技术领域Technical Field
本发明属于医疗美容技术领域，具体涉及一种注射用再生丝素蛋白/重组人源化胶原蛋白凝胶及其制备方法。The present invention belongs to the technical field of medical cosmetology, and in particular relates to an injectable regenerated silk fibroin/recombinant humanized collagen gel and a preparation method thereof.
背景技术Background Art
随着医疗美容行业的快速发展，注射填充类材料越来越受到广泛关注。作为最常使用的注射填充类材料透明质酸钠凝胶虽然具有较好的即刻填充和塑形效果，但是由于透明质酸钠在组织环境中降解速度较快，同时，透明质酸钠本身又不具备刺激组织自身细胞生长和胶原蛋白产生的组织再生功能，导致患者通常每6个月左右就需要进行重复注射，大大增加了患者的治疗费用和不良感受。近年来，再生医美填充材料逐渐兴起，随着胶原蛋白填充剂、聚左旋乳酸面部填充剂、聚己内酯填充剂相继获批，医美填充材料也从传统的物理填充材料向组织再生填充材料方向发展。With the rapid development of the medical beauty industry, injectable filling materials are receiving more and more attention. As the most commonly used injectable filling material, sodium hyaluronate gel has good immediate filling and shaping effects. However, due to the rapid degradation of sodium hyaluronate in the tissue environment, sodium hyaluronate itself does not have the tissue regeneration function to stimulate the growth of tissue cells and collagen production, patients usually need repeated injections every 6 months or so, which greatly increases the patient's treatment costs and adverse experiences. In recent years, regenerative medical beauty filling materials have gradually emerged. With the approval of collagen fillers, poly-L-lactic acid facial fillers, and polycaprolactone fillers, medical beauty filling materials have also developed from traditional physical filling materials to tissue regeneration filling materials.
胶原蛋白是人体组织器官的主要结构蛋白，约占人体蛋白质总量的30～40％，胶原蛋白是人体细胞外基质最主要的成分，主要存在于皮肤、软骨、骨、肌腱、韧带、角膜、器官被膜、硬脑膜等部位的结缔组织。人类目前已发现28种型别的胶原蛋白，其中最常见的类型是：I型(真皮、骨骼、肌腱、韧带等，含量最多，约占胶原总量的80％～90％)、II型(软骨、玻璃体等)、III型(皮肤、血管、肠等)、IV型(基底膜等)、V型(骨、真皮、角膜、胎盘等)。从来源上看，可分为天然胶原蛋白和人工合成胶原蛋白；从是否能成纤维看，可分为成纤维胶原和非成纤维胶原两大类。动物源胶原蛋白：从牛皮、猪皮、鱼皮等胶原含量高的动物组织中提取纯化而来，具有生物活性和完整三螺旋结构，主要用于止血海绵、凝胶剂、生物敷料、人工骨及护肤品等产品，是当前的主要制备技术。重组胶原蛋白：采用重组DNA技术，对编码所需人胶原蛋白质的基因进行遗传操作和修饰，利用质粒或病毒载体将目的基因带入适当的宿主细胞(细胞、酵母或其他真核细胞等)中，表达翻译成胶原蛋白或类似胶原蛋白的多肽，再通过提取和纯化等步骤制备而成。Collagen is the main structural protein of human tissues and organs, accounting for about 30-40% of the total protein in the human body. Collagen is the most important component of the human extracellular matrix and is mainly found in connective tissues in the skin, cartilage, bone, tendon, ligament, cornea, organ capsule, dura mater, etc. Humans have discovered 28 types of collagen, of which the most common types are: type I (dermis, bones, tendons, ligaments, etc., with the highest content, accounting for about 80%-90% of the total collagen), type II (cartilage, vitreous, etc.), type III (skin, blood vessels, intestines, etc.), type IV (basement membrane, etc.), type V (bone, dermis, cornea, placenta, etc.). From the source, it can be divided into natural collagen and artificially synthesized collagen; from the perspective of whether it can form fibers, it can be divided into two categories: fibroblastic collagen and non-fibroblastic collagen. Animal collagen: extracted and purified from high-collagen animal tissues such as cowhide, pig skin, and fish skin. It has biological activity and a complete triple helix structure. It is mainly used in hemostatic sponges, gels, biological dressings, artificial bones, and skin care products. It is the main preparation technology at present. Recombinant collagen: using recombinant DNA technology, genetic manipulation and modification of genes encoding the required human collagen protein are performed, and plasmids or viral vectors are used to bring the target gene into appropriate host cells (cells, yeast or other eukaryotic cells, etc.), and the expression is translated into collagen or collagen-like polypeptides, and then prepared through extraction and purification steps.
根据重组胶原蛋白的来源，将其细分为三种类型：重组人胶原蛋白、重组人源化胶原蛋白、重组类胶原蛋白三个类别。“重组人胶原蛋白”是指由DNA重组技术制备的人胶原蛋白特定型别基因编码的全长氨基酸序列、且具有三螺旋结构的重组胶原蛋白材料。如重组胶原蛋白材料是由DNA重组技术制备的人胶原蛋白特定型别基因编码的全长氨基酸序列、但不具有三螺旋结构，也不属于“重组人胶原蛋白”。“重组人源化胶原蛋白”由DNA重组技术制备的人胶原蛋白特定型别基因编码的全长或部分氨基酸序列片段，或是含人胶原蛋白功能片段的组合。“重组类胶原蛋白”由DNA重组技术制备的经设计、修饰后的特定基因编码的氨基酸序列或其片段，或是这类功能性氨基酸序列片段的组合。其基因编码序列或氨基酸序列与人胶原蛋白的基因编码序列或氨基酸序列同源性低。According to the source of recombinant collagen, it is subdivided into three types: recombinant human collagen, recombinant humanized collagen, and recombinant collagen-like proteins. "Recombinant human collagen" refers to a recombinant collagen material with a full-length amino acid sequence encoded by a specific type of human collagen gene prepared by DNA recombinant technology and having a triple helix structure. If the recombinant collagen material is a full-length amino acid sequence encoded by a specific type of human collagen gene prepared by DNA recombinant technology, but does not have a triple helix structure, it does not belong to "recombinant human collagen". "Recombinant humanized collagen" is a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombinant technology, or a combination of functional fragments of human collagen. "Recombinant collagen-like proteins" are amino acid sequences or fragments encoded by specific genes that are designed and modified and prepared by DNA recombinant technology, or a combination of such functional amino acid sequence fragments. Its gene coding sequence or amino acid sequence has low homology with the gene coding sequence or amino acid sequence of human collagen.
普通的胶原蛋白，如动物胶原蛋白，重组胶原蛋白较于传统动物胶原蛋白，具有无病毒性隐患、优异的生物学相容性和功效性、低免疫原性的特点，有效规避了传统动物源胶原蛋白的病毒隐患、排异反应，并克服了传统动物胶原蛋白因动物年龄差异、种属差异导致的终端产品临床疗效不确切、质量不稳定的弊端。Ordinary collagen, such as animal collagen, and recombinant collagen have the characteristics of no viral risks, excellent biological compatibility and efficacy, and low immunogenicity compared to traditional animal collagen. They effectively avoid the viral risks and rejection reactions of traditional animal-derived collagen, and overcome the shortcomings of traditional animal collagen, such as uncertain clinical efficacy and unstable quality of the end product due to differences in animal age and species.
