技术领域technical field
本发明涉及医学生物复合材料领域，具体地指一种壳聚糖—丝胶蛋白复合生物支架和搭载神经生长因子的复合生物支架及其制备方法和应用。The invention relates to the field of medical biological composite materials, in particular to a chitosan-sericin composite biological scaffold, a composite biological scaffold loaded with nerve growth factor, and a preparation method and application thereof.
背景技术Background technique
外周神经卡压病变是临床外科的常见疾病，在人群的患病率高达3.8%，其主要包括腕管综合征、肘管综合征、桡管综合征、腓总神经卡压综合征等。神经卡压可以发生于身体的任意部位，最常见于关节附近的骨性纤维管，特别是神经行径中的肥厚的肌腱弓、变异韧带及占位性病变（腱鞘囊肿、脂肪瘤、血肿或肿瘤等）均可对外周神经进行挤压。传统的外周神经卡压治疗方法有手术治疗和保守治疗两种, 少部分病人症状轻微，可采取保守治疗，如局部注射类固醇药物、夹板固定、瑜伽等；大部分卡压病人具备手术治疗指征，通常采取卡压神经松解术进行手术治疗。近年来有研究表明，手术治疗的失败率达到25%，部分患者接受手术治疗后不能实现长期疗效。这种现象被认为与手术后局部瘢痕组织形成以及神经卡压后病理损伤有关。如何进一步改善手术治疗的效果是近年来手外科领域的重要课题和热点问题。Peripheral nerve entrapment is a common disease in clinical surgery, with a prevalence rate as high as 3.8% in the population. It mainly includes carpal tunnel syndrome, cubital tunnel syndrome, radial tunnel syndrome, and common peroneal nerve entrapment syndrome. Nerve entrapment can occur anywhere in the body, most commonly in bony fibrous tubes near joints, especially in hypertrophic tendon arches, aberrant ligaments, and space-occupying lesions (ganglion cysts, lipomas, hematomas, or tumors) in the course of nerves etc.) can squeeze the peripheral nerve. Traditional peripheral nerve entrapment treatment methods include surgical treatment and conservative treatment. A small number of patients have mild symptoms and can be treated conservatively, such as local injection of steroid drugs, splinting, yoga, etc.; most entrapment patients have surgical treatment indications , usually with entrapped nerve release for surgical treatment. Studies in recent years have shown that the failure rate of surgical treatment reaches 25%, and some patients cannot achieve long-term curative effect after surgical treatment. This phenomenon is considered to be related to the formation of local scar tissue after surgery and pathological damage after nerve entrapment. How to further improve the effect of surgical treatment is an important topic and hot issue in the field of hand surgery in recent years.
组织工程方法近年来被广泛应用于外周神经离断损伤的治疗，并取得了理想的效果，但尚未被应用于外周神经卡压治疗领域。壳聚糖（Chitosan）是一种天然生物材料，由甲壳素脱乙酰基所得，而甲壳素广泛存在蟹壳、虾壳和节肢动物的外壳中，也存在藻类等低等植物的细胞壁中，自然界每年生物合成的甲壳素有数十亿吨之多，所以壳聚糖是一种十分丰富的自然资源。因其有杀菌、抑菌和促进伤口愈合的功能，在临床上已经被开发应用为伤口敷料。此外，壳聚糖具有良好的生物相容性，免疫原性弱，在体内可降解代谢。目前壳聚糖已在食物保鲜、生物医学、环保、化妆品、农业等众多领域广泛应用。通过化学修饰作用，在壳聚糖分子结构中引入各种功能团或将壳聚糖与其他生物材料共混，改善壳聚糖的物化性质，从而使其各自具有不同的功能及功效，是壳聚糖以后研究的一个重要方面。Tissue engineering methods have been widely used in the treatment of peripheral nerve transection injuries in recent years, and have achieved ideal results, but have not been applied to the field of peripheral nerve entrapment. Chitosan (Chitosan) is a natural biological material obtained by deacetylation of chitin, and chitin is widely found in crab shells, shrimp shells and arthropod shells, and also in the cell walls of algae and other lower plants. There are billions of tons of chitin biosynthesized every year, so chitosan is a very rich natural resource. Because of its bactericidal, antibacterial and wound healing functions, it has been clinically developed and applied as a wound dressing. In addition, chitosan has good biocompatibility, weak immunogenicity, and can be degraded and metabolized in vivo. At present, chitosan has been widely used in food preservation, biomedicine, environmental protection, cosmetics, agriculture and many other fields. Through chemical modification, various functional groups are introduced into the molecular structure of chitosan or chitosan is blended with other biological materials to improve the physical and chemical properties of chitosan, so that each has different functions and effects. An important aspect of future research on glycans.
丝胶蛋白（Silk Sericin）是包裹在丝素纤维表层的一种天然大分子粘性蛋白，约占蚕茧含量的20~30%，由分子量为24~400 kDa的多肽组成，其分子由丝氨酸、天门冬氨酸和甘氨酸等18种氨基酸组成。长期以来由于人们对丝胶蛋白认识的不足和研究的局限性，导致丝胶在缫丝工业中被当作废物处理，浪费了大量宝贵的天然资源。近年来人们发现丝胶蛋白具有保湿、抗菌、抗氧化、抗凝血以及促进细胞粘附和增殖等生物特性。不仅如此，丝胶具有亲水性和可降解性，是理想的生物医用材料。已有的报道显示丝胶常用于与其他材料（如弹性蛋白，聚乙烯醇等）共聚或简单交联，混合制作生物支架以获得性能优良的生物材料。充分发挥丝胶蛋白在组织工程学中的应用，具有重要的社会意义和广阔的应用前景。Silk Sericin (Silk Sericin) is a natural macromolecular viscous protein wrapped on the surface of silk fibers, accounting for about 20-30% of the cocoon content, composed of polypeptides with a molecular weight of 24-400 kDa, and its molecules are composed of serine, tianmen Composed of 18 kinds of amino acids such as aspartic acid and glycine. For a long time, due to the lack of understanding of sericin and the limitations of research, sericin has been treated as waste in the silk reeling industry, wasting a lot of valuable natural resources. In recent years, it has been found that sericin has biological properties such as moisturizing, antibacterial, anti-oxidation, anti-coagulation, and promoting cell adhesion and proliferation. Not only that, sericin is hydrophilic and degradable, and it is an ideal biomedical material. Existing reports have shown that sericin is often used for copolymerization or simple crosslinking with other materials (such as elastin, polyvinyl alcohol, etc.), and mixed to make bioscaffolds to obtain biomaterials with excellent properties. It is of great social significance and broad application prospect to give full play to the application of sericin in tissue engineering.
本发明以家蚕丝素缺失型突变品种蚕茧为材料，采用溴化锂提取法提取纯丝胶蛋白，利用化学共价键交联的方式将壳聚糖与丝胶蛋白结合，成功制备了壳聚糖—丝胶蛋白复合支架。该复合支架兼具壳聚糖和丝胶蛋白各自的优点，并且弥补了单一材料应用存在的不足。在治疗外周神经卡压疾病的应用中，复合材料的壳聚糖成分可发挥其杀菌、抑菌、抗组织粘连的作用；而丝胶蛋白成分可以改善壳聚糖的机械性能，丝胶蛋白的降解产物可发挥神经营养与保护作用。此外，本发明针对外周神经受卡压后的主要病理改变（髓鞘厚度变薄及脱髓鞘改变、轴突数量减少、轴突直径变细等），利用复合生物支架搭载和缓释神经生长因子（NGF），NGF可以促进轴突的再生，调节髓鞘蛋白的表达，促进髓鞘的再生和髓鞘厚度的恢复。The present invention uses cocoons of silkworm silk fibroin-deficient mutant varieties as materials, extracts pure sericin protein by lithium bromide extraction method, combines chitosan and sericin protein by chemical covalent bond cross-linking, and successfully prepares chitosan— Sericin composite scaffold. The composite scaffold has both the advantages of chitosan and sericin, and makes up for the shortcomings of single material application. In the application of treating peripheral nerve entrapment diseases, the chitosan component of the composite material can exert its bactericidal, antibacterial, and anti-tissue adhesion effects; while the sericin component can improve the mechanical properties of chitosan, and the sericin component can Degradation products can exert neurotrophic and protective effects. In addition, the present invention aims at the main pathological changes (thinning of myelin sheath and change of demyelination, reduction of number of axons, thinning of axon diameter, etc.) after peripheral nerves are entrapped, and uses composite biological scaffolds to carry and release nerve growth NGF can promote the regeneration of axons, regulate the expression of myelin protein, and promote the regeneration of myelin and the recovery of myelin thickness.
