疾病传播等问题。20世纪90年代提出了组织工程，随后骨组织工程支架材料的研究得到了快速的发展。生物陶瓷类材料具有生物体骨骼和牙齿的重要组成成分相同的羟基磷灰石，具有优良的生物活性和生物力学强度，但其脆性高、韧性差，不利于新生骨的重塑。合成有机高分子材料来源广、物化性质可调控、优异加工成型性等优点，如聚酯、聚酸酐、聚原酸酯、聚膦腈、聚乳酸，聚羟基乙酸及其共聚物等多种合成高分子，但材料缺乏生物活性，细胞相容性较差，不降解或降解产物影响组织微环境。
天然有机高分子由于具有优异的生物活性在组织工程材料研究中广受青睐，如胶原、明胶、海藻酸盐、透明质酸、壳聚糖、蚕丝蛋白等。与其它天然高分子尤其是蛋白质高分子不同的是：蚕丝由丝素纤维(重量占比70～80％)和外覆的丝胶蛋白(重量占比20～30％)组成，组成独特，蛋白纯度很高，制备方法可以比较简单温和，即可以控制对蚕丝蛋白大分子较小的破坏，不像胶原蛋白等天然高分子纯化复杂、分子量小且分布散、易变性等。丝素蛋白大分子由6条约390kDa的重链和6条约26kDa轻链以二硫键连接，再与另一种约25kDa的糖蛋白/P25疏水作用结合，其中重链具有形成致密结构、高性能材料 的序列结构基础。丝胶蛋白的分子量也比较大，丝胶蛋白有9种之多，涉川明朗从组织学角度指出丝胶蛋白分外中内三层，小松计一提出外覆丝胶蛋白由外及内存在4种，即丝胶I、丝胶II、丝胶III和丝胶IV，且由外及内溶解性能越来越低，β构象增加。
丝素蛋白和丝胶蛋白具有优异的细胞、血液和组织相容性以及可控成型，作为组织工程支架材料已在皮肤、血管、骨等组织工程应用中开展了广泛研究。再生丝素蛋白溶液都以强碱弱酸盐或酶脱胶盐溶解获得，这些方法造成了丝素蛋白大分子的破坏；再生丝胶蛋白溶液主要来源于蚕茧，两种溶液一般独立分离制备。如CN02138129.1公开了一种丝素丝胶蛋白复合组织工程支架的制备，复合材料采用预先制备好的再生丝素蛋白与再生丝胶蛋白按比例进行混合再制备而成，其中丝素纤维的脱胶方法采用了强碱弱酸盐溶剂，丝胶蛋白提取的是蚕茧茧丝外覆丝胶的较外层的部分。又如CN201510353363.8公开了一种含丝素蛋白和丝胶蛋白的骨组织工程支架材料及其制备方法，也是分步分别获得再生丝素蛋白与再生丝胶蛋白后再加以混合制备支架材料，其中丝素蛋白的制备没有公开具体工艺条件，丝胶蛋白的获得来自于蚕茧，并经醇等有机溶剂处理。
但上述研究的再生蚕丝(丝素，丝胶)蛋白材料同样力学性能较差，难以满足骨、牙齿等硬组织替换的要求，其问题的关键是现有的脱胶方法和抽提方法对丝素蛋白和丝胶蛋白大分子结构破坏较大，而高分子的聚合度(分子量)是决定高分子性能的极其重要的内在因素。
发明内容
本发明解决的技术问题在于提供一种仿生复合骨架的制备方法，该仿生复合骨架具有高强度和模量。
有鉴于此，本申请提供了一种仿生复合骨支架的制备方法，包括以下步骤：
A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液；
C)将所述丝素丝胶蛋白复合溶液浓缩后冷冻干燥，将得到的初始骨架置于小分子一元醇中处理后冻干，得到仿生复合骨支架。
优选的，所述温水的温度为35～60℃，所述预处理的时间为1～10min。
优选的，步骤A)中，所述置于65～95℃的水中处理的时间为1～8h。
优选的，步骤A)中，所述蚕丝与温水的比例为1g：(40～60)ml，预处理后的蚕丝与水的比例为1g：(40～60)ml。
优选的，步骤A)中，所述预处理之后还包括：将预处理后的蚕丝取出后于25～30℃的水中洗净、脱水；
所述处理后干燥之前还包括：将处理后的蚕丝于25～30℃的水中清洗后脱水；所述干燥采用40～60℃的热风干燥。
优选的，所述中性盐溶液为8～10M的溴化锂水溶液，所述初处理的蚕丝与所述溴化锂水溶液的比例为1g：(20～30)ml。
优选的，步骤B)中，所述处理的温度为50～80℃。
优选的，所述纯化的方法具体为：
将得到的丝素丝胶蛋白复合溶解液灌注于截留分子量10～50kDa的透析袋内，置于去离子水中持续透析1～3天。
优选的，步骤C)中，所述小分子一元醇为浓度为70～80％的乙醇溶液，所述处理的时间为10～60min；所述冷冻干燥的冷冻温度为-10～-80℃，时间为12～36h，干燥的时间为24～72h。
本申请还提供了一种仿生复合骨支架，由丝素丝胶蛋白复合溶液制备得到，所述丝素丝胶蛋白复合溶液的制备方法，包括以下步骤：