丝素蛋白至1993年被美国食品和药物管理局(FDA)批准为生物材料以来，由于其出色的机械性能，良好的生物相容性，生物降解性以及结构调整的多功能性而被广泛应用于生物医药和组织工程领域。丝素蛋白可以被加工成溶液、丝膜、微球/微粒、水凝胶、支架和电纺膜等具有不同结构的材料，应用于创伤修复、药物载体、组织填充和人造器官等领域。近年来，已上市的丝素蛋白产品包括艾尔健公司开发的SERI外科支架，用于腹壁重建和整形外科。美国Sofregen公司开发了一种SilkVoice天然蚕丝蛋白注射剂，用于治疗声带介质化和声带功能不全。苏州苏豪生物材料公司开发了一种丝素蛋白的创面敷料，用于烫伤及烧伤创面的愈合修复。目前，将丝素蛋白制备成用于面部整形填充产品尚属空白。根据相关研究，作为一种具有优良生物相容性的材料，丝素蛋白可以促进皮肤成纤维细胞的生长、分化和增值，同时，丝素蛋白在体内降解后所释放的多种氨基酸又可加速细胞新陈代谢，促进胶原蛋白合成，增强皮肤张力和弹性及色素分解。相比常用的胶原蛋白凝胶，丝素蛋白水凝胶在支持原代人真皮成纤维细胞的增殖和角质形成细胞的迁移方面表现出优越性。因此，如果将丝素蛋白和胶原蛋白制备成一种可注射的软组织填充材料用于面部整形，将具有极大的应用潜力。丝素蛋白在作为可注射软组织填充材料应用于面部整形使用时，需要将其制备成可通过注射器注射的水凝胶，进而将其注入到皮肤组织，实现美容填充的功效。Since silk fibroin was approved as a biomaterial by the U.S. Food and Drug Administration (FDA) in 1993, it has been widely used in the fields of biomedicine and tissue engineering due to its excellent mechanical properties, good biocompatibility, biodegradability and versatility of structural adjustment. Silk fibroin can be processed into materials with different structures such as solutions, silk films, microspheres/microparticles, hydrogels, scaffolds and electrospun membranes, and used in the fields of wound repair, drug carriers, tissue filling and artificial organs. In recent years, silk fibroin products that have been launched on the market include the SERI surgical stent developed by Allergan for abdominal wall reconstruction and plastic surgery. Sofregen in the United States has developed a SilkVoice natural silk protein injection for the treatment of vocal cord medialization and vocal cord insufficiency. Suzhou Soho Biomaterials Co., Ltd. has developed a silk fibroin wound dressing for the healing and repair of scald and burn wounds. At present, there is still a blank in the preparation of silk fibroin for facial plastic surgery and filling products. According to relevant research, as a material with excellent biocompatibility, silk fibroin can promote the growth, differentiation and proliferation of skin fibroblasts. At the same time, the various amino acids released by silk fibroin after degradation in the body can accelerate cell metabolism, promote collagen synthesis, enhance skin tension and elasticity, and pigment decomposition. Compared with commonly used collagen gels, silk fibroin hydrogels show superiority in supporting the proliferation of primary human dermal fibroblasts and the migration of keratinocytes. Therefore, if silk fibroin and collagen are prepared into an injectable soft tissue filling material for facial plastic surgery, it will have great application potential. When silk fibroin is used as an injectable soft tissue filling material for facial plastic surgery, it needs to be prepared into a hydrogel that can be injected through a syringe, and then injected into the skin tissue to achieve the effect of cosmetic filling.
丝素蛋白是由桑蚕后部丝腺合成分泌的纤维状蚕丝蛋白。桑蚕丝纤维中的核心蛋白质由理论分子量约为391kDa的重链(H-chain)、25kDa的轻链(L-chain)和30kDa或27kDa纤维六聚体/P25组成，H链蛋白、L链蛋白和P25蛋白的物质的量比是6：6：1。重链和轻链通过单个二硫键连接，P25为含有ASN寡糖链的糖蛋白，P25与二硫键连接的重链和轻链通过非共价的疏水相互作用连接。再生丝蛋白，是蚕丝核心纤维(丝素蛋白纤维)溶解除盐后的蛋白质混合物，其基本的氨基酸基序(motif)与丝素蛋白相同。再生丝蛋白在脱胶和溶解过程导致其分子链无序降解，分子量下降至不低于10kDa分子量，且分子链的构效关系与丝素蛋白纤维中的构效关系存在较大差别，通常以微黄色半透明的水溶液的形式存在。按成型和干燥方法的不同，也可制成水溶性(聚集态结构主要为Silk I)和水不溶性(聚集态结构主要为Silk II)的白色粉末。Fibroin is a fibrous silk protein synthesized and secreted by the silk gland at the rear of the silkworm. The core protein in the silk fiber is composed of a heavy chain (H-chain) with a theoretical molecular weight of about 391kDa, a light chain (L-chain) of 25kDa, and a 30kDa or 27kDa fiber hexamer/P25. The molar ratio of H-chain protein, L-chain protein and P25 protein is 6:6:1. The heavy chain and the light chain are connected by a single disulfide bond. P25 is a glycoprotein containing an ASN oligosaccharide chain. P25 is connected to the heavy chain and the light chain connected by the disulfide bond through non-covalent hydrophobic interactions. Regenerated silk protein is a protein mixture after the silk core fiber (fibroin fiber) is dissolved and desalted. Its basic amino acid motif is the same as that of fibroin. The degumming and dissolution process of regenerated silk protein leads to disordered degradation of its molecular chain, and the molecular weight drops to no less than 10kDa. The structure-activity relationship of the molecular chain is quite different from that of silk fibroin fiber, and it usually exists in the form of a slightly yellow translucent aqueous solution. According to different molding and drying methods, it can also be made into water-soluble (aggregate structure is mainly Silk I) and water-insoluble (aggregate structure is mainly Silk II) white powder.
目前，制备再生丝素蛋白凝胶的方法主要包括化学交联法和物理交联法。化学交联法制备的丝素蛋白水凝胶所要用到的交联剂戊二醛、京尼平、辣根过氧化酶等大多具有较高的毒性，因此水凝胶植入组织后，由于交联剂残留造成的毒性问题难以解决。与化学交联相比较，物理交联的丝素蛋白水凝胶具有更好的生物安全性，更适合作为组织填充材料应用在医疗美容领域。目前，已报道的丝素蛋白物理凝胶的制备方法包括，水溶性有机溶剂诱导、表面活性剂诱导、pH值诱导、超声/震荡诱导和电场诱导等。但是，上述方法制备的丝素蛋白物理凝胶的共同的特点是，凝胶内部分子分布都较为松散，交联结构稳定性差，导致其在植入机体组织后，极易被组织微环境及各种蛋白酶快速分解，从而达不到长周期的组织填充效果。重组人源化胶原蛋白是具有高级结构和功能的胶原蛋白，不仅可以替代传统动物胶原，还可以解决高端注射生物材料的免疫原性，其三螺旋结构与水溶性并存的特性，胶原蛋白具有多样性，目前人类已识别并被鉴定的共有28种，每种型别分布在人体不同的部位，发挥着重要的修复再生功能。重组人源化胶原蛋白氨基酸序列与人自身胶原蛋白100％一致，具有正确稳定的三螺旋结构，无免疫原性及病毒污染，细胞粘附性高，可全方位全生命周期参与人体组织器官的修复再生，广泛应用于人体皮肤、骨、软骨、心血管系统、口腔及管腔组织等医疗健康领域。At present, the methods for preparing regenerated silk fibroin gel mainly include chemical crosslinking and physical crosslinking. Most of the crosslinking agents used in the silk fibroin hydrogel prepared by the chemical crosslinking method, such as glutaraldehyde, genipin, horseradish peroxidase, etc., have high toxicity. Therefore, after the hydrogel is implanted into the tissue, the toxicity problem caused by the crosslinking agent residue is difficult to solve. Compared with chemical crosslinking, the physically crosslinked silk fibroin hydrogel has better biosafety and is more suitable for use as a tissue filling material in the field of medical cosmetology. At present, the reported preparation methods of silk fibroin physical gel include water-soluble organic solvent induction, surfactant induction, pH induction, ultrasound/oscillation induction and electric field induction. However, the common characteristics of the silk fibroin physical gel prepared by the above methods are that the internal molecular distribution of the gel is relatively loose, and the crosslinking structure has poor stability, which leads to it being easily decomposed by the tissue microenvironment and various proteases after being implanted into the body tissue, thereby failing to achieve a long-term tissue filling effect. Recombinant humanized collagen is a collagen with advanced structure and function. It can not only replace traditional animal collagen, but also solve the immunogenicity of high-end injectable biomaterials. Its triple helix structure and water solubility coexist. Collagen is diverse. At present, there are 28 types that have been identified and identified by humans. Each type is distributed in different parts of the human body and plays an important role in repair and regeneration. The amino acid sequence of recombinant humanized collagen is 100% consistent with human collagen. It has a correct and stable triple helix structure, no immunogenicity and virus contamination, high cell adhesion, and can participate in the repair and regeneration of human tissues and organs in all aspects and throughout the life cycle. It is widely used in medical and health fields such as human skin, bone, cartilage, cardiovascular system, oral cavity and luminal tissue.