本发明壳聚糖—丝胶蛋白复合生物支架结合了两种成分各自的优点，具有良好的生物相容性、优异的机械性能，在体内易于降解，可作为生长因子，药物以及细胞的载体。本发明实例将壳聚糖—丝胶蛋白复合生物支架用作神经生长因子NGF的缓释载体，分别利用复合生物支架的优点及NGF的作用，治疗外周神经卡压疾病，促进受损神经组织学及功能学的修复。The chitosan-sericin composite bioscaffold of the present invention combines the respective advantages of the two components, has good biocompatibility, excellent mechanical properties, is easy to degrade in vivo, and can be used as a carrier of growth factors, medicines and cells. In the example of the present invention, the chitosan-sericin composite biological scaffold is used as the slow-release carrier of nerve growth factor NGF, and the advantages of the composite biological scaffold and the effect of NGF are used respectively to treat peripheral nerve entrapment diseases and promote damaged nerve histology. and functional restoration.
发明内容Contents of the invention
本发明提供一种壳聚糖—丝胶蛋白复合生物支架，具有良好生物活性和生物相容性，可以在体内自然降解并促进外周神经卡压损伤的修复，同时所述复合支架兼具壳聚糖和丝胶蛋白各自的优点，弥补单一材料的不足，如复合支架具有壳聚糖的杀菌、抑菌、抗组织粘连的作用，丝胶蛋白的神经营养与保护作用，支架中的丝胶蛋白成分可改善壳聚糖的机械性能。The invention provides a chitosan-sericin composite biological scaffold, which has good biological activity and biocompatibility, can be naturally degraded in vivo and can promote the repair of peripheral nerve entrapment damage, and at the same time, the composite scaffold has chitosan The respective advantages of sugar and sericin make up for the deficiency of a single material. For example, the composite scaffold has the functions of sterilization, antibacterial and anti-tissue adhesion of chitosan, the neurotrophic and protective effects of sericin, and the sericin in the scaffold Components improve the mechanical properties of chitosan.
本发明还提供一种上述壳聚糖—丝胶蛋白复合生物支架的制备方法，工艺简单，成本低廉，同时有利于产品的质量控制以及大规模生产。The invention also provides a preparation method of the above-mentioned chitosan-sericin composite biological scaffold, which has simple process and low cost, and is beneficial to product quality control and large-scale production.
本发明还提供了上述用于外周神经卡压治疗的搭载NGF的壳聚糖—丝胶蛋白复合生物支架的制备方法。The present invention also provides a preparation method for the above-mentioned chitosan-sericin composite biological scaffold loaded with NGF for the treatment of peripheral nerve entrapment.
本发明首先提供一种壳聚糖—丝胶蛋白复合生物支架，其成分为壳聚糖与丝胶蛋白。壳聚糖又称脱乙酰甲壳素，是由自然界广泛存在的几丁质经过脱乙酰作用得到的。壳聚糖具有灭菌、促进伤口愈合、吸收伤口渗出物、抗组织粘连、不易脱水收缩等作用，已用于制备外科敷料。壳聚糖能被生物体内的溶菌酶降解为低聚糖，具有无毒、能被生物体完全吸收代谢的特点。丝胶蛋白是包裹在丝素纤维表层的一种天然大分子蛋白，具有良好的生物相容性和生物降解性，可在体内降解，且降解产物为氨基酸（主要为丝氨酸、天冬氨酸和甘氨酸），降解产物不仅对机体无毒，能被机体代谢，且具有神经营养作用。本发明人研究发现复合支架兼具有壳聚糖与丝胶蛋白各自的优点，是用于外周神经卡压治疗的理想生物材料。The invention firstly provides a chitosan-sericin composite bio-scaffold whose components are chitosan and sericin. Chitosan, also known as deacetylated chitin, is obtained through deacetylation of chitin that exists widely in nature. Chitosan has the functions of sterilizing, promoting wound healing, absorbing wound exudate, resisting tissue adhesion, not easy to syneresis, etc., and has been used to prepare surgical dressings. Chitosan can be degraded into oligosaccharides by lysozyme in organisms, and it is non-toxic and can be completely absorbed and metabolized by organisms. Sericin is a natural macromolecular protein wrapped on the surface of silk fibres. It has good biocompatibility and biodegradability, and can be degraded in vivo, and the degradation products are amino acids (mainly serine, aspartic acid and Glycine), the degradation products are not only non-toxic to the body, can be metabolized by the body, but also have neurotrophic effects. The inventors found that the composite scaffold has both the advantages of chitosan and sericin, and is an ideal biological material for peripheral nerve entrapment treatment.
进一步地，本发明提供的壳聚糖—丝胶蛋白复合生物支架是采用丝素缺失型突变品种家蚕的蚕茧作为原料提取丝胶蛋白，并与壳聚糖经共价交联剂交联成型后获得复合水凝胶，进一步冷冻干燥制备为复合支架。其中，壳聚糖购自SIGMA公司，纯度大于75%，家蚕丝素缺失型突变品种蚕茧购自中国农业科学院蚕业研究所，该类品种资源保存于中国农业科学院蚕业研究所国家蚕资源保存中心。Further, the chitosan-sericin composite bioscaffold provided by the present invention uses silkworm cocoons of a silk fibroin-deficient mutant variety silkworm as a raw material to extract sericin, and cross-links chitosan with a covalent cross-linking agent to form The composite hydrogel was obtained, which was further freeze-dried to prepare a composite scaffold. Among them, chitosan was purchased from SIGMA company, with a purity greater than 75%, and silkworm cocoons of silk fibroin-deficient mutant varieties were purchased from the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences, and the resources of such varieties are preserved in the National Silkworm Resource Conservation of the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences center.
由于复合支架优异的生物相容性和生物活性，壳聚糖成分可发挥其杀菌、抑菌、抗组织粘连的作用；而丝胶蛋白成分可以改善壳聚糖的机械性能，丝胶蛋白的降解产物可发挥神经营养与保护作用。本发明的一个实例将壳聚糖—丝胶蛋白复合生物支架制备为缓释载体，搭载神经生长因子（NGF），支架的尺寸为直径1cm，厚度2mm，薄圆片状，用于大鼠坐骨神经卡压的治疗。Due to the excellent biocompatibility and bioactivity of the composite scaffold, the chitosan component can exert its bactericidal, antibacterial, and anti-tissue adhesion effects; while the sericin component can improve the mechanical properties of chitosan, and the degradation of sericin The product can exert neurotrophic and protective effects. In an example of the present invention, the chitosan-sericin composite bioscaffold was prepared as a slow-release carrier, loaded with nerve growth factor (NGF), and the size of the scaffold was 1cm in diameter, 2mm in thickness, thin disc shape, and used for rat sciatic nerve Compression treatment.
本发明还提供了一种上述壳聚糖—丝胶蛋白复合生物支架的制备方法，包括以下步骤：The present invention also provides a preparation method of the above-mentioned chitosan-sericin composite biological scaffold, comprising the following steps:
1）采用丝素缺失型突变品种家蚕的蚕茧作为原料，将其粉碎并经紫外线或乙醇灭菌，然后使用LiBr或LiCl水溶液进行丝胶蛋白的提取，将提取物制成浓度为质量体积比0.005~0.2g/mL的丝胶蛋白水溶液；1) Use silkworm cocoons of silk fibroin-deficient mutant silkworm as raw materials, crush them and sterilize them with ultraviolet light or ethanol, then use LiBr or LiCl aqueous solution to extract sericin, and make the extract with a concentration of mass volume ratio of 0.005 ~0.2g/mL sericin aqueous solution;
2）采用1％（v/v）的乙酸溶液作为溶剂溶解壳聚糖，调pH至5.90~5.95，0.22 μm滤器过滤除菌，得到0.01 g/mL的壳聚糖乙酸溶液；2) Use 1% (v/v) acetic acid solution as a solvent to dissolve chitosan, adjust the pH to 5.90-5.95, filter and sterilize with a 0.22 μm filter, and obtain a 0.01 g/mL chitosan acetic acid solution;
3）将上述丝胶蛋白水溶液与壳聚糖乙酸溶液体积比按照80:20～20:80比例混匀，加入共价交联剂，再混匀后注入模具中成型，得到壳聚糖—丝胶蛋白复合水凝胶，所述共价交联剂的使用量为每1 ml壳聚糖—丝胶蛋白混合溶液加入333μL浓度为质量体积比0.01 g/ml的共价交联剂；3) Mix the above-mentioned sericin aqueous solution and chitosan acetic acid solution in a volume ratio of 80:20 to 20:80, add a covalent cross-linking agent, and then inject it into a mold for molding to obtain chitosan-silk For the colloid composite hydrogel, the amount of the covalent cross-linking agent is to add 333 μL of a covalent cross-linking agent with a concentration of 0.01 g/ml per 1 ml of the chitosan-sericin mixed solution;
4）将所述壳聚糖—丝胶蛋白水凝胶冷冻干燥，得到所述壳聚糖—丝胶蛋白复合支架。4) freeze-drying the chitosan-sericin hydrogel to obtain the chitosan-sericin composite scaffold.