A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液。
本申请提供了一种仿生复合骨架的制备方法，采用温和脱胶、一步溶解法制备了丝素丝胶蛋白复合溶液，在温水中的预处理以及后续的特定温度水中的处理，使得蚕丝表面的丝胶蛋白部分脱除或全部脱除，丝胶蛋白的存在增加了复合膜分子链之间的强氢键结合和网络结构，提高了复合骨架的强度和模量， 或采用特定温度水中脱胶、丝素纤维后续脱胶过程中丝素蛋白大分子结构得到保护，而使得再生丝素蛋白分子量高以提高复合骨架的强度；丝素纤维外层的丝胶蛋白随着包覆层的由外及内，丝胶蛋白的种类、结构和性能也不同，通过控制脱胶程度，得到了不同组成、力学性能可控的高强高韧丝素蛋白复合三维支架，解决了骨再生修复的天然生物聚合物组织工程三维支架力学性能不足的卡脖子技术难题，且复合支架具有原位快速诱导羟基磷灰石沉积的骨生成活性。
具体实施方式
为了进一步理解本发明，下面结合实施例对本发明优选实施方案进行描述，但是应当理解，这些描述只是为进一步说明本发明的特征和优点，而不是对本发明权利要求的限制。
针对现有技术中，丝素蛋白大分子破坏严重或丝素丝胶复合溶液分别制备而影响丝素丝胶蛋白复合骨架强度和模量的问题，本申请提供了一种仿生复合骨架，该种复合骨架是由丝素丝胶复合溶液制备得到，在丝素丝胶仿生复合骨架制备过程中，采用去离子水温和脱胶、一步溶解法制备了大分子量高粘度丝素丝胶蛋白复合溶液，该方法可以避免丝素纤维大分子的破坏，最终使制备的丝素丝胶蛋白复合溶液中的丝素蛋白具有较高的粘度和大分子量，进而由其制备得到的丝素丝胶仿生复合骨架具有较好的抗压模量和强度。具体的，本发明实施例公开了一种仿生复合骨支架的制备方法，包括以下步骤：
A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液；
C)将所述丝素丝胶蛋白复合溶液浓缩后冷冻干燥，将得到的初始骨架置于小分子一元醇中处理后冻干，得到仿生复合骨支架。
在上述丝素丝胶蛋白仿生复合骨架制备过程中，本申请首先将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，即得到初处理的蚕丝；在此过程中，蚕丝的丝胶蛋白全部或部分溶解，而丝素蛋白仍然是纤维状态， 若有未溶解的丝胶，则未溶解的丝胶包裹在丝素纤维的外表面，溶解掉的丝胶则存在于水中，呈溶液状态。所述温水的温度为35～60℃，即所述蚕丝可在35℃、40℃、45℃、50℃、55℃或60℃的温水中进行预处理，所述蚕丝与温水的比例为1g：(40～60)ml，更具体地，所述蚕丝与所述温水的比例为1g：42ml、1g：45ml、1g：48ml、1g：52ml、1g：55ml、1g：58ml。所述预处理在上述恒温的温水中进行，所述预处理的时间为1～10min，且上述预处理一直在搅拌中进行，以保证蚕丝可以初步、完全浸渍于水中。在预处理完成后，则将预处理后的蚕丝取出后于25～30℃的去离子水中洗净、脱水。脱水后的蚕丝则在65～95℃的水中处理，更具体地，脱水后的蚕丝可在68℃、70℃、72℃、75℃、78℃、80℃、82℃、84℃、87℃、90℃或93℃的水中处理。本申请蚕丝先在水中预处理再在水中处理的方式，可以在保护丝素蛋白大分子结构不被破坏的同时溶解丝胶蛋白。所述预处理后的蚕丝与水的比例为1g：(40～60)ml，更具体地，所述预处理后的蚕丝与水的比例为1g：42ml、1g：45ml、1g：48ml、1g：50ml、1g：52ml、1g：55ml、1g：58ml或1g：60ml。根据水的不同温度以及时间可以调整蚕丝丝胶蛋白的脱胶率，以实现不同粘度和不同分子量丝素丝胶蛋白复合溶液的制备；在本申请中，所述水中处理的时间优选为1～8h，以使蚕丝部分或全部脱胶；更具体地，所述水中处理的时间为1.5h、2h、2.5h、3h、3.2h、3.6h、4.2h、4.5h、5.0h、5.5h、6.0h、6.5h、6.8h、7.0h或7.5h。在处理之后，则将处理后的蚕丝于25～30℃的水中清洗后脱水、干燥；所述干燥采用40～60℃的热风干燥。蚕丝经过上述处理，得到初处理的蚕丝，该蚕丝仍然呈现纤维状态，实现了丝素蛋白大分子的保护，以利于保证丝素丝胶蛋白的粘度。