特别地，制备再生丝素蛋白/重组人源化胶原蛋白双网络凝胶在作为面部填充剂使用时，具备重组人源化胶原蛋白的良好的生物相容性和修复特性，其中重组人源化胶原蛋白具有三螺旋结构，无免疫原性及病毒污染，细胞粘附性高，相比其它动物源胶原蛋白或于人胶原蛋白的基因编码序列或氨基酸序列同源性低的重组类胶原蛋白，作为注射用的生物材料，具有与人体胶原蛋白同源性高的明显优势，同时还兼具有再生丝素蛋白的促进皮肤成纤维细胞的生长、分化和增值，降解周期长，可以刺激组织内的成纤维细胞生长及胶原蛋白的分泌的功效，改善了传统透明质酸钠凝胶填充剂维持时间短，无法刺激组织再生的缺陷。这项研究，具有明显的创新优势和前瞻性。In particular, the regenerated silk protein/recombinant humanized collagen double network gel has good biocompatibility and repair properties of recombinant humanized collagen when used as a facial filler. Recombinant humanized collagen has a triple helix structure, no immunogenicity and virus contamination, and high cell adhesion. Compared with other animal collagen or recombinant collagen with low homology in gene coding sequence or amino acid sequence to human collagen, as an injectable biomaterial, it has the obvious advantage of high homology with human collagen. At the same time, it also has the effect of promoting the growth, differentiation and proliferation of skin fibroblasts of regenerated silk protein, and has a long degradation cycle, which can stimulate the growth of fibroblasts and the secretion of collagen in tissues, improving the defects of traditional sodium hyaluronate gel fillers with short maintenance time and inability to stimulate tissue regeneration. This research has obvious innovative advantages and foresight.
发明内容Summary of the invention
针对现有技术的不足，本发明提供一种注射用再生丝素蛋白/重组人源化胶原蛋白凝胶及其制备方法。In view of the deficiencies in the prior art, the present invention provides an injectable regenerated silk fibroin/recombinant humanized collagen gel and a preparation method thereof.
本发明的技术方案如下：一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，组成成分按重量份数计包括：1.01～10.21份再生丝素蛋白、1.01～10.12份重组III型人源化胶原蛋白、0～0.52份润湿剂、0.1～1.01份表面活性剂和100～1000份PBS磷酸缓冲液。The technical scheme of the present invention is as follows: a regenerated silk fibroin/recombinant humanized collagen double network gel, the components of which include, by weight: 1.01 to 10.21 parts of regenerated silk fibroin, 1.01 to 10.12 parts of recombinant type III humanized collagen, 0 to 0.52 parts of a wetting agent, 0.1 to 1.01 parts of a surfactant and 100 to 1000 parts of PBS phosphate buffer.
其中所述再生丝素蛋白的分子量为10-40KDa，优选为10-20KDa。The molecular weight of the regenerated silk fibroin is 10-40 KDa, preferably 10-20 KDa.
其中所述润湿剂选自丙二醇、甘油或丁二醇；优选为甘油。The wetting agent is selected from propylene glycol, glycerol or butylene glycol; preferably glycerol.
其中所述表面活性剂选自油酸酸钠或十二烷基硫酸钠，优选油酸钠。The surfactant is selected from sodium oleate or sodium lauryl sulfate, preferably sodium oleate.
其中所述PBS磷酸缓冲液的浓度为0.01M，pH为7.2～7.4，优选pH为7.4。The concentration of the PBS phosphate buffer is 0.01 M, and the pH is 7.2-7.4, preferably 7.4.
其中所述0.01M pH7.4的PBS磷酸缓冲液配置方法为：8.50g氯化钠、2.20g磷酸氢二钠、0.20g磷酸二氢钠和1L纯化水搅拌溶解即得。The 0.01M pH 7.4 PBS phosphate buffer solution is prepared by stirring and dissolving 8.50 g of sodium chloride, 2.20 g of disodium hydrogen phosphate, 0.20 g of sodium dihydrogen phosphate and 1 L of purified water.
优选地，所述再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，组成成分按重量份数计包括：10.21份再生丝素蛋白、10.12份重组III型人源化胶原蛋白、0.52份甘油、1.01份油酸钠和1000份PBS磷酸缓冲液。Preferably, the regenerated silk fibroin/recombinant humanized collagen double network gel comprises the following components by weight: 10.21 parts of regenerated silk fibroin, 10.12 parts of recombinant humanized type III collagen, 0.52 parts of glycerol, 1.01 parts of sodium oleate and 1000 parts of PBS phosphate buffer.
本发明还提供了所述再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，包括以下步骤：The present invention also provides a method for preparing the regenerated silk fibroin/recombinant humanized collagen double network gel, comprising the following steps:
1)取配制好的PBS磷酸缓冲液，加入再生丝素蛋白，重组III型人源化胶原蛋白，润湿剂，搅拌溶解；1) Take the prepared PBS phosphate buffer, add regenerated silk fibroin, recombinant type III humanized collagen, and wetting agent, and stir to dissolve;
2)将表面活性剂加入到步骤1)的溶液中，过滤膜，滤液灌装在预灌封注射器中，附上泡罩盒和封口膜，于30℃～40℃孵化24～36h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) adding a surfactant to the solution of step 1), filtering the membrane, filling the filtrate into a prefilled syringe, attaching a blister box and a sealing film, and incubating at 30° C. to 40° C. for 24 to 36 hours to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
本发明提供的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶在制备组织再生医疗美容填充材料中的应用。The invention provides a method for preparing a tissue regeneration medical cosmetic filling material using a regenerated silk fibroin/recombinant humanized collagen double network gel.
本发明的有益效果：根据本发明实施例的方案，通过油酸钠或十二烷基硫酸钠诱导丝素蛋白物理交联形成再生丝素蛋白/重组III型人源化胶原蛋白双网络凝胶，结合过滤除菌和无菌加工技术，制备出无菌的再生丝素蛋白/重组III型人源化胶原蛋白双网络凝胶。作为成一种可注射的面部填充剂，在医美领域，运用中胚层疗法，可以注射到皮肤真皮层，达到面部除皱的效果。本发明实施例具有创新性、前瞻性和革命性的特点。目前没有公布再生丝素蛋白/重组III型人源化胶原蛋白自交联凝胶的技术，相比重组III型人源化胶原蛋白溶液，油酸钠或十二烷基硫酸钠诱导再生丝素蛋白物理交联形成的再生丝素蛋白/重组III型人源化胶原蛋白双网络凝胶，不仅有重组III型人源化胶原蛋白自身的优势，还运用了美容性脂肪组织膨胀和美容性脂肪组织修复的生理行可接受的代谢脂质：油酸钠或十二烷基硫酸钠，再结合生物可降解和生物相容性较好的再生丝素蛋白，补充胶原蛋白的同时，刺激纤维细胞和胶原蛋白的再生，具有加速细胞新陈代谢，延缓皮肤衰老，增强美容性脂肪组织膨胀，加速美容性脂肪组织修复，是一种具有创新性的组织再生医疗美容填充材料，彻底解决皮肤衰老的问题。Beneficial effects of the present invention: According to the scheme of the embodiment of the present invention, physical cross-linking of silk fibroin is induced by sodium oleate or sodium dodecyl sulfate to form a regenerated silk fibroin/recombinant type III humanized collagen double network gel, and sterile regenerated silk fibroin/recombinant type III humanized collagen double network gel is prepared by combining filtration sterilization and aseptic processing technology. As an injectable facial filler, in the field of medical beauty, mesotherapy can be used to inject into the dermis of the skin to achieve the effect of facial wrinkle removal. The embodiments of the present invention are innovative, forward-looking and revolutionary. At present, there is no published technology for self-cross-linking gel of regenerated silk fibroin/recombinant type III humanized collagen. Compared with recombinant type III humanized collagen solution, the regenerated silk fibroin/recombinant type III humanized collagen double network gel formed by physical cross-linking of regenerated silk fibroin induced by sodium oleate or sodium dodecyl sulfate not only has the advantages of recombinant type III humanized collagen itself, but also uses physiologically acceptable metabolic lipids for cosmetic adipose tissue expansion and cosmetic adipose tissue repair: sodium oleate or sodium dodecyl sulfate, combined with biodegradable and biocompatible regenerated silk fibroin, while supplementing collagen, stimulates the regeneration of fibroblasts and collagen, accelerates cell metabolism, delays skin aging, enhances cosmetic adipose tissue expansion, and accelerates cosmetic adipose tissue repair. It is an innovative tissue regeneration medical cosmetic filling material that completely solves the problem of skin aging.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1示出了根据本发明实施例1的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的照片；FIG1 shows a photograph of a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 1 of the present invention;
图2示出了根据本发明实施例1的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的红外光谱图；FIG2 shows an infrared spectrum of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 1 of the present invention;
图3示出了根据本发明实施例1的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的粒度图谱；FIG3 shows the particle size spectrum of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 1 of the present invention;
图4示出了根据本发明实施例1的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'和相位角δ与频率f的坐标图；FIG4 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 1 of the present invention, at (25±0.2)° C., using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz, and selecting a frequency scan to plot the viscous modulus G”, the elastic modulus G’ and the phase angle δ versus the frequency f;
图5示出了根据本发明实施例1的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25士0.2)℃，采用流变仪在剪切速率：0.001s-1～1000s-1进行蠕动扫描，分别绘制剪切应力、粘度η与剪切速率的坐标图；FIG5 shows the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 1 of the present invention. The shear stress, viscosity η and shear rate are plotted respectively at (25±0.2)°C and the peristaltic scanning is performed at a shear rate of 0.001s-1 to 1000s-1 using a rheometer. Coordinate diagram of
图6示出了根据本发明实施例2的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的粒度图谱；FIG6 shows the particle size spectrum of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 2 of the present invention;
图7示出了根据本发明实施例2的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'和相位角δ与频率f的坐标图；FIG7 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 2 of the present invention, at (25±0.2)° C., using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz, and selecting a frequency scan to plot the viscous modulus G”, elastic modulus G’ and phase angle δ versus frequency f;
图8示出了根据本发明实施例2的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25士0.2)℃，采用流变仪在剪切速率：0.001s-1～1000s-1进行蠕动扫描，分别绘制剪切应力、粘度η与剪切速率的坐标图；FIG8 shows the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 2 of the present invention. The shear stress, viscosity η and shear rate are plotted respectively at (25±0.2)°C and the peristaltic scanning is performed using a rheometer at a shear rate of 0.001s-1 to 1000s-1. Coordinate diagram of
图9示出了根据本发明实施例3的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'和相位角δ与频率f的坐标图。FIG9 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 3 of the present invention. At (25±0.2)° C., a rheometer is used to draw a coordinate diagram of the viscous modulus G”, elastic modulus G’, phase angle δ and frequency f by frequency scanning at a shear rate ranging from 0.1 Hz to 100 Hz.