其中，粉碎为可以采用剪刀将蚕茧剪碎或其他能使其破碎的方法。灭菌方法可采用紫外照射法，把蚕茧粉碎成1 mm2左右，用紫外线分别照射正反面至少5 min；或者采用乙醇浸泡法，将蚕茧粉碎成1 mm2左右，用70%~80%体积浓度的乙醇至少浸泡5 min，在3500 r/min条件下离心5分钟，去掉乙醇；还可以将上述紫外照射法和乙醇浸泡法联合应用，先用紫外线照射正反面，再用乙醇浸泡，然后离心去掉乙醇。上述三种方法均可达到满意的灭菌效果。Wherein, crushing refers to cutting silkworm cocoons with scissors or other methods capable of breaking them. Sterilization method can adopt ultraviolet irradiation method, crush silkworm cocoons into about 1 mm2 , and irradiate the front and back sides with ultraviolet rays for at least 5 minutes respectively; or use ethanol immersion method, crush silkworm cocoons into about1 mm Soak in ethanol with a certain concentration for at least 5 minutes, centrifuge at 3500 r/min for 5 minutes, and remove the ethanol; you can also combine the above-mentioned ultraviolet irradiation method and ethanol soaking method, first irradiate the front and back with ultraviolet rays, then soak in ethanol, and then centrifuge Remove ethanol. The above three methods can achieve satisfactory sterilization effect.
进一步地，制取所述丝胶蛋白水溶液的方法包括：Further, the method for preparing the sericin aqueous solution includes:
1）采用丝素缺失型突变品种家蚕的蚕茧作为原料，将其粉碎并灭菌，在25~50℃，采用浓度为6~8 mol/L的LiBr或LiCl水溶液，在25~50℃进行提取处理5~24小时，每克蚕茧碎片采用20~100 mL 6~8 mol/L的LiBr或LiCl水溶液进行提取；1) Use silkworm cocoons of silk fibroin-deficient mutant silkworm as raw materials, crush and sterilize them, and extract them at 25-50°C with LiBr or LiCl aqueous solution with a concentration of 6-8 mol/L at 25-50°C After treatment for 5-24 hours, each gram of cocoon fragments is extracted with 20-100 mL of 6-8 mol/L LiBr or LiCl aqueous solution;
2）将步骤1）得到的提取物离心，去除不溶性物质，得到澄清溶液；2) Centrifuge the extract obtained in step 1) to remove insoluble substances to obtain a clear solution;
3）向步骤2）得到的澄清溶液中加入0.5~3mol/L、pH 8.0~11.0的Tris-HCl缓冲液，并在超纯水中进行透析；3) Add 0.5~3mol/L Tris-HCl buffer solution with pH 8.0~11.0 to the clarified solution obtained in step 2), and dialyze in ultrapure water;
4）将步骤3）得到的透析液离心去除沉淀，浓缩得到浓度为0.005~0.2g/mL的丝胶蛋白水溶液。在本发明的一个实施例中，可使所述丝胶蛋白水溶液的浓度为0.04g/mL，以取得更好的交联效果。4) Centrifuge the dialysate obtained in step 3) to remove the precipitate, and concentrate to obtain a sericin aqueous solution with a concentration of 0.005-0.2 g/mL. In one embodiment of the present invention, the concentration of the sericin aqueous solution may be 0.04 g/mL to achieve better cross-linking effect.
上述丝胶蛋白水溶液的制备过程在无菌环境中操作，所用原料和设备均经灭菌处理，例如，所用的超纯水、透析袋用前均采用高压灭菌，所用的LiBr水溶液、三羟甲基氨基甲烷盐酸盐（Tris-HCl）均经过0.22 μm滤器过滤除菌，乙醇为经过亲水性0.22 μm滤器过滤后，用灭菌的超纯水配置而成。The preparation process of the above-mentioned sericin aqueous solution is operated in a sterile environment, and the raw materials and equipment used are all sterilized. For example, the ultrapure water used and the dialysis bag are autoclaved before use, and the used LiBr aqueous solution, trihydroxy Methylaminomethane hydrochloride (Tris-HCl) was sterilized by filtration through a 0.22 μm filter, and ethanol was prepared by sterilizing ultrapure water after being filtered through a hydrophilic 0.22 μm filter.
进一步地，制备所述壳聚糖乙酸溶液的方法包括：Further, the method for preparing described chitosan acetic acid solution comprises:
1）双蒸水配制体积比1％冰醋酸溶液；1) Prepare a 1% glacial acetic acid solution with a volume ratio of double distilled water;
2）称取壳聚糖溶于步骤1）所得1％乙酸溶液，放置于37oC摇床中充分溶解30分钟；用1mol/L NaOH溶液调pH值至5.90~5.95；经过0.22 μm滤器过滤除菌得到0.01 g/mL壳聚糖乙酸溶液。2) Weigh chitosan and dissolve it in the 1% acetic acid solution obtained in step 1), place it in a shaker at 37o C for 30 minutes to fully dissolve; adjust the pH value to 5.90~5.95 with 1mol/L NaOH solution; filter through a 0.22 μm filter Sterilize to obtain 0.01 g/mL chitosan acetic acid solution.
上述共价交联剂可选择戊二醛、丙二醛和京尼平等中的一种或多种。例如，本发明的一个实施例中可使用浓度为1wt%的京尼平。The above-mentioned covalent cross-linking agent can be selected from one or more of glutaraldehyde, malondialdehyde and genipal. For example, genipin at a concentration of 1 wt% can be used in one embodiment of the present invention.
进一步地，将上述丝胶蛋白水溶液与壳聚糖乙酸溶液混匀，在所述壳聚糖—丝胶蛋白混合溶液中加入所述共价交联剂，混匀后注入模具，在37oC下维持至少8小时以上使之成型，得到壳聚糖—丝胶蛋白水凝胶。在一个具体实施方式中，可将其置于室温下过夜成型。在此过程中，壳聚糖与丝胶蛋白在交联剂的作用下发生交联反应，得到壳聚糖—丝胶蛋白水凝胶。Further, mix the above-mentioned sericin aqueous solution and chitosan acetic acid solution, add the covalent cross-linking agent into the chitosan-sericin mixed solution, inject the mold into the mold after mixing, and heat maintain at least 8 hours or more to form it, and obtain chitosan-sericin hydrogel. In one embodiment, it can be left to form overnight at room temperature. During this process, chitosan and sericin undergo a cross-linking reaction under the action of a cross-linking agent to obtain chitosan-sericin hydrogel.
为了利于增加复合支架的孔隙度，可将壳聚糖—丝胶蛋白水凝胶冷冻干燥之前，在零度以下冷冻成型，例如，置于-20oC至-190oC下（一般可以置于液氮中）冷冻至少4小时，可更有利于负载生长因子和药物等。In order to increase the porosity of the composite scaffold, the chitosan-sericin hydrogel can be freeze-dried before freeze-drying, for example, placed at -20o C to -190o C (generally can be placed liquid nitrogen) for at least 4 hours, which is more conducive to loading growth factors and drugs.
上述壳聚糖—丝胶蛋白复合支架（尺寸可通过模具的选择而控制），例如可以制成直径1cm，厚度2~3mm，薄圆片状的缓释载体，以适应不同程度的外周神经损伤。获得的复合支架应在无菌条件下保存。The above-mentioned chitosan-sericin composite scaffold (the size can be controlled by the choice of the mold), for example, can be made into a slow-release carrier in the shape of a thin disc with a diameter of 1cm and a thickness of 2-3mm, so as to adapt to different degrees of peripheral nerve injury . The obtained composite scaffold should be stored under sterile conditions.