按照本发明，然后将初处理的蚕丝在中性盐溶液中处理，纯化，即得到丝素丝胶蛋白复合溶液。在此过程中，将初处理的纤维状的蚕丝溶解，即得到丝素丝胶蛋白复合溶液，此时的蚕丝内芯为丝素纤维，外围包裹有不同含量的丝胶。所述中性盐溶液在本申请中具体选自溴化锂水溶液，其浓度为8～10M，所述初处理的蚕丝与所述溴化锂水溶液的比例为1g：(20～30)ml，更具体地，所述初处理的蚕丝雨所述溴化锂水溶液的比例为1g：22ml、1g：24ml、1g：25ml、1g：27ml或1g：29ml。所述处理的温度具体为50～80℃，更具体地， 所述处理的温度具体为52℃、57℃、62℃、65℃、67℃、68℃、72℃、75℃或78℃。
在上述处理之后，则将得到的丝素丝胶溶解液进行纯化，以去除溴化锂和小分子量丝素丝胶蛋白；所述纯化的方式具体为将得到的丝素丝胶蛋白复合溶解液灌注于截留分子量10～50kDa的透析袋内，置于去离子水持续透析1～3天。
本申请最后是利用制备得到的丝素丝胶蛋白复合溶液成骨架的过程，其具体是将丝素丝胶蛋白复合溶液浓缩至一定程度，脱除气泡后于-20～-80℃预冷冻12～48h后再冷冻干燥24～72h，得到的初始复合骨架再置于小分子一元醇溶液中处理10～60min，以使得到的复合骨架不溶于水以及组织液，最后于水中浸泡，恒温恒湿风干，再冻干即得到丝素丝胶蛋白仿生复合骨架；更具体地，所述处理的时间为15min、20min、22min、26min、32min、36min、47min、46min、52min、54min、56min或58min。
本发明采用温和脱胶、一步溶解法制备了丝素丝胶蛋白复合溶液，突破了传统的丝素蛋白和丝胶蛋白分别制备、分别溶解后混合再用于制备复合材料的方法。重要的是，本发明从高分子的分子量这个影响聚合物性能的根本因素出发，提供了高性能再生丝素丝胶蛋白复合材料的制备技术。温和脱胶一步溶解法丝蛋白溶液制备过程中，采用热水法脱胶，使得丝素蛋白大分子链(尤其是重链)在脱胶过程中不会被破坏，该复合材料中丝蛋白分子量集中在150kDa以上的高分子量区域，而报导的常规方法制备的丝素蛋白或丝素与丝胶混合后制备的材料分子量大小呈现连续分布、主要分布在85kDa以下直至15kDa甚至更小，因此本发明提供的丝素蛋白分子量大且分子量分散程度很小，提高了复合三维骨架的强度和模量。丝胶蛋白的存在增加了复合骨架分子链之间的强氢键结合，也提高了复合骨架的强度和模量。丝素纤维外层的丝胶蛋白随着包覆层的由外及内，丝胶的种类、结构和性能(尤其是力学性能)也不同。本发明通过控制脱胶程度，得到了不同组成、力学性能可控的高强高韧丝素丝胶蛋白复合三维支架，解决了骨再生修复的天然生物聚合物组织工程三维支架力学性能不足的卡脖子技术难题，且复合支架具有原位快速诱导羟基磷灰石沉积的骨生成活性。
同时，本申请还提供了一种仿生复合骨支架，由丝素丝胶蛋白复合溶液制 备得到，所述丝素丝胶蛋白复合溶液的制备方法，包括以下步骤：
A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液。
上述步骤A)和步骤B)为丝素丝胶蛋白复合溶液制备的过程，此处不进行赘述。本申请所述丝素丝胶蛋白仿生复合骨支架由丝素丝胶蛋白复合溶液制备得到。
为了进一步理解本发明，下面结合实施例对本发明提供的丝素丝胶蛋白复合支架的制备方法进行详细说明，本发明的保护范围不受以下实施例的限制。
实施例1
1.以经过煮茧缫制的家蚕生丝为原料，将家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水、40～60℃的热风进行干燥；
2.干燥后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮3小时，取出含有丝胶的蚕丝用25～30℃的去离子充分清洗干净，脱水后用40～60℃的热风进行干燥；
3.测得蚕丝的脱胶率14％；
4.称取干燥后的蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得到家蚕丝素丝胶蛋白复合溶解液；
5.将丝素丝胶蛋白复合溶解液灌注于截留分子量为50kDa的透析袋内，透析袋壁是半透膜，将灌注了丝素丝胶蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素丝胶蛋白复合水溶液；
6.采用旋转蒸发器浓缩得50mg/mL的复合溶液，搅拌均匀，去除溶液中的气泡，加入到圆柱形或方形的器皿中，将样品-10～-80℃冷冻12～36h，然后立即放进冷冻干燥机中干燥24～72h，取出后用80％的乙醇溶液浸泡样品20min左右，然后在恒温恒湿间风干得家蚕丝素丝胶蛋白复合支架。