图10示出了根据本发明实施例3的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25士0.2)℃，采用流变仪在剪切速率：0.001s-1～1000s-1进行蠕动扫描，分别绘制剪切应力、粘度η与剪切速率的坐标图；FIG10 shows the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 3 of the present invention. The shear stress, viscosity η and shear rate are plotted respectively at (25±0.2)°C and the peristaltic scanning is performed at a shear rate of 0.001s-1 to 1000s-1 using a rheometer. Coordinate diagram of
图11示出了根据本发明实施例4的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的红外光谱图；FIG11 shows an infrared spectrum of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 4 of the present invention;
图12示出了根据本发明实施例4的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'与频率f的坐标图；FIG12 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 4 of the present invention, at (25±0.2)° C., using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz, and selecting a frequency scan to plot a coordinate diagram of the viscous modulus G”, the elastic modulus G’ and the frequency f;
图13在示出了根据本发明实施例5的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'和相位角8与频率f的坐标图；FIG13 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 5 of the present invention, at (25±0.2)° C., using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz, and selecting a frequency scan to plot the viscous modulus G”, elastic modulus G’ and phase angle 8 versus frequency f;
图14示出了根据本发明实施例6的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'与频率f的坐标图；FIG14 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 6 of the present invention, at (25±0.2)° C., using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz, and selecting a frequency scan to plot a coordinate diagram of the viscous modulus G”, the elastic modulus G’ and the frequency f;
图15示出了根据本发明实施例7的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'与频率f的坐标图；FIG15 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 7 of the present invention, at (25±0.2)° C., using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz, and selecting a frequency scan to plot a coordinate diagram of the viscous modulus G”, the elastic modulus G’ and the frequency f;
图16示出了根据本发明实施例8的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'与频率f的坐标图；FIG16 shows a graph of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 8 of the present invention, wherein the viscous modulus G”, the elastic modulus G’ and the frequency f are plotted by using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz at (25±0.2)° C. and selecting a frequency scan;
图17示出了根据本发明实施例9的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'与频率f的坐标图；FIG17 shows a graph of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 9 of the present invention, wherein the viscous modulus G”, the elastic modulus G’ and the frequency f are plotted by using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz at (25±0.2)° C. and selecting a frequency scan;
图18示出了根据本发明实施例10的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'与频率f的坐标图；FIG18 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 10 of the present invention, at (25±0.2)° C., a rheometer is used to draw a coordinate diagram of the viscous modulus G”, the elastic modulus G’ and the frequency f at a shear rate ranging from 0.1 Hz to 100 Hz by frequency scanning;
图19示出了根据本发明实施例11的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'与频率f的坐标图；FIG19 shows a graph of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 11 of the present invention, where a rheometer is used at (25±0.2)° C. and a frequency scan is performed at a shear rate of 0.1 Hz to 100 Hz to plot the viscous modulus G”, the elastic modulus G’ and the frequency f;
图20示出了根据本发明实施例12的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'与频率f的坐标图；FIG20 shows a graph of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 12 of the present invention, wherein the viscous modulus G”, the elastic modulus G’ and the frequency f are plotted by using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz at (25±0.2)° C. and selecting a frequency scan;
图21示出了根据本发明实施例13的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25士0.2)℃，采用流变仪在剪切速率：0.001s-1～1000s-1进行蠕动扫描，分别绘制剪切应力、粘度η与剪切速率的坐标图；FIG21 shows the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 13 of the present invention. The shear stress, viscosity η and shear rate are plotted respectively at (25±0.2)°C and the peristaltic scanning is performed at a shear rate of 0.001s-1 to 1000s-1 using a rheometer. Coordinate diagram of
图22示出了根据本发明实施例14的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'和相位角δ与频率f的坐标图；FIG22 shows a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 14 of the present invention, at (25±0.2)° C., using a rheometer at a shear rate ranging from 0.1 Hz to 100 Hz, and selecting a frequency scan to plot the viscous modulus G”, elastic modulus G’ and phase angle δ versus frequency f;
图23示出了根据本发明实施例14的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，在(25士0.2)℃，采用流变仪在剪切速率：0.001s-1～1000s-1进行蠕动扫描，分别绘制剪切应力、粘度η与剪切速率的坐标图；FIG23 shows the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 14 of the present invention. The shear stress, viscosity η and shear rate are plotted respectively at (25±0.2)°C and the peristaltic scanning is performed using a rheometer at a shear rate of 0.001s-1 to 1000s-1. Coordinate diagram of
图24示出了根据本发明实施例14的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的粒度数据；FIG24 shows the particle size data of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 14 of the present invention;
图25示出了根据本发明实施例14的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下12个月的解刨照片；FIG25 shows an anatomical photograph of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 14 of the present invention injected subcutaneously into rats for 12 months;
图26了根据本发明实施例14的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下不同时间周期组织切片的H&E染色照片；FIG26 shows H&E staining photos of tissue sections of rats injected with regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 14 of the present invention at different time periods;
图27了根据本发明实施例14的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下不同时间周期组织切片的Masson染色照片。FIG27 shows Masson staining photographs of rat subcutaneous tissue sections injected with the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 14 of the present invention at different time periods.
图28了根据本发明实施例14的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下不同时间周期组织切片的CD31标记照片；FIG28 shows CD31 labeled photos of rat tissue sections injected with the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 14 of the present invention at different time periods under the skin;
图29了根据本发明实施例14的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下不同时间周期组织切片的α-SMA标记照片。FIG29 shows α-SMA labeled photographs of rat subcutaneous tissue sections at different time periods after the regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of Example 14 of the present invention was injected.
具体实施方式DETAILED DESCRIPTION
下面的实施例可以使本专业的本领域技术人员更全面地理解本发明，但并不因此将本发明限制在所述的实施例范围之中。The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.