在一个具体实施方式中，壳聚糖—丝胶蛋白复合生物支架的制备方法及应用具体可包括以下步骤：In a specific embodiment, the preparation method and application of chitosan-sericin composite bio-scaffold may specifically include the following steps:
1）称取家蚕丝素缺失型突变品种蚕茧，并将其剪成1 mm2碎片，清洗3次，去除水分，先将蚕茧铺在一个无菌平板，用紫外线照射五分钟后，用另一个无菌平板覆盖在其上面，然后反过来，拿掉起始的无菌平板暴露出蚕茧碎片的另一面用紫外线再照射五分钟，或者可以用镊子将蚕茧碎片逐个翻面，实现用紫外线分别照射正反面各5分钟；1) Weigh silkworm silk fibroin-deficient mutant cocoons, cut them into 1 mm2 pieces, wash 3 times, remove water, first spread the cocoons on a sterile plate, irradiate with ultraviolet light for five minutes, and then use another Cover it with a sterile plate, and then turn it over, remove the initial sterile plate to expose the other side of the cocoon fragments and irradiate with ultraviolet light for five minutes, or you can use tweezers to turn the cocoon fragments one by one to achieve UV irradiation separately 5 minutes for each side;
2）将步骤1）的蚕茧碎片浸泡于无菌的浓度为约6 mol/L的LiBr或LiCl水溶液中（每克蚕茧碎片加入55 mL的LiBr或LiCl水溶液）充分浸泡后（必要时进行搅拌），置于35℃水浴24小时左右，使原料中的丝胶蛋白被提取并溶解于LiBr或LiCl水溶液；2) Soak the silkworm cocoon fragments in step 1) in a sterile LiBr or LiCl aqueous solution with a concentration of about 6 mol/L (add 55 mL of LiBr or LiCl aqueous solution per gram of silkworm cocoon fragments) and fully soak (stir if necessary) , placed in a 35°C water bath for about 24 hours, so that the sericin in the raw material is extracted and dissolved in LiBr or LiCl aqueous solution;
3）然后将步骤2）中的提取物在3500 r/min离心，分离去除不溶性物质，得到澄清的溶液；3) Then centrifuge the extract in step 2) at 3500 r/min to separate and remove insoluble substances to obtain a clear solution;
4）向步骤3）得到的澄清溶液中加入四分之一体积无菌的1 mol/L，pH 9.0的Tris-HCl缓冲液，并将其进行透析，得到丝胶蛋白水溶液；4) Add a quarter volume of sterile 1 mol/L, pH 9.0 Tris-HCl buffer solution to the clarified solution obtained in step 3), and dialyze it to obtain a sericin aqueous solution;
5）将步骤4）得到丝胶蛋白水溶液离心去除沉淀，浓缩得到浓度为0.005~0.2g/mL的丝胶蛋白水溶液，优选浓度为4%，置于4℃保存备用；5) Centrifuge the sericin aqueous solution obtained in step 4) to remove the precipitate, concentrate to obtain a sericin aqueous solution with a concentration of 0.005-0.2g/mL, preferably 4%, and store it at 4°C for later use;
6）称取壳聚糖溶于体积比1%乙酸溶液，放置于37℃摇床中充分溶解30分钟；用1 mol/LNaOH溶液调pH值至5.90~5.95；经过0.22 μm滤器过滤除菌得到0.01 g/ml壳聚糖乙酸溶液，置于4℃保存备用；6) Weigh chitosan and dissolve it in 1% acetic acid solution by volume, and place it in a shaker at 37°C for 30 minutes to fully dissolve; adjust the pH value to 5.90~5.95 with 1 mol/L NaOH solution; filter and sterilize through a 0.22 μm filter to obtain 0.01 g/ml chitosan acetic acid solution, stored at 4°C for later use;
7）将步骤5）得到的无菌丝胶蛋白水溶液与步骤6）得到的无菌壳聚糖溶液充分混匀，得到壳聚糖—丝胶蛋白混合溶液；7) fully mixing the sterile sericin aqueous solution obtained in step 5) and the sterile chitosan solution obtained in step 6) to obtain a chitosan-sericin mixed solution;
8）向步骤7）得到的壳聚糖—丝胶蛋白混合溶液中加入无菌京尼平，加入量为每1 ml混合溶液中加入0.33 mL浓度为 0.01 mg/mL的京尼平，充分混匀后注入无菌模具中，置于室温12小时，得到壳聚糖—丝胶蛋白水凝胶；8) Add sterile genipin to the chitosan-sericin mixed solution obtained in step 7). Inject into aseptic mold after evenly, place at room temperature for 12 hours, obtain chitosan-sericin hydrogel;
9）将制成的壳聚糖—丝胶蛋白水凝胶放入无菌平皿中，再置于-80℃冷冻4小时。9) Put the prepared chitosan-sericin hydrogel into a sterile plate, and freeze at -80°C for 4 hours.
10）将9）中冷冻后半成品放入冷冻真空干燥机中，在零度以下过夜冻干，冻干后得到的支架从模具中取出。10) Put the frozen semi-finished product in 9) into a freeze-vacuum dryer, freeze-dry overnight at below zero, and take out the stent obtained after freeze-drying from the mold.
11）使步骤7）得到的壳聚糖—丝胶蛋白混合溶液中壳聚糖与丝胶蛋白的体积比为1：1，加入20 uL浓度为10 ng/uL的NGF溶液，充分混匀，后续步骤不变，得到搭载NGF的壳聚糖—丝胶蛋白复合生物支架。11) Make the volume ratio of chitosan and sericin in the chitosan-sericin mixed solution obtained in step 7) to be 1:1, add 20 uL of NGF solution with a concentration of 10 ng/uL, mix well, Subsequent steps remain unchanged, and a chitosan-sericin composite bioscaffold loaded with NGF is obtained.
本发明还提供上述壳聚糖—丝胶蛋白复合生物支架在外周神经卡压治疗中的应用。本发明提供的壳聚糖—丝胶蛋白复合生物支架可用于制备体内可降解移植物，可用于搭载神经生长因子NGF，用于外周神经卡压手术解压后的神经修复。The present invention also provides the application of the above-mentioned chitosan-sericin composite biological scaffold in the treatment of peripheral nerve entrapment. The chitosan-sericin composite bioscaffold provided by the invention can be used to prepare biodegradable grafts in vivo, can be used to carry nerve growth factor NGF, and can be used for nerve repair after peripheral nerve entrapment surgery and decompression.
本发明的有益效果在于：The beneficial effects of the present invention are:
1、本发明采用丝素缺失型突变品种家蚕的蚕茧作为原料提取丝胶蛋白，并与壳聚糖经交联成型，首次利用京尼平做为交联剂制备出壳聚糖—丝胶蛋白复合水凝胶，并进一步获得壳聚糖—丝胶蛋白复合生物支架，更进一步地，首次将该类复合支架应用于外周神经卡压疾病修复领域。1. The present invention uses silkworm cocoons of silkworm lacking mutant species as raw material to extract sericin, and forms it with chitosan through cross-linking, and for the first time uses genipin as a cross-linking agent to prepare chitosan-sericin Composite hydrogel, and further obtain chitosan-sericin composite bioscaffold, and further, for the first time, this kind of composite scaffold is applied to the field of peripheral nerve entrapment disease repair.
2、本发明提供的壳聚糖—丝胶蛋白复合生物支架具有良好的生物相容性，在损伤神经修复完成后可以完全降解。壳聚糖降解产物为低聚糖，具有抗菌、防粘连作用，可预防手术解压操作后组织粘连与瘢痕形成；丝胶蛋白降解产物为氨基酸，能够支持神经细胞存活和增殖，具有神经营养和保护作用。壳聚糖—丝胶蛋白复合生物支架是一种优良的生物材料应用于外周神经卡压的治疗。2. The chitosan-sericin composite bioscaffold provided by the present invention has good biocompatibility and can be completely degraded after repairing damaged nerves. The degradation products of chitosan are oligosaccharides, which have antibacterial and anti-adhesion effects, and can prevent tissue adhesion and scar formation after surgical decompression; the degradation products of sericin are amino acids, which can support the survival and proliferation of nerve cells, and have neurotrophic and protective properties. effect. Chitosan-sericin composite bioscaffold is an excellent biomaterial used in the treatment of peripheral nerve entrapment.
3、本发明提供的上述壳聚糖—丝胶蛋白复合生物支架还具有良好的机械性能，如强度和柔韧性，在体内不会对组织造成过度挤压。3. The above-mentioned chitosan-sericin composite bioscaffold provided by the present invention also has good mechanical properties, such as strength and flexibility, and will not cause excessive extrusion to tissues in vivo.
4、按照本发明方法制成的上述壳聚糖—丝胶蛋白复合生物支架还具有孔隙多、比表面积大的特点，具有良好的药物或细胞因子缓释能力，支架可以搭载细胞因子或者药物，使其缓慢释放，以促进卡压神经损伤的修复。本发明提供的实例为复合生物支架搭载神经生长因子NGF用于外周神经卡压的治疗。4. The above-mentioned chitosan-sericin composite bioscaffold made according to the method of the present invention also has the characteristics of many pores and a large specific surface area, and has good drug or cytokine slow-release ability, and the scaffold can carry cytokines or drugs. It is released slowly to promote the repair of entrapped nerve damage. The example provided by the present invention is that the composite biological scaffold carrying nerve growth factor NGF is used for the treatment of peripheral nerve entrapment.
5、与传统单纯手术解除坐骨神经压迫相比，本发明方法首次将神经生长因子NGF用于外周神经卡压的治疗，NGF可以促进轴突的再生，调节髓鞘蛋白的表达，促进髓鞘的再生和髓鞘厚度的恢复，针对神经卡压损伤的病理变化进行治疗。5. Compared with the traditional simple operation to relieve sciatic nerve compression, the method of the present invention uses nerve growth factor NGF for the treatment of peripheral nerve entrapment for the first time. NGF can promote the regeneration of axons, regulate the expression of myelin protein, and promote the regeneration of myelin sheath and the recovery of myelin sheath thickness, and treat the pathological changes of nerve entrapment injury.