7.使用质构仪测得丝素丝胶蛋白复合支架的抗压强度和抗压模量分别可 达3MPa和101MPa。
实施例2
1.以经过煮茧缫制的家蚕生丝为原料，将家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水、40～60℃的热风进行干燥；
2.干燥后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮6小时，取出含有丝胶的蚕丝用25～30℃的去离子充分清洗干净，脱水后用40～60℃的热风进行干燥；
3.测得蚕丝的脱胶率21.8％；
4.称取干燥后的蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得到家蚕丝素丝胶蛋白复合溶解液；
5.将丝素丝胶蛋白复合溶解液灌注于截留分子量为50kDa的透析袋内，透析袋壁是半透膜，将灌注了丝素丝胶蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素丝胶蛋白复合水溶液；
6.采用旋转蒸发器浓缩得50mg/mL的复合溶液，搅拌均匀，去除溶液中的气泡，加入到圆柱形或方形的器皿中，将样品-10～-80℃冷冻12～36h，然后立即放进冷冻干燥机中干燥24～72h，取出后用80％的乙醇溶液浸泡样品20min左右，然后在恒温恒湿间风干得家蚕丝素丝胶蛋白复合支架。
7.使用质构仪测得丝素丝胶蛋白复合支架的抗压缩强度和抗压模量分别可达4MPa和116MPa。
实施例3
1.以经过煮茧缫制的家蚕生丝为原料，将家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水、40～60℃的热风进行干燥；
2.干燥后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮6小时，取出含有丝胶的蚕丝用25～30℃的去离子充分清洗干净，脱水后用40～60℃的热风进行干燥；
3.测得蚕丝的脱胶率21.8％；
4.称取干燥后的蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得到家蚕丝素丝胶蛋白复合溶解液；
5.将丝素丝胶蛋白复合溶解液灌注于截留分子量为50kDa的透析袋内，透析袋壁是半透膜，将灌注了丝素丝胶蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素丝胶蛋白复合水溶液；
6.采用旋转蒸发器浓缩得80mg/mL的复合溶液，搅拌均匀，去除溶液中的气泡，加入到圆柱形或方形的器皿中，将样品-10～-80℃冷冻12～36h，然后立即放进冷冻干燥机中干燥24～72h，取出后用80％的乙醇溶液浸泡样品20min左右，然后在恒温恒湿间风干得家蚕丝素丝胶蛋白复合支架。
7.使用质构仪测得丝素丝胶蛋白复合支架的抗压强度和抗压模量分别可达5MPa和134MPa。
比较例1常规纯丝素溶液制备方法
1.以经过煮茧缫制的家蚕生丝为原料，将家蚕生丝按照浴比1:50(g/mL)放入浓度为0.1～0.06％的碳酸钠水溶液中，于98～100℃处理三次，每次处理30分钟，然后用25～30℃的去离子水将丝充分清洗干净，脱水后用40～60℃的热风进行干燥；
2.测得蚕丝的脱胶率23％；
3.称取干燥后的家蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得家蚕丝素蛋白溶解液；
4.将丝素蛋白溶解液灌注于截留分子量为50kDa的透析袋内，透析袋壁是半透膜，将灌注了丝素蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液；
5.采用旋转蒸发器浓缩得80mg/mL的丝素蛋白溶液，搅拌均匀，去除溶液中的气泡，加入到圆柱形或方形的器皿中，将样品-10～-80℃冷冻12～36h，然后立即放进冷冻干燥机中干燥24～72h，取出后用80％的乙醇溶液浸泡样品20min左右，然后在恒温恒湿间风干得家蚕丝素蛋白支架；
6.使用质构仪测得丝素蛋白支架的抗压缩性能：抗压强度可达1.65MPa， 抗压模量可达40.5MPa。
比较例2常规纯丝素溶液制备方法再与丝胶混合的复合支架
1.以经过煮茧缫制的家蚕生丝为原料，将家蚕生丝按照浴比1:50(g/mL)放入浓度为0.1～0.06％的碳酸钠水溶液中，于98～100℃处理三次，每次处理30分钟，然后用25～30℃的去离子水将丝充分清洗干净，脱水后用40～60℃的热风进行干燥；