配制0.01M PBS磷酸缓冲液pH 7.2：氯化钠：8.50g，磷酸氢二钠：2.20g，磷酸二氢钠：0.30g，纯化水：1L；Prepare 0.01M PBS phosphate buffer pH 7.2: sodium chloride: 8.50 g, disodium hydrogen phosphate: 2.20 g, sodium dihydrogen phosphate: 0.30 g, purified water: 1 L;
配制0.01M PBS磷酸缓冲液pH 7.4：氯化钠：8.50g，磷酸氢二钠：2.20g，磷酸二氢钠：0.20g，纯化水：1L；Prepare 0.01M PBS phosphate buffer pH 7.4: sodium chloride: 8.50 g, disodium hydrogen phosphate: 2.20 g, sodium dihydrogen phosphate: 0.20 g, purified water: 1 L;
实施例1Example 1
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取300ml配制好的0.01M pH 7.2PBS磷酸缓冲液，加入6.01g可溶性再生丝素蛋白，3.01g重组III型人源化胶原蛋白，搅拌30min后。1) Take 300 ml of prepared 0.01 M pH 7.2 PBS phosphate buffer, add 6.01 g of soluble regenerated silk fibroin and 3.01 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.31g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在3ml预灌封注射器中，附上泡罩盒和封口膜，于32±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，可顺利通过30G(0.3*13RWLB)以下注射针，不堵针头。2) Add 0.31 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 3 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 32±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel, which can pass through an injection needle below 30G (0.3*13RWLB) without clogging the needle.
图1示出了根据本发明实施例的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的照片。FIG. 1 shows a photograph of a regenerated silk fibroin/recombinant humanized collagen double network gel obtained according to the preparation method of an embodiment of the present invention.
图2示出了根据本发明实施例的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的红外光谱数据。由图2可知，丝素蛋白和重组人源化胶原蛋白酰胺丝素蛋白在1700cm-1～1600cm-1，1590cm-1～1460cm-1，以及1280cm-1～1190cm-1处存在特征峰，分别对应丝素蛋白和重组人源化胶原蛋白的酰胺I带、酰胺II带以及酰胺III带。Figure 2 shows the infrared spectrum data of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained by the preparation method according to an embodiment of the present invention. As shown in Figure 2, silk fibroin and recombinant humanized collagen amide silk fibroin have characteristic peaks at 1700cm-1 ~1600cm-1 , 1590cm-1 ~1460cm-1 , and 1280cm-1 ~1190cm-1 , corresponding to the amide I band, amide II band and amide III band of silk fibroin and recombinant humanized collagen, respectively.
图3示出了根据本发明实施例的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的粒度数据。由图3可知，再生生丝素蛋白/重组人源化胶原蛋白双网络凝胶的D50：83μm，D90：188μm。Figure 3 shows the particle size data of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained by the preparation method according to an embodiment of the present invention. As shown in Figure 3, the D50 of the regenerated silk fibroin/recombinant humanized collagen double network gel is 83 μm and the D90 is 188 μm.
图4在(25±0.2)℃，采用流变仪在剪切速率从0.1Hz～100Hz下，选行频率扫描绘制黏性模量G”、弹性模量G'和相位角δ与频率f的坐标图。由图4可知，在剪切速率从0.1Hz下，弹性模量G'：≥3000Pa，黏性模量G”：≥250Pa，对于强凝胶，黏性模量G”几乎等于弹性模量G’，凝胶抵抗变形的度量；复模量越高，凝胶的变形越小，结果显示再生丝素蛋白/重组人源化胶原蛋白双网络凝胶具有良好的粘弹性。FIG4 shows a graph of the viscous modulus G”, elastic modulus G’ and phase angle δ versus frequency f at (25±0.2)°C using a rheometer with a shear rate ranging from 0.1 Hz to 100 Hz. As shown in FIG4 , at a shear rate of 0.1 Hz, the elastic modulus G’: ≥3000 Pa, the viscous modulus G”: ≥250 Pa. For strong gels, the viscous modulus G” is almost equal to the elastic modulus G’, which is a measure of the gel’s resistance to deformation. The higher the complex modulus, the smaller the deformation of the gel. The results show that the regenerated silk fibroin/recombinant humanized collagen double network gel has good viscoelasticity.
图5出了本实施例中制备的凝胶成品，在(25士0.2)℃，采用流变仪在剪切速率：0.001s-1～1000s-1进行蠕动扫描，分别绘制剪切应力、粘度η与剪切速率的坐标图。FIG5 shows the finished gel product prepared in this embodiment. The shear stress, viscosity η and shear rate are plotted respectively at (25±0.2)°C and the shear rate is 0.001s-1 to 1000s-1 using a rheometer for creep scanning. Coordinate diagram of .
由图5可知，在剪切速率：0.001s-1～1000s-1下，剪切粘度≥30mPa·s。As shown in FIG5 , at a shear rate of 0.001 s-1 to 1000 s-1 , the shear viscosity is ≥ 30 mPa·s.
实施例2Example 2
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入2.51g可溶性再生丝素蛋白，1.01g重组III型人源化胶原蛋白，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 2.51 g of soluble regenerated silk fibroin and 1.01 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.10g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在3ml预灌封注射器中，附上泡罩盒和封口膜，于32±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.10 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 3 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 32±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图6可知，再生生丝素蛋白/重组人源化胶原蛋白双网络凝胶的D50：88μm，D50：218μm。As shown in FIG6 , the D50 of the regenerated silk fibroin/recombinant humanized collagen double network gel is 88 μm and D50 is 218 μm.
由图7可知，在剪切速率从0.1Hz下，弹性模量G'：≥5000Pa，黏性模量G”：≥250Pa，。It can be seen from Figure 7 that when the shear rate is from 0.1Hz, the elastic modulus G': ≥5000Pa, and the viscous modulus G": ≥250Pa.
由图8可知，在剪切速率：0.001s-1～1000s-1下，剪切粘度≥50mPa·s。As can be seen from FIG8 , at a shear rate of 0.001 s-1 to 1000 s-1 , the shear viscosity is ≥ 50 mPa·s.
实施例3Example 3
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取300ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入3.01g可溶性再生丝素蛋白，1.50g重组III型人源化胶原蛋白，搅拌30min后。1) Take 300 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 3.01 g of soluble regenerated silk fibroin and 1.50 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.31g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在3ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化48±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.31 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 3 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 48±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图9可知，在剪切速率从0.1Hz下，弹性模量G'：≥400Pa，黏性模量G”：≥70Pa，。It can be seen from Figure 9 that when the shear rate is from 0.1Hz, the elastic modulus G': ≥400Pa, and the viscous modulus G": ≥70Pa.
由图10可知，在剪切速率：0.001s-1～1000s-1下，剪切粘度≥200mPa·s。As shown in FIG10 , at a shear rate of 0.001 s-1 to 1000 s-1 , the shear viscosity is ≥ 200 mPa·s.
实施例4Example 4
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.01g可溶性再生丝素蛋白，1.05g重组III型人源化胶原蛋白，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.01 g of soluble regenerated silk fibroin and 1.05 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.12g十二烷基硫酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.12 g of sodium dodecyl sulfate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图11可知，丝素蛋白和重组人源化胶原蛋白酰胺丝素蛋白在1700cm-1～1600cm-1，1590cm-1～1460cm-1，以及1280cm-1～1190cm-1处存在特征峰，分别对应丝素蛋白和重组人源化胶原蛋白的酰胺I带、酰胺II带以及酰胺III带。As shown in Figure 11, silk fibroin and recombinant humanized collagen amide silk fibroin have characteristic peaks at 1700cm- 1-1600cm-1 ,1590cm -1-1460cm-1 , and1280cm -1-1190cm-1 , corresponding to the amide I band, amide II band and amide III band of silk fibroin and recombinant humanized collagen, respectively.
由图12可知，在剪切速率从0.1Hz下，弹性模量G'：≥160Pa，黏性模量G”：≥10Pa。It can be seen from Figure 12 that when the shear rate is from 0.1 Hz, the elastic modulus G': ≥160 Pa, and the viscous modulus G": ≥10 Pa.
实施例5Example 5
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.01g可溶性再生丝素蛋白，1.05g重组III型人源化胶原蛋白，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.01 g of soluble regenerated silk fibroin and 1.05 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.52g十二烷基硫酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.52 g of sodium dodecyl sulfate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图13可知，在剪切速率从0.1Hz下，弹性模量G'：≥80Pa，黏性模量G”：≥9Pa。It can be seen from Figure 13 that when the shear rate is from 0.1 Hz, the elastic modulus G': ≥80 Pa, and the viscous modulus G": ≥9 Pa.