6、本发明提供的上述壳聚糖—丝胶蛋白复合生物支架制备工艺简单，成本低廉，同时有利于产品的质量控制以及大规模生产。6. The above-mentioned chitosan-sericin composite bioscaffold provided by the present invention has a simple preparation process, low cost, and is beneficial to product quality control and large-scale production.
附图说明Description of drawings
其中，CS代表壳聚糖，SS代表丝胶蛋白。Among them, CS stands for chitosan, and SS stands for sericin.
图1为不同比例（体积比）无菌壳聚糖、丝胶蛋白混合溶液形成水凝胶效果图；标尺为1厘米。Figure 1 is a diagram showing the effect of hydrogel formation from sterile chitosan and sericin mixed solutions in different proportions (volume ratio); the scale bar is 1 cm.
图2为本发明实施例制备的不同比例壳聚糖—丝胶蛋白复合水凝胶冷冻并低温真空干燥得到的冻干支架外观图；标尺为1厘米。Fig. 2 is an appearance view of a freeze-dried scaffold obtained by freezing and vacuum-drying low-temperature vacuum-dried chitosan-sericin composite hydrogels with different proportions prepared in the embodiment of the present invention; the scale bar is 1 cm.
图3为本发明实施例制备的不同比例壳聚糖—丝胶蛋白复合生物支架的横截面扫描电镜图片；标尺均为500微米。Fig. 3 is a cross-sectional scanning electron microscope picture of chitosan-sericin composite bioscaffolds with different proportions prepared in the embodiment of the present invention; the scales are all 500 microns.
图4为本发明实施例制备的不同比例壳聚糖—丝胶蛋白复合生物支架吸水膨胀率随时间的变化曲线。Fig. 4 is the variation curve of water swelling rate with time of different proportions of chitosan-sericin composite bioscaffold prepared in the embodiment of the present invention.
图5为本发明实施例制备的不同比例壳聚糖—丝胶蛋白复合生物支架的傅氏转换红外线光谱分析。Fig. 5 is the Fourier transform infrared spectrum analysis of chitosan-sericin composite bioscaffolds with different proportions prepared in the embodiment of the present invention.
图6为本发明实施例制备的不同比例壳聚糖—丝胶蛋白复合生物支架中施万细胞的增殖情况对比。Figure 6 is a comparison of the proliferation of Schwann cells in different proportions of chitosan-sericin composite biological scaffolds prepared in the embodiment of the present invention.
图7A为搭载神经营养因子（NGF）后不同比例壳聚糖—丝胶蛋白复合生物支架NGF每日释放量情况对比。图7B为搭载神经营养因子（NGF）后不同比例壳聚糖—丝胶蛋白复合生物支架NGF累积释放量情况对比。Figure 7A is a comparison of the daily release of NGF from different proportions of chitosan-sericin composite bioscaffolds loaded with neurotrophic factor (NGF). Figure 7B is a comparison of the cumulative release of NGF in different proportions of chitosan-sericin composite bioscaffolds loaded with neurotrophic factor (NGF).
图8A为不同比例壳聚糖—丝胶蛋白复合生物支架缓释NGF促未分化PC12细胞分化显微镜相差图；其中a为阴性对照组（未加入NGF），b为CS100/SS0组，c为CS80/SS20组，d为CS50/SS50组，e为CS20/SS80组，f为CS0/SS100组，标尺均为50微米。图8B为不同比例壳聚糖—丝胶蛋白复合生物支架缓释NGF促未分化PC12细胞分化情况柱状统计图。Figure 8A is the microscopic phase contrast image of different proportions of chitosan-sericin composite biological scaffolds slow-release NGF to promote the differentiation of undifferentiated PC12 cells; where a is the negative control group (without adding NGF), b is the CS100/SS0 group, and c is CS80 /SS20 group, d is CS50/SS50 group, e is CS20/SS80 group, f is CS0/SS100 group, and the scale bar is 50 microns. Fig. 8B is a histogram of different proportions of chitosan-sericin composite bio-scaffold slow-release NGF promoting differentiation of undifferentiated PC12 cells.
图9为大鼠坐骨神经卡压模型解压术后置入搭载NGF复合生物支架，复合生物支架在不同时间点体内降解情况。Figure 9 shows the decompression of the rat sciatic nerve entrapment model after implantation of a composite biological scaffold equipped with NGF, and the degradation of the composite biological scaffold at different time points.
图10A、图10B分别为大鼠坐骨神经卡压模型手术解压后不同处理组第2周、第4周卡压部位坐骨神经MBP、β-3 tubulin、DAPI染色图，标尺为50微米。Figure 10A and Figure 10B are the MBP, β-3 tubulin, and DAPI staining images of the sciatic nerve at the entrapment site in different treatment groups after decompression of the rat sciatic nerve compression model at the 2nd week and the 4th week respectively, and the scale bar is 50 microns.
图11为大鼠坐骨神经卡压模型手术解压后不同处理组第2周、第4周腓肠肌肌纤维Masson染色图，标尺为20微米。Figure 11 is the Masson staining diagram of gastrocnemius muscle fibers in different treatment groups at the 2nd week and the 4th week after decompressing the rat sciatic nerve entrapment model, and the scale bar is 20 microns.
图12为大鼠坐骨神经卡压模型手术解压后不同处理组第2周、第4周腓肠肌细胞直径统计。Figure 12 shows the statistics of gastrocnemius muscle cell diameters in different treatment groups at the 2nd and 4th weeks after the sciatic nerve entrapment model was decompressed by surgery.
具体实施方式detailed description
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, and Not all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
本发明首先制备了无菌壳聚糖溶液，同时以家蚕丝素缺失型突变品种蚕茧为原料，应用无菌抽提的方法，采用溴化锂（LiBr）或氯化锂（LiCl）提取法成功提取纯丝胶蛋白，并利用化学交联和冷冻抽干的方式，成功地制备了壳聚糖—丝胶蛋白复合生物支架，该复合支架具有防止组织粘连、抗菌作用，同时其降解产物对机体无毒无害且具有神经营养作用。In the present invention, a sterile chitosan solution is firstly prepared, and at the same time, the cocoons of silkworm silk fibroin-deficient mutant varieties are used as raw materials, and the aseptic extraction method is used to successfully extract pure Sericin, and by means of chemical cross-linking and freeze-drying, a chitosan-sericin composite biological scaffold was successfully prepared. The composite scaffold can prevent tissue adhesion and antibacterial effects, and its degradation products are non-toxic to the body Harmless and neurotrophic.
本发明提供的壳聚糖—丝胶蛋白复合生物支架可用于制备体内的可降解的移植物，本发明利用壳聚糖—丝胶蛋白复合生物支架搭载神经生长因子NGF，能有效促进外周神经卡压疾病损伤的修复。The chitosan-sericin composite bioscaffold provided by the present invention can be used to prepare degradable grafts in the body. The present invention uses the chitosan-sericin composite bioscaffold to carry nerve growth factor NGF, which can effectively promote peripheral nerve carding. Repair of pressure disease damage.
实施例1 壳聚糖—丝胶蛋白复合生物支架的制备Example 1 Preparation of chitosan-sericin composite bioscaffold
1、蚕茧的选择:1. Selection of silkworm cocoons:
选用家蚕丝素缺失突变品种的蚕茧（购于中国农业科学院蚕业研究所，该家蚕丝素缺失突变品种保存于中国农业科学院蚕业研究所国家蚕资源保存中心，保藏编号（或商品编号）185Nd-s, 140Nd-s,139Nd-s为原材料，主要的化学成分为：丝胶蛋白。Silkworm cocoons of silkworm silk fibroin-deficient mutant varieties were selected (purchased from the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences, and the silkworm silk fibroin-deficient mutant varieties were preserved in the National Silkworm Resource Preservation Center of the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences, and the preservation number (or product number)185Nd -s, 140Nd-s, 139Nd-s are raw materials, the main chemical composition is: sericin.
2、壳聚糖的选择：2. Choice of chitosan:
壳聚糖购自Sigma公司，纯度大于75%。Chitosan was purchased from Sigma with a purity greater than 75%.
3、丝胶的提取与分离3. Extraction and separation of sericin
1）分别称取家蚕丝素缺失突变品种的蚕茧1g并剪成1 mm2左右的碎片，置于洁净的烧杯中。将上述蚕茧碎片用超纯水清洗3次，3500 r/min离心5分钟去除水分。1) Weigh 1 g of silkworm cocoons of silkworm silk fibroin-deficient mutant varieties, cut them into pieces of about 1 mm2 , and place them in a clean beaker. The cocoon fragments were washed three times with ultrapure water, and centrifuged at 3500 r/min for 5 minutes to remove water.
在无菌环境中，将蚕茧碎片使用75%酒精分别浸泡5分钟，然后在3500r/min下离心5分钟去掉酒精后备用。In a sterile environment, the cocoon fragments were soaked in 75% alcohol for 5 minutes respectively, and then centrifuged at 3500r/min for 5 minutes to remove the alcohol and set aside.