2.测得蚕丝的脱胶率23％；
3.称取干燥后的家蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得家蚕丝素蛋白溶解液；
4.将丝素蛋白溶解液灌注于截留分子量为50kDa的透析袋内，透析袋壁是半透膜，将灌注了丝素蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液；
5.另取经过煮茧缫制的家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子水预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水；
6.脱水后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮7小时，弃去纤维，收集丝胶水溶液冷冻干燥得固体丝胶；
7.然后采用旋转蒸发器浓缩步骤4得到的丝素蛋白水溶液、添加与实施例3等同含量、步骤6制备的丝胶、溶解混合均匀，调制丝素丝胶复合溶液浓度80mg/mL，去除溶液中的气泡，加入到圆柱形或方形的器皿中，将样品-10～-80℃冷冻12～36h，然后立即放进冷冻干燥机中干燥24～72h，取出后用80％的乙醇溶液浸泡样品20min左右，然后在恒温恒湿间风干得家蚕丝素蛋白支架。
8.使用质构仪测得丝素蛋白支架的抗压缩性能如下：抗压强度可达1.4MPa，抗压初始模量可达33MPa。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的 保护范围内。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种仿生复合骨支架的制备方法，包括以下步骤：
    A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
    B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液；
    C)将所述丝素丝胶蛋白复合溶液浓缩后冷冻干燥，将得到的初始骨架置于小分子一元醇中处理后冻干，得到仿生复合骨支架。
  2. 根据权利要求1所述的制备方法，其特征在于，所述温水的温度为35～60℃，所述预处理的时间为1～10min。
  3. 根据权利要求1所述的制备方法，其特征在于，步骤A)中，所述置于65～95℃的水中处理的时间为1～8h。
  4. 根据权利要求1所述的制备方法，其特征在于，步骤A)中，所述蚕丝与温水的比例为1g：(40～60)ml，预处理后的蚕丝与水的比例为1g：(40～60)ml。
  5. 根据权利要求1所述的制备方法，其特征在于，步骤A)中，所述预处理之后还包括：将预处理后的蚕丝取出后于25～30℃的水中洗净、脱水；
    所述处理后干燥之前还包括：将处理后的蚕丝于25～30℃的水中清洗后脱水；所述干燥采用40～60℃的热风干燥。
  6. 根据权利要求1所述的制备方法，其特征在于，所述中性盐溶液为8～10M的溴化锂水溶液，所述初处理的蚕丝与所述溴化锂水溶液的比例为1g：(20～30)ml。
  7. 根据权利要求1所述的制备方法，其特征在于，步骤B)中，所述处理的温度为50～80℃。
  8. 根据权利要求1所述的制备方法，其特征在于，所述纯化的方法具体为：
    将得到的丝素丝胶蛋白复合溶解液灌注于截留分子量10～50kDa的透析袋内，置于去离子水中持续透析1～3天。
  9. 根据权利要求1所述的制备方法，其特征在于，步骤C)中，所述小分子一元醇为浓度为70～80％的乙醇溶液，所述处理的时间为10～60min；所述冷冻干燥的冷冻温度为-10～-80℃，时间为12～36h，干燥的时间为24～72h。
  10. 一种仿生复合骨支架，由丝素丝胶蛋白复合溶液制备得到，所述丝素丝胶蛋白复合溶液的制备方法，包括以下步骤：
    A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
    B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液。
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