实施例6Example 6
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.52g可溶性再生丝素蛋白，1.56g重组III型人源化胶原蛋白，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.52 g of soluble regenerated silk fibroin and 1.56 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.52g十二烷基硫酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.52 g of sodium dodecyl sulfate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图14可知，在剪切速率从0.1Hz下，弹性模量G'：≥280Pa，黏性模量G”：≥20Pa。It can be seen from Figure 14 that when the shear rate is from 0.1 Hz, the elastic modulus G': ≥280 Pa, and the viscous modulus G": ≥20 Pa.
实施例7Example 7
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.52g可溶性再生丝素蛋白，1.56g重组III型人源化胶原蛋白，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.52 g of soluble regenerated silk fibroin and 1.56 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.52g十二烷基硫酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于32±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.52 g of sodium dodecyl sulfate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 32±1° C. for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图15可知，在剪切速率从0.1Hz下，弹性模量G'：≥140Pa，黏性模量G”：≥20Pa。It can be seen from Figure 15 that when the shear rate is from 0.1 Hz, the elastic modulus G': ≥140 Pa, and the viscous modulus G": ≥20 Pa.
实施例8Example 8
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.01g可溶性再生丝素蛋白，1.06g重组III型人源化胶原蛋白，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.01 g of soluble regenerated silk fibroin and 1.06 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.10g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.10 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图16可知，在剪切速率从0.1Hz下，弹性模量G'：≥330Pa，黏性模量G”：≥20Pa。It can be seen from Figure 16 that when the shear rate is from 0.1 Hz, the elastic modulus G': ≥330 Pa, and the viscous modulus G": ≥20 Pa.
实施例9Example 9
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.01g可溶性再生丝素蛋白，1.06g重组III型人源化胶原蛋白，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.01 g of soluble regenerated silk fibroin and 1.06 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.52g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.52 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图17可知，在剪切速率从0.1Hz下，弹性模量G'：≥240Pa，黏性模量G”：≥20Pa。It can be seen from Figure 17 that when the shear rate is from 0.1 Hz, the elastic modulus G': ≥240 Pa, and the viscous modulus G": ≥20 Pa.
实施例10Example 10
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.51g可溶性再生丝素蛋白，1.52g重组III型人源化胶原蛋白，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.51 g of soluble regenerated silk fibroin and 1.52 g of recombinant type III humanized collagen, and stir for 30 minutes.
2)将0.52g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.52 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图18可知，在剪切速率从0.1Hz下，弹性模量G'：≥690Pa，黏性模量G”：≥39Pa。It can be seen from Figure 18 that when the shear rate is from 0.1 Hz, the elastic modulus G': ≥690 Pa, and the viscous modulus G": ≥39 Pa.
实施例11Embodiment 11
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.51g可溶性再生丝素蛋白，1.52g重组III型人源化胶原蛋白，0.52g甘油，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.51 g of soluble regenerated silk fibroin, 1.52 g of recombinant type III humanized collagen, and 0.52 g of glycerol, and stir for 30 minutes.
2)将0.51g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.51 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图19可知，在剪切速率从0.1Hz下，弹性模量G'：≥800Pa，黏性模量G”：≥50Pa。It can be seen from Figure 19 that when the shear rate is 0.1 Hz, the elastic modulus G': ≥800 Pa, and the viscous modulus G": ≥50 Pa.
实施例12Example 12
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.51g可溶性再生丝素蛋白，1.52g重组III型人源化胶原蛋白，0.52g丁二醇，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.51 g of soluble regenerated silk fibroin, 1.52 g of recombinant type III humanized collagen, and 0.52 g of butanediol, and stir for 30 minutes.
2)将0.51g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于37±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.51 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 37±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图20可知，在剪切速率从0.1Hz下，弹性模量G'：≥600Pa，黏性模量G”：≥30Pa。It can be seen from Figure 20 that when the shear rate is 0.1 Hz, the elastic modulus G': ≥600 Pa, and the viscous modulus G": ≥30 Pa.
实施例13Example 13
本发明实施例提供了一种再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a regenerated silk fibroin/recombinant humanized collagen double network gel, and the preparation method comprises the following steps:
1)取100ml配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入1.51g可溶性再生丝素蛋白，1.52g重组III型人源化胶原蛋白，0.52g丁二醇，搅拌30min后。1) Take 100 ml of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 1.51 g of soluble regenerated silk fibroin, 1.52 g of recombinant type III humanized collagen, and 0.52 g of butanediol, and stir for 30 minutes.
2)将0.51g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在1ml预灌封注射器中，附上泡罩盒和封口膜，于32±1℃孵化24±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 0.51 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 1 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 32±1°C for 24±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
由图21可知，在剪切速率从0.1Hz下，弹性模量G'：≥200Pa，黏性模量G”：≥19Pa。It can be seen from Figure 21 that when the shear rate is 0.1 Hz, the elastic modulus G': ≥200 Pa, and the viscous modulus G": ≥19 Pa.
实施例14Embodiment 14
本发明实施例提供了一种无菌的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶的制备方法，该制备方法包括如下步骤：The embodiment of the present invention provides a method for preparing a sterile regenerated silk fibroin/recombinant humanized collagen double network gel, the preparation method comprising the following steps:
1)取1L配制好的0.01M pH 7.4的PBS磷酸缓冲液，加入10.21g可溶性再生丝素蛋白，10.12g重组III型人源化胶原蛋白，0.51g甘油，搅拌30min后。1) Take 1 L of prepared 0.01 M pH 7.4 PBS phosphate buffer, add 10.21 g of soluble regenerated silk fibroin, 10.12 g of recombinant type III humanized collagen, and 0.51 g of glycerol, and stir for 30 minutes.
2)将1.01g油酸钠加入到上述溶液中，过0.45μm和0.22μm滤膜，滤液灌装在3ml预灌封注射器中，附上泡罩盒和封口膜，于32±1℃孵化36±1h，得到再生丝素蛋白/重组人源化胶原蛋白双网络凝胶。2) Add 1.01 g of sodium oleate to the above solution, filter through 0.45 μm and 0.22 μm filter membranes, fill the filtrate into a 3 ml prefilled syringe, attach a blister box and a sealing film, and incubate at 32±1°C for 36±1 h to obtain a regenerated silk fibroin/recombinant humanized collagen double network gel.
取凝胶样品适量，按照YY/T 0471.1-2014中3.6弥散特性的方法检测，试验结果：产品为不可溶，可弥散。Take an appropriate amount of gel sample and test it according to the method of 3.6 Dispersion Characteristics in YY/T 0471.1-2014. The test result is: the product is insoluble but dispersible.
取凝胶样品适量，试验温度25±0.1℃，采用粘度仪(NDJ-5SC)或同等设备，按《中华人民共和国药典》(2020年版四部)0633粘度测量方法中旋转法的方法测定，试验结果：产品的动力黏度应在≥10000mPa·s。Take an appropriate amount of gel sample, test temperature 25±0.1℃, use viscometer (NDJ-5SC) or equivalent equipment, and measure according to the rotation method in 0633 Viscosity Measurement Method of "Pharmacopoeia of the People's Republic of China" (Volume 4 of 2020 Edition). Test results: The dynamic viscosity of the product should be ≥10000mPa·s.
取样品适量，按照GB/T 16886.5-2017《医疗器械生物学评价第5部分：体外细胞毒性试验》规定的浸提液法(MTT法)进行，浸提介质为细胞培养液。结果显示该凝胶在MTT细胞毒性试验条件下无细胞毒性。Take an appropriate amount of sample and perform the extraction method (MTT method) specified in GB/T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test", with the extraction medium being cell culture medium. The results showed that the gel had no cytotoxicity under the conditions of the MTT cytotoxicity test.
取样品适量，按照GB/T 16886.10-2017《医疗器械生物学评价第10部分：刺激与皮肤致敏试验》推荐的皮肤致敏试验(最大剂量法)的要求，对试验样品在试验条件下使豚鼠产生皮肤致敏反应的潜能做出评定。结果显示试验样品未引起皮肤致敏反应，阳性激发结果的发生率为0％。Take an appropriate amount of sample and evaluate the potential of the test sample to cause skin sensitization reaction in guinea pigs under the test conditions according to the skin sensitization test (maximum dose method) recommended in GB/T 16886.10-2017 "Biological Evaluation of Medical Devices Part 10: Irritation and Skin Sensitization Test". The results showed that the test sample did not cause skin sensitization reaction, and the incidence of positive stimulation results was 0%.