2）向灭菌后的蚕茧碎片中加入55 mL的浓度为6 mol/L的LiBr水溶液，将烧杯放入恒温水浴锅内35℃水浴24 小时，溶解丝胶蛋白；2) Add 55 mL of LiBr aqueous solution with a concentration of 6 mol/L to the sterilized cocoon fragments, put the beaker in a constant temperature water bath at 35°C for 24 hours to dissolve sericin;
3）将步骤2）得到的提取物转入离心管内3500 r/min离心5分钟，去除不溶性物质，得到澄清的溶液；3) Transfer the extract obtained in step 2) into a centrifuge tube and centrifuge at 3500 r/min for 5 minutes to remove insoluble substances and obtain a clear solution;
4）向步骤3）得到的澄清溶液中加入四分之一体积的 Tris-HCl 缓冲液（1 mol/L，pH9.0）；4) Add a quarter volume of Tris-HCl buffer (1 mol/L, pH9.0) to the clear solution obtained in step 3);
5）将步骤4）中获得的溶液转入到预处理好的透析袋（MWCO 3500）中，然后放置于含有超纯水的烧杯中；将该烧杯置于搅拌器上慢速搅拌透析，每隔2小时换一次水，共透析24小时；5) Transfer the solution obtained in step 4) into a pretreated dialysis bag (MWCO 3500), and then place it in a beaker containing ultrapure water; place the beaker on a stirrer for slow stirring and dialysis, every Change the water every 2 hours for a total of 24 hours of dialysis;
6）将步骤5）中获得的透析液转到离心管中，3500 r/min离心5分钟，去除沉淀；6) Transfer the dialysate obtained in step 5) to a centrifuge tube, and centrifuge at 3500 r/min for 5 minutes to remove the precipitate;
7）将步骤6）获得的溶液再装入到透析袋中并将透析袋两端用夹子夹紧，再将透析袋置于质量百分浓度为20%的PEG6000溶液（使用前经高压灭菌）中浓缩，将丝胶蛋白水溶液浓缩到0.005~0.2 g/mL。7) Put the solution obtained in step 6) into the dialysis bag and clamp both ends of the dialysis bag with clips, and then place the dialysis bag in a PEG6000 solution with a concentration of 20% by mass (autoclaved before use) ), concentrate the sericin aqueous solution to 0.005~0.2 g/mL.
4、无菌壳聚糖溶液的制备4. Preparation of sterile chitosan solution
1）双蒸水稀释100%冰醋酸配制成体积比1％的乙酸溶液；1) Dilute 100% glacial acetic acid with double distilled water to make 1% acetic acid solution by volume;
2）称取壳聚糖溶于步骤1）所得1％乙酸溶液，得到0.01 g/mL壳聚糖乙酸溶液，放置于37℃摇床中充分溶解30分钟；2) Weigh chitosan and dissolve it in the 1% acetic acid solution obtained in step 1) to obtain a 0.01 g/mL chitosan acetic acid solution, and place it in a shaker at 37°C for 30 minutes to fully dissolve;
3）配制1 mol/L NaOH溶液，加入步骤2）所得0.01 g/mL壳聚糖乙酸溶液，调节溶液pH值至5.90~5.95；3) Prepare 1 mol/L NaOH solution, add the 0.01 g/mL chitosan acetic acid solution obtained in step 2), and adjust the pH value of the solution to 5.90~5.95;
4）步骤3）得到的壳聚糖溶液经过0.22 μm滤器过滤除菌，即可得到无菌壳聚糖溶液，备用。4) The chitosan solution obtained in step 3) is filtered and sterilized with a 0.22 μm filter to obtain a sterile chitosan solution for later use.
5、壳聚糖—丝胶蛋白复合生物支架的制备5. Preparation of chitosan-sericin composite bioscaffold
1）取5个洁净烧杯，分别按照体积比：壳聚糖：丝胶蛋白=1：0、壳聚糖：丝胶蛋白=4：1、壳聚糖：丝胶蛋白=1：1、壳聚糖：丝胶蛋白=1：4、壳聚糖：丝胶蛋白=0：1将无菌壳聚糖乙酸溶液与丝胶蛋白水溶液加入到烧杯中，充分混匀，得到CS100/SS0、CS80/SS20、CS50/SS50、CS20/SS80、CS0/SS100五种不同浓度比例的壳聚糖—丝胶蛋白混合溶液；1) Take 5 clean beakers, according to the volume ratio: chitosan: sericin = 1:0, chitosan: sericin = 4:1, chitosan: sericin = 1:1, shell Polycan: Sericin = 1: 4, Chitosan: Sericin = 0: 1 Add sterile chitosan acetic acid solution and sericin aqueous solution into a beaker and mix well to obtain CS100/SS0, CS80 /SS20, CS50/SS50, CS20/SS80, CS0/SS100 five kinds of chitosan-sericin mixed solutions with different concentration ratios;
2）每种比例加入京尼平水溶液，加入量为每1 mL混合溶液中加入330 uL 0.01 g/mL的京尼平水溶液，充分混匀后注入无菌模具中，在37oC下反应8小时，得到壳聚糖—丝胶蛋白复合水凝胶，其外观如图1所示（标尺为1厘米）；将2）中得到的壳聚糖—丝胶蛋白复合水凝胶放入无菌培养皿中，放入-80℃冷冻4小时，再放入冷冻真空干燥机中过夜冻干，冻干后从模具中取出得到无菌壳聚糖—丝胶蛋白复合生物支架，其外观如图2所示（标尺为1厘米）；2) Add genipin aqueous solution in each ratio, the amount added is 330 uL 0.01 g/mL genipin aqueous solution per 1 mL of the mixed solution, mix well, pour into a sterile mold, and react at 37o C for 8 hours, the chitosan-sericin composite hydrogel was obtained, and its appearance is shown in Figure 1 (the scale is 1 cm); the chitosan-sericin composite hydrogel obtained in 2) was put into a sterile Put it in a petri dish, freeze it at -80°C for 4 hours, then put it in a freezer vacuum dryer for overnight freeze-drying, take it out from the mold after freeze-drying to get a sterile chitosan-sericin composite biological scaffold, its appearance is shown in the figure 2 (the scale is 1 cm);
实施例2 壳聚糖—丝胶蛋白复合生物支架性能分析Example 2 Performance analysis of chitosan-sericin composite bioscaffold
对实施例1中制备的壳聚糖—丝胶蛋白复合生物支架进行以下性能分析，具体为：The chitosan-sericin composite bioscaffold prepared in embodiment 1 is carried out following performance analysis, specifically:
1、宏观结构1. Macro structure
使用选定的模具，使得到的壳聚糖—丝胶蛋白复合支架被制备成直径1cm，高2~3mm的薄片状结构，如图2所示。Using the selected mold, the obtained chitosan-sericin composite scaffold was prepared into a sheet-like structure with a diameter of 1 cm and a height of 2-3 mm, as shown in Fig. 2 .
2、微观结构。2. Microstructure.
在扫描电子显微镜（ULTRA PLUS-43-13，Zeiss, 德国产）下观察壳聚糖—丝胶蛋白复合生物支架。如图3所示，五种不同比例壳聚糖—丝胶蛋白复合生物支架横截面的SEM图，标尺为500微米，支架内部为多空隙结构，CS100/SS0、CS80/SS20、CS50/SS50、CS20/SS80、CS0/SS100五种复合支架孔径平均值分别为157.69 μm、130.55 μm、146.27 μm、136.35 μm、151.99 μm，大量微孔可很好地支持细胞生长和促进营养物质及代谢物质交换。The chitosan-sericin composite bioscaffold was observed under a scanning electron microscope (ULTRA PLUS-43-13, Zeiss, Germany). As shown in Figure 3, the SEM images of the cross-sections of five chitosan-sericin composite bioscaffolds with different ratios, the scale is 500 microns, and the interior of the scaffold is a multi-void structure, CS100/SS0, CS80/SS20, CS50/SS50, The average pore diameters of the five composite scaffolds of CS20/SS80 and CS0/SS100 are 157.69 μm, 130.55 μm, 146.27 μm, 136.35 μm, and 151.99 μm, respectively. A large number of micropores can well support cell growth and promote the exchange of nutrients and metabolic substances.
3、力学性能3. Mechanical properties
利用微型万能测试机（Instron5848 MicroTester，Instron, USA）在常温下测试壳聚糖—丝胶蛋白复合生物支架的力学性能。测得CS100/SS0、CS80/SS20、CS50/SS50、CS20/SS80、CS0/SS100五种复合支架的弹性模量分别为0.0254 ± 0.0019 MPa、0.0310 ±0.0011 MPa、0.0347 ± 0.0022 MPa、0.0360 ± 0.0008 MPa、0.0478 ± 0.0023 MPa。可见在五种复合生物支架中，随着丝胶蛋白浓度的增加，复合支架的机械性能得到改善。其中，CS50/SS50比例复合生物支架具有足够合适的机械性能能够支持神经损伤修复。The mechanical properties of chitosan-sericin composite bioscaffolds were tested at room temperature using a miniature universal testing machine (Instron5848 MicroTester, Instron, USA). The measured elastic modulus of the five composite scaffolds of CS100/SS0, CS80/SS20, CS50/SS50, CS20/SS80, and CS0/SS100 are 0.0254 ± 0.0019 MPa, 0.0310 ± 0.0011 MPa, 0.0347 ± 0.0022 MPa, 0.0360 ± 0.0008 MPa, respectively , 0.0478 ± 0.0023 MPa. It can be seen that among the five composite biological scaffolds, with the increase of sericin concentration, the mechanical properties of the composite scaffolds are improved. Among them, the CS50/SS50 ratio composite bioscaffold has sufficient and suitable mechanical properties to support nerve injury repair.
4、膨胀率4. Expansion rate
将冻干的复合生物支架浸泡于与体内pH近似的磷酸盐缓冲溶液（pH7.4）中，在不同时间点取出，按以下公式测定其吸水膨胀率（其中Ws为膨胀状态下的重量，Wd为干重）。Soak the freeze-dried composite bioscaffold in a phosphate buffer solution (pH7.4) that is similar to the pH in the body, take it out at different time points, and measure its water swelling rate according to the following formula (where Ws is the weight in the expanded state, Wd is the dry weight).
实验结果如图4所示，以CS50/SS50为例，由图4可知，在浸入PBS溶液24小时之后，丝胶蛋白神经导管膨胀了9.6倍，48小时之后趋于稳定，在14倍左右，能够适用于体内环境。The experimental results are shown in Figure 4. Taking CS50/SS50 as an example, it can be seen from Figure 4 that after immersion in PBS solution for 24 hours, the sericin nerve conduit expanded by 9.6 times, and stabilized after 48 hours, at about 14 times. Can be applied to the internal environment.
5、孔隙率5. Porosity
将冻干后的复合生物支架称重记为M0，测量其体积记为Vm，浸泡到无水乙醇中24小时后，称重记为Mt，根据以下公式计算丝胶蛋白神经导管的孔隙率（无水乙醇密度=0.789 g/cm3）Weigh the freeze-dried composite biological scaffold as M0 , measure its volume as Vm , soak it in absolute ethanol for 24 hours, and record it as Mt , and calculate the sericin nerve guide according to the following formula: Porosity (absolute ethanol density =0.789 g/cm3 )
实施例1中制备的CS100/SS0、CS80/SS20、CS50/SS50、CS20/SS80、CS0/SS100五种复合生物支架孔隙率分别为83.30%、84.13%、85.34%、82.47%、90.73%，含有比较多的孔径，有利于细胞因子的包埋以及细胞的搭载。The porosities of the five composite biological scaffolds prepared in Example 1, CS100/SS0, CS80/SS20, CS50/SS50, CS20/SS80, and CS0/SS100, were 83.30%, 84.13%, 85.34%, 82.47%, and 90.73%, respectively, containing More pore size is conducive to the embedding of cytokines and loading of cells.
6、红外光谱分析6. Infrared spectral analysis
利用傅里叶变换红外光谱仪（Nexus, Thermal Nicolet, USA）分别测定五种复合生物支架在4000-650 cm-1的特征峰，谱图如图5所示。Fourier transform infrared spectrometer (Nexus, Thermal Nicolet, USA) was used to measure the characteristic peaks of the five composite biological scaffolds at 4000-650 cm-1 , and the spectra are shown in Fig. 5 .
图5中可见CS0/SS100中丝胶蛋白独特的酰胺Ⅰ峰（1600-1690cm-1）、酰胺Ⅱ峰（1480-1575 cm-1）和酰胺Ⅲ峰（1229-1301 cm1）；CS100/SS0中壳聚糖位于1656 cm1、1490cm1两个特征性的吸收峰。两种成分混合后，按照S100/SS0、CS80/SS20、CS50/SS50、CS20/SS80、CS0/SS100比例顺序，随着丝胶蛋白浓度的增加，丝胶蛋白特征性吸收峰从无到有并逐渐增强，而壳聚糖的特征性吸收峰则逐渐减弱最后消失，说明复合生物支架确实具备壳聚糖和丝胶蛋白两种成分，且混合后的复合生物支架兼具了壳聚糖和丝胶蛋白两者的优点。Figure 5 shows the unique amide I peak (1600-1690cm-1 ), amide II peak (1480-1575 cm-1 ) and amide III peak (1229-1301 cm1 ) of sericin in CS0/SS100; CS100/SS0 There are two characteristic absorption peaks of chitosan at 1656 cm1 and 1490 cm1 . After the two components are mixed, according to the ratio sequence of S100/SS0, CS80/SS20, CS50/SS50, CS20/SS80, CS0/SS100, with the increase of the concentration of sericin, the characteristic absorption peak of sericin changes from nothing to existing. gradually strengthened, while the characteristic absorption peak of chitosan gradually weakened and finally disappeared, indicating that the composite bioscaffold did have two components of chitosan and sericin, and the mixed composite bioscaffold had both chitosan and silk Colloids have the advantages of both.
实施例3 复合生物支架支持神经细胞存活和增殖能力的评估Example 3 Evaluation of Composite Biological Scaffold Supporting Nerve Cell Survival and Proliferation Ability
使用实施例1中制备的无菌壳聚糖溶液及丝胶蛋白水溶液按照CS100/SS0、CS80/SS20、CS50/SS50、CS20/SS80、CS0/SS100五种比例直接加到细胞孔板中，每种比例加入京尼平水溶液，加入量为每1 mL混合溶液中加入330 uL浓度为0.01 g/mL的京尼平水溶液，在37oC下反应8小时，接着在-80℃下冷冻4小时，然后放入真空冷冻干燥机中冻干，得到冻干的壳聚糖—丝胶蛋白复合生物支架；将从细胞培养瓶收集的大鼠施万细胞（RSC96）经悬浮，吹散，种植于铺有冻干支架的细胞培养板上。细胞培养所用培养基为DMEM（Dulbecco's ModifiedEagle's Medium）培养基，培养基中加入5%胎牛血清，细胞放于细胞培养箱（37℃，CO2浓度为5％，湿度为100％）中培养。Use the aseptic chitosan solution prepared in Example 1 and the sericin aqueous solution to directly add in the cell well plate according to CS100/SS0, CS80/SS20, CS50/SS50, CS20/SS80, CS0/SS100 five kinds of ratios, each Add genipin aqueous solution in different proportions, the addition amount is 330 uL genipin aqueous solution with a concentration of 0.01 g/mL per 1 mL of mixed solution, react at 37o C for 8 hours, and then freeze at -80 °C for 4 hours , and then freeze-dried in a vacuum freeze dryer to obtain a freeze-dried chitosan-sericin composite biological scaffold; the rat Schwann cells (RSC96) collected from the cell culture flask were suspended, blown, and planted in Cell culture plates lined with lyophilized scaffolds. The medium used for cell culture was DMEM (Dulbecco's Modified Eagle's Medium) medium, in which 5% fetal bovine serum was added, and the cells were cultured in a cell culture incubator (37°C,CO2 concentration 5%, humidity 100%).
如图6所示，在种植后，采用CCK-8方法检测细胞在种植1，3，5，7和9天后细胞的增殖情况。由图6可知，大鼠施万细胞（RSC96）的数量随时间增长，因此冻干的壳聚糖—丝胶蛋白复合生物支架能够支持RSC96细胞的长期存活和增殖，并且CS50/SS50浓度比例组优于其他四种浓度比例组。As shown in FIG. 6 , after planting, the cell proliferation was detected 1, 3, 5, 7 and 9 days after planting by using the CCK-8 method. It can be seen from Figure 6 that the number of rat Schwann cells (RSC96) increases with time, so the freeze-dried chitosan-sericin composite biological scaffold can support the long-term survival and proliferation of RSC96 cells, and the CS50/SS50 concentration ratio group Better than the other four concentration ratio groups.
实施例4 复合生物支架搭载NGF的控释检测Example 4 Controlled Release Detection of NGF on Composite Biological Scaffold
使用实施例1中制备的无菌壳聚糖溶液及丝胶蛋白水溶液按照CS100/SS0、CS80/SS20、CS50/SS50、CS20/SS80、CS0/SS100五种比例混合均匀，加入20 uL浓度为10 ng/uL的无菌NGF溶液，每种比例加入京尼平水溶液，加入量为每1 mL混合溶液中加入0.33mL质量浓度为0.01 g/mL的京尼平水溶液，混合均匀后注入模具中，在37oC下反应8小时，接着在-80℃下冷冻4小时，然后放入真空冷冻干燥机中冻干，得到搭载NGF的壳聚糖—丝胶蛋白复合生物支架。Use the aseptic chitosan solution and sericin aqueous solution prepared in Example 1 to mix evenly according to five ratios of CS100/SS0, CS80/SS20, CS50/SS50, CS20/SS80, CS0/SS100, and add 20 uL of concentration to 10 ng/uL sterile NGF solution, add genipin aqueous solution in each ratio, the addition amount is 0.33mL of genipin aqueous solution with a mass concentration of 0.01 g/mL per 1 mL of mixed solution, mix well and inject into the mold, React at 37o C for 8 hours, then freeze at -80 ° C for 4 hours, and then freeze-dry in a vacuum freeze dryer to obtain a chitosan-sericin composite bioscaffold loaded with NGF.