取样品适量，按照GB/T 16886.10-2017《医疗器械生物学评价第10部分：刺激与皮肤致敏试验》》中推荐的皮内反应的要求对样品浸提液进行生物相容性试验。试验样品采用极性浸提液(0.9％氯化钠注射液)和非极性浸提液(棉籽油)浸提，所得浸提液通过皮内注射途径给予新西兰白化兔，对材料在试验条件下产生的刺激反应的潜能做出评定。结果显示试验样品极性浸提液皮内反应最终记分为0.00；非极性浸提液皮内反应最终记分为0.00，最终结果显示皮内反应良好。Take an appropriate amount of sample and conduct biocompatibility test on the sample extract according to the requirements of intradermal reaction recommended in GB/T 16886.10-2017 "Biological Evaluation of Medical Devices Part 10: Irritation and Skin Sensitization Test". The test samples were extracted with polar extract (0.9% sodium chloride injection) and non-polar extract (cottonseed oil). The obtained extract was given to New Zealand albino rabbits by intradermal injection to evaluate the potential of the material to produce irritation under the test conditions. The results showed that the final score of intradermal reaction of the polar extract of the test sample was 0.00; the final score of intradermal reaction of the non-polar extract was 0.00, and the final result showed that the intradermal reaction was good.
取样品适量，按照GB/T 16886.11-2021《医疗器械生物学评价第11部分：全身毒性试验》中推荐的方法进行试验。直接使用样品作为试验液，对小鼠进行皮下注射，结果显示试验样品对小鼠无急性全身毒性反应。Take an appropriate amount of sample and conduct the test according to the method recommended in GB/T 16886.11-2021 "Biological Evaluation of Medical Devices Part 11: Systemic Toxicity Test". Use the sample directly as the test solution and inject it subcutaneously into mice. The results show that the test sample has no acute systemic toxicity reaction to mice.
取样品适量，按照GB/T 16886.11-2021《医疗器械生物学评价第11部分：全身毒性试验》中推荐的方法进行试验。采用0.9％氯化钠注射液对试验样品进行浸提，取浸提上清液对新西兰兔进行耳缘静脉注射，结果显示试验样品凝胶的试验液热原检查符合规定。Take an appropriate amount of sample and conduct the test according to the method recommended in GB/T 16886.11-2021 "Biological Evaluation of Medical Devices Part 11: Systemic Toxicity Test". The test sample was extracted with 0.9% sodium chloride injection, and the supernatant was injected into the ear vein of New Zealand rabbits. The results showed that the test solution pyrogen test of the test sample gel met the requirements.
由图22可知，在剪切速率从0.1Hz下，弹性模量G'：≥700Pa，黏性模量G”：≥50Pa。It can be seen from Figure 22 that when the shear rate is 0.1 Hz, the elastic modulus G': ≥700 Pa, and the viscous modulus G": ≥50 Pa.
由图23可知，在剪切速率：0.001s-1～1000s-1下，剪切粘度≥20mPa·s。As can be seen from FIG. 23 , at a shear rate of 0.001 s-1 to 1000 s-1 , the shear viscosity is ≥ 20 mPa·s.
由图24可知，再生生丝素蛋白/重组人源化胶原蛋白双网络凝胶的D50：54μm，D90：116μm。As shown in FIG. 24 , the D50 of the regenerated silk fibroin/recombinant humanized collagen double network gel is 54 μm and the D90 is 116 μm.
图25示出了根据本发明实施例的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下，在第3个月的解刨图片。由图25可知，双网络凝胶植入组织3个月后没有完成降解，注射部位仍有一定体积的凝胶存在，证明了可注射凝胶不但具有长周期组织填充效果。Figure 25 shows a dissection picture of the regenerated silk fibroin/recombinant humanized collagen double network gel obtained by the preparation method of an embodiment of the present invention injected into the subcutaneous tissue of a rat at the third month. As shown in Figure 25, the double network gel has not been completely degraded after being implanted in the tissue for 3 months, and a certain volume of gel still exists at the injection site, proving that the injectable gel not only has a long-term tissue filling effect.
图26示出了根据本发明实施例的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下，在第3个月组织切片的苏木精-伊红(hematoxylin-eosinstaining，H&E)染色照片。由图26可知，大鼠的皮下注射组织及其周边细胞状态良好，注射3个月后，组织部位无明显细胞萎缩现象。Figure 26 shows a hematoxylin-eosin (H&E) staining photograph of tissue sections of rats injected subcutaneously with the regenerated silk fibroin/recombinant humanized collagen double network gel prepared according to the preparation method of an embodiment of the present invention at the third month. As shown in Figure 26, the subcutaneous injected tissue of the rat and its surrounding cells are in good condition, and there is no obvious cell atrophy in the tissue site after 3 months of injection.
图27示出了根据本发明实施例的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下，在第3个月组织切片的胶原纤维马森(Masson)染色照片。由图27可知，注射3个月后，大鼠的皮下注射组织及其周边胶原增生效果显著，组织切片染色呈现深蓝色(由于附图为灰度图，因此无法看出深蓝色，但是实际效果图中，深蓝色是遍布在除图中圆形细胞外的其他区域中)，说明再生丝素蛋白/重组人源化胶原蛋白双网络凝胶具有加速组织自体胶原蛋白分泌，刺激组织再生的有益效果。Figure 27 shows a Masson staining photo of collagen fibers in tissue sections of rats injected subcutaneously with a double network gel of regenerated silk fibroin/recombinant humanized collagen obtained by the preparation method of an embodiment of the present invention. As shown in Figure 27, 3 months after the injection, the collagen proliferation effect of the subcutaneous injected tissue and its surrounding areas of the rats was significant, and the tissue sections were stained dark blue (because the attached figure is a grayscale image, the dark blue cannot be seen, but in the actual effect diagram, the dark blue is distributed in other areas except the circular cells in the figure), indicating that the double network gel of regenerated silk fibroin/recombinant humanized collagen has the beneficial effect of accelerating the secretion of autologous collagen in the tissue and stimulating tissue regeneration.
图28示出了根据本发明实施例的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下，在第3个月组织切片的CD31(CD31又称为血小板-内皮细胞黏附分子(Platelet endothelial cell adhesion molecule-1，PECAM-1/CD31)染色照片。由图28可知，注射3个月后，大鼠的皮下注射组织及其周边皮内细胞增殖显著，组织切片标记呈现深蓝色(由于附图为灰度图，因此无法看出深蓝色，但是实际效果图中，深蓝色是遍布在除图中圆形细胞外的其他区域中)，说明再生丝素蛋白/重组人源化胶原蛋白双网络凝胶具有加速皮内细胞增殖，刺激皮内细胞迁移。FIG28 shows a CD31 (CD31 is also known as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) staining photograph of tissue sections at the third month after the regenerated silk fibroin/recombinant humanized collagen double network gel obtained by the preparation method of an embodiment of the present invention was injected subcutaneously into rats. As shown in FIG28 , three months after the injection, the subcutaneous injected tissue of the rats and its surrounding intradermal cells proliferated significantly, and the tissue section markings appeared dark blue (since the attached figure is a grayscale image, the dark blue cannot be seen, but in the actual effect figure, the dark blue is distributed in other areas except the circular cells in the figure), indicating that the regenerated silk fibroin/recombinant humanized collagen double network gel has the ability to accelerate the proliferation of intradermal cells and stimulate the migration of intradermal cells.
图29示出了根据本发明实施例的制备方法获得的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶注射到大鼠皮下，在第3个月组织切片的α-SMA(平滑肌肌动蛋白)染色照片。由图29可知，注射3个月后，大鼠的皮下注射组织及其周边肌上皮细胞增殖显著，组织切片标记呈现深蓝色(由于附图为灰度图，因此无法看出深蓝色，但是实际效果图中，深蓝色是遍布在除图中圆形细胞外的其他区域中)，说明再生丝素蛋白/重组人源化胶原蛋白双网络凝胶具有加速肌上皮细胞增殖，刺激肌上细胞迁移。Figure 29 shows the α-SMA (smooth muscle actin) staining photos of tissue sections at the third month after the regenerated silk fibroin/recombinant humanized collagen double network gel obtained by the preparation method of an embodiment of the present invention was injected into the subcutaneous part of rats. As shown in Figure 29, 3 months after the injection, the subcutaneous injection tissue of the rat and its surrounding myoepithelial cells proliferated significantly, and the tissue section marks showed dark blue (because the attached figure is a grayscale image, the dark blue cannot be seen, but in the actual effect figure, the dark blue is distributed in other areas except the circular cells in the figure), indicating that the regenerated silk fibroin/recombinant humanized collagen double network gel has the ability to accelerate the proliferation of myoepithelial cells and stimulate the migration of myoepithelial cells.