小心将支架从模具中取出，放入洁净的六孔板中，在孔中加入PBS (pH 7.4) 1 mL充分浸没冻干支架，置于37oC。Carefully take the scaffold out of the mold, put it into a clean six-well plate, add 1 mL of PBS (pH 7.4) to the well to fully submerge the freeze-dried scaffold, and place it at 37o C.
在第1、2、3、4、5、6、7、8、9、10、12、14、16、20、25、30、35和40天，小心取出上清液，并重新加入1 mL PBS (pH 7.4)。On days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 25, 30, 35, and 40, carefully remove the supernatant and add back 1 mL PBS (pH 7.4).
采用酶联免疫法(ELISA)测量上清液中NGF的含量，图7A所示为各检测时间点NGF的释放量，图7B所示为NGF累计释放率，通过计算各个时间点上清液中累计的NGF含量与总缓释量的比值即得到NGF累计释放率。由图7A、图7B结果可知搭载了NGF的五种复合生物支架均可缓释出NGF，缓释的平台期每日缓释出的NGF量约为0.2 ng，可以达到治疗外周神经损伤所需的治疗量。五种复合生物支架的缓释性能之间相比较没有明显的差异。The content of NGF in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Figure 7A shows the release amount of NGF at each detection time point, and Figure 7B shows the cumulative release rate of NGF. By calculating the amount of NGF in the supernatant at each time point The ratio of the accumulated NGF content to the total sustained-release amount is the cumulative release rate of NGF. From the results in Figure 7A and Figure 7B, it can be seen that the five composite bioscaffolds loaded with NGF can release NGF slowly, and the daily slow-release NGF amount in the plateau stage of slow release is about 0.2 ng, which can meet the needs for the treatment of peripheral nerve injury. amount of treatment. There was no significant difference in the sustained-release properties of the five composite bioscaffolds.
实施例5 搭载NGF的壳聚糖—丝胶蛋白复合生物支架修复卡压神经能力的评估Example 5 Evaluation of the ability of chitosan-sericin composite bioscaffold loaded with NGF to repair entrapped nerves
选取32只200-250 g的SD大鼠，按照每100g注射0.3 ml 10%水合氯醛的剂量将32只大鼠麻醉后，取右侧卧位，暴露右后肢坐骨神经，在坐骨神经上卡套一个内径1.3 mm外径2.0mm长1.0 cm的硅胶套管。左侧坐骨神经作假手术处理，即小心暴露并游离坐骨神经。然后用7-0尼龙线进行缝合。卡压处理1个月后将32只大鼠随机平均分成四个组：单纯手术解压组、空载支架组、卡压部位直接注射NGF溶液组、搭载NGF复合生物支架组。仍按照每100 g注射0.3 mL10%水合氯醛的剂量将32只大鼠麻醉后，取右侧卧位，暴露右后肢坐骨神经。单纯手术解压组仅去除卡压在神经上的硅胶套管不做其他处理；手术解压后置入空载支架组为：去除卡压在神经上的硅胶套管后放置未搭载NGF的空载复合生物支架；卡压部位直接注射NGF溶液组为：去除卡压在神经上的硅胶套管后直接注射NGF溶液；搭载NGF复合生物支架组为：去除卡压在神经上的硅胶套管后放置搭载NGF的壳聚糖—丝胶蛋白复合生物支架，然后用7-0尼龙线进行缝合。其中，壳聚糖—丝胶蛋白复合生物支架规格为直径1.0 cm，高2 mm，搭载NGF量为200 ng，所用壳聚糖与丝胶蛋白体积比为1：1，即CS50/SS50。Select 32 SD rats of 200-250 g, inject 0.3 ml of 10% chloral hydrate per 100 g to anesthetize the 32 rats, take the right side lying position, expose the sciatic nerve of the right hind limb, and insert a clamp on the sciatic nerve A silicone sleeve with an inner diameter of 1.3 mm and an outer diameter of 2.0 mm and a length of 1.0 cm. The left sciatic nerve was sham-operated by carefully exposing and mobilizing the sciatic nerve. Then suture with 7-0 nylon suture. After 1 month of entrapment treatment, 32 rats were randomly divided into four groups: simple surgical decompression group, empty stent group, direct injection of NGF solution at the entrapment site group, and NGF composite biological stent group. 32 rats were still anesthetized by injecting 0.3 mL of 10% chloral hydrate per 100 g, and placed in the right lateral position to expose the sciatic nerve of the right hind limb. In the simple surgical decompression group, only the silicone sleeve pressed on the nerve was removed without other treatment; in the unloaded stent group after surgical decompression, the unloaded stent without NGF was placed after removing the silicone sleeve pressed on the nerve. Biological stents; direct injection of NGF solution at the compression site: remove the silicone sleeve pressed on the nerve and inject NGF solution directly; carry NGF composite biological scaffold group: remove the silicone sleeve pressed on the nerve and place it on the The chitosan-sericin composite bioscaffold of NGF was then sutured with 7-0 nylon thread. Among them, the chitosan-sericin composite bioscaffold has a diameter of 1.0 cm, a height of 2 mm, and the amount of NGF loaded is 200 ng. The volume ratio of chitosan and sericin used is 1:1, that is, CS50/SS50.
图9为大鼠坐骨神经卡压模型解压术后置入搭载NGF复合生物支架，复合生物支架在不同时间点体内降解情况。Figure 9 shows the decompression of the rat sciatic nerve entrapment model after implantation of a composite biological scaffold equipped with NGF, and the degradation of the composite biological scaffold at different time points.
在手术解压并进行上述4种分组处理2周、4周之后，对治疗部位坐骨神经做MBP和β3-微管蛋白免疫组化染色，MBP为髓鞘相关蛋白，β3-微管蛋白为神经轴突微管的标志，结果分别如图10A、图10B所示。与其他三个处理组相比，手术解压后置入搭载NGF复合生物支架组治疗部位坐骨神经髓鞘相关蛋白和神经轴突微管结构密度明显增大，说明手术解压后置入搭载NGF复合生物支架组能更好地在手术解压后促进受损神经的修复。Immunohistochemical staining of MBP and β3-tubulin was performed on the sciatic nerve at the treatment site after surgical decompression and the above four grouping treatments for 2 and 4 weeks. MBP is myelin-associated protein and β3-tubulin is axon The marks of microtubules are shown in Figure 10A and Figure 10B respectively. Compared with the other three treatment groups, the density of sciatic nerve myelin-associated protein and nerve axon microtubule structure in the treatment site of the group implanted with NGF composite biological scaffold after surgical decompression was significantly increased, indicating that after surgical decompression, the implantation of NGF composite biological scaffold The group was better able to promote the repair of damaged nerves after surgical decompression.
同时取坐骨神经支配的腓肠肌做马松染色统计肌纤维直径，评估肌纤维萎缩情况。如图11所示，在四个处理组中手术解压后置入搭载NGF复合生物支架组肌纤维直径最大，与假手术组无明显差异，说明手术解压后置入搭载NGF复合生物支架组能更好地修复受损神经并减少腓肠肌细胞的萎缩。图12为图11的统计图片。综上，在大鼠坐骨神经卡压模型中，手术解除卡压后使用搭载NGF的壳聚糖—丝胶蛋白复合生物支架治疗，具有良好的神经损伤修复效果。At the same time, the gastrocnemius muscle innervated by the sciatic nerve was collected for Masson staining to count the muscle fiber diameter and evaluate the muscle fiber atrophy. As shown in Figure 11, among the four treatment groups, the muscle fiber diameter in the group equipped with NGF composite bioscaffold after surgical decompression was the largest, and there was no significant difference with the sham operation group, indicating that the group after surgical decompression and implanted with NGF composite bioscaffold had better performance. effectively repair damaged nerves and reduce the atrophy of gastrocnemius muscle cells. FIG. 12 is a statistical picture of FIG. 11 . In conclusion, in the rat sciatic nerve entrapment model, the use of chitosan-sericin composite bioscaffold loaded with NGF after surgery to relieve entrapment has a good effect on nerve injury repair.
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.
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| CN201610477686.2ACN106039416B (en) | 2016-06-27 | 2016-06-27 | Chitosan-sericin compound bio bracket and its preparation method and application |
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