实施例1-3，4-7列举了两种表面活性剂：十二烷基硫酸钠和油酸钠，对比制备的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，油酸钠相比十二烷基硫酸钠诱导的凝胶粘弹性数据较好，另外油酸钠相比十二烷基硫酸钠生物安全性数据较好，故本发明优选油酸钠作为发明的表面活性剂。Examples 1-3 and 4-7 list two surfactants: sodium dodecyl sulfate and sodium oleate. Comparing the prepared regenerated silk fibroin/recombinant humanized collagen double network gel, sodium oleate has better gel viscoelasticity data than sodium dodecyl sulfate. In addition, sodium oleate has better biosafety data than sodium dodecyl sulfate. Therefore, the present invention prefers sodium oleate as the surfactant of the invention.
实施例4-5和8-9，筛选了两种表面活性剂用量对再生丝素蛋白/重组人源化胶原蛋白双网络凝胶制备的影响，表面活性剂用量为1mg/ml和5mg/ml，表面活性剂用量少时，制备的凝胶粘弹性数据较好，表面活性剂用量越小，对于皮肤组织刺激性越友好，故本发明中表面活性剂用量优选1mg/ml。Examples 4-5 and 8-9 screened the effects of two surfactant dosages on the preparation of regenerated silk fibroin/recombinant humanized collagen double network gels. The surfactant dosages were 1 mg/ml and 5 mg/ml. When the surfactant dosage was small, the viscoelastic data of the prepared gel was better. The smaller the surfactant dosage, the more friendly it is to skin tissue irritation. Therefore, the surfactant dosage in the present invention is preferably 1 mg/ml.
实施例10～12，筛选润湿剂：丁二醇和甘油对再生丝素蛋白/重组人源化胶原蛋白双网络凝胶制备的影响，加入丁二醇或甘油相比不加润湿剂，制备的凝胶粘弹性数据较好。另外，润湿剂对皮肤组织相容性较好，对提高保湿具有更好的优势，故本发明优选添加润湿剂。Examples 10-12, screening of wetting agents: the effects of butylene glycol and glycerol on the preparation of regenerated silk fibroin/recombinant humanized collagen double network gels. Compared with no wetting agent, the viscoelastic data of the prepared gels with the addition of butylene glycol or glycerol are better. In addition, the wetting agent has better compatibility with skin tissues and has a better advantage in improving moisturizing, so the present invention preferably adds a wetting agent.
实施例11-12，筛选润湿剂种类对再生丝素蛋白/重组人源化胶原蛋白双网络凝胶制备的影响，加入甘油相比丁二醇，制备的备的凝胶粘弹性数据较好，故本发明优选甘油作为润湿剂。In Example 11-12, the effect of the type of wetting agent on the preparation of regenerated silk fibroin/recombinant humanized collagen double network gel was screened. Compared with butanediol, the viscoelastic data of the prepared gel was better when glycerol was added. Therefore, glycerol is preferably used as the wetting agent in the present invention.
实施例6-7和12-13，筛选了再生丝素蛋白/重组人源化胶原蛋白双网络凝胶制备时温度的选择，成胶温度选择32±1℃和37±1℃，成胶温度越高，制备的凝胶粘弹性数据较好，但是重组人源化胶原蛋白不耐热，故本发明中成胶温度选择32±1℃。In Examples 6-7 and 12-13, the selection of temperature for the preparation of regenerated silk fibroin/recombinant humanized collagen double network gel was screened. The gelation temperatures were 32±1°C and 37±1°C. The higher the gelation temperature, the better the viscoelastic data of the prepared gel. However, recombinant humanized collagen is not heat-resistant, so the gelation temperature in the present invention is selected to be 32±1°C.
实施例3、11、14，筛选了再生丝素蛋白/重组人源化胶原蛋白双网络凝胶制备时成胶时间的选择，选择24±1h、36±1h，48±1h，成胶时间大于24±1h，满足凝胶制备的要求，故本发明中成胶时间选择≥24±1h。In Examples 3, 11, and 14, the gelation time for preparing the regenerated silk fibroin/recombinant humanized collagen double network gel was screened, and 24±1h, 36±1h, and 48±1h were selected. The gelation time was greater than 24±1h, which met the requirements for gel preparation. Therefore, the gelation time in the present invention was selected to be ≥24±1h.
实施例1-2和8，筛选了再生丝素蛋白用量对再生丝素蛋白/重组人源化胶原蛋白双网络凝胶制备的影响，再生丝素蛋白用量选择10～25mg/ml时，再生丝素蛋白用量越高，制备的凝胶粘弹性数据较好，另外，再生丝素蛋白用量为10mg/ml，亦可满足对成胶的要求，故本发明中再生丝素蛋白用量可以选择10～25mg/ml。Examples 1-2 and 8 screened the effect of the amount of regenerated silk fibroin on the preparation of regenerated silk fibroin/recombinant humanized collagen double network gel. When the amount of regenerated silk fibroin was selected to be 10-25 mg/ml, the higher the amount of regenerated silk fibroin was, the better the viscoelastic data of the prepared gel was. In addition, the amount of regenerated silk fibroin was 10 mg/ml, which could also meet the requirements for gelation. Therefore, the amount of regenerated silk fibroin in the present invention can be selected to be 10-25 mg/ml.
实施例3和8，筛选了重组人源化胶原蛋白用量对再生丝素蛋白/重组人源化胶原蛋白双网络凝胶制备的影响，重组人源化胶原蛋白用量选择10～15mg/ml时，重组人源化胶原蛋白用量越高，制备的凝胶粘弹性数据较好，而重组人源化胶原蛋白用量选择产品的临床适用性相关，故本发明中重组人源化胶原蛋白用量可以选择10～15mg/ml。In Examples 3 and 8, the effect of the amount of recombinant humanized collagen on the preparation of regenerated silk fibroin/recombinant humanized collagen double network gel was screened. When the amount of recombinant humanized collagen was selected to be 10-15 mg/ml, the higher the amount of recombinant humanized collagen, the better the viscoelastic data of the prepared gel. The amount of recombinant humanized collagen selected is related to the clinical applicability of the product. Therefore, the amount of recombinant humanized collagen in the present invention can be selected to be 10-15 mg/ml.
实施例1-14，列举多个试验条件，其中所述0.01M PBS磷酸缓冲液pH为7.2～7.4，均满足对再生丝素蛋白/重组人源化胶原蛋白双网络凝胶制备的要求。其中实施例14，采用最优的试验方案：再生丝素蛋白用量为10mg/ml，重组人源化胶原蛋白用量10mg/ml，油酸钠：1mg/ml，甘油：5mg/ml，成胶温度：32±1℃，成胶时间：36±1h，制备得到的再生丝素蛋白/重组人源化胶原蛋白双网络凝胶，测试结果中，动力黏度较高，短期的生物安全性较好，粘弹性数据较好，凝胶的粒度大小适中。另外，动物试验数据中，表现出再生丝素蛋白/重组人源化胶原蛋白双网络凝胶具有加速组织自体胶原蛋白分泌，促进细胞增殖，刺激细胞迁移，刺激组织再生的功能。Examples 1-14 list multiple test conditions, wherein the pH of the 0.01M PBS phosphate buffer is 7.2-7.4, which all meet the requirements for the preparation of the regenerated silk fibroin/recombinant humanized collagen double network gel. Among them, Example 14 adopts the optimal test scheme: the amount of regenerated silk fibroin is 10 mg/ml, the amount of recombinant humanized collagen is 10 mg/ml, sodium oleate: 1 mg/ml, glycerol: 5 mg/ml, gelation temperature: 32±1°C, gelation time: 36±1h, and the prepared regenerated silk fibroin/recombinant humanized collagen double network gel has a high dynamic viscosity, good short-term biosafety, good viscoelasticity data, and a moderate particle size of the gel. In addition, the animal test data show that the regenerated silk fibroin/recombinant humanized collagen double network gel has the function of accelerating the secretion of autologous collagen in the tissue, promoting cell proliferation, stimulating cell migration, and stimulating tissue regeneration.
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