CN116948207A - Modified collagen and its preparation method, collagen composite material and its application - Google Patents
Modified collagen and its preparation method, collagen composite material and its application Download PDFInfo
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- CN116948207A CN116948207A CN202210417050.4A CN202210417050A CN116948207A CN 116948207 A CN116948207 A CN 116948207A CN 202210417050 A CN202210417050 A CN 202210417050A CN 116948207 A CN116948207 A CN 116948207A
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Abstract
本发明涉及生物打印技术领域，公开了一种改性胶原蛋白及其制备方法、胶原蛋白复合材料及其应用。所述改性胶原蛋白具有三股螺旋结构，并且在所述改性胶原蛋白的至少部分氨基上连接有功能基团；其中，所述功能基团选自如下所示的一价残基中的一种或多种，式中，R1、R2、R3和R4各自独立地为H、甲基、乙基或丙基。本发明的改性胶原蛋白在生物打印后也能维持胶原蛋白的结构和功能。
The invention relates to the technical field of bioprinting and discloses a modified collagen and its preparation method, collagen composite material and its application. The modified collagen has a triple helix structure, and functional groups are connected to at least part of the amino groups of the modified collagen; wherein the functional group is selected from one of the monovalent residues shown below One or more, in the formula, R 1 , R 2 , R 3 and R 4 are each independently H, methyl, ethyl or propyl. The modified collagen of the present invention can also maintain the structure and function of collagen after bioprinting.
Description
技术领域Technical field
本发明涉及高分子化学、材料化学、增材制造等技术领域，具体涉及一种改性胶原蛋白及其制备方法、胶原蛋白复合材料及其应用、以及生物材料的制备方法。The invention relates to the technical fields of polymer chemistry, material chemistry, additive manufacturing and other technical fields, and specifically relates to a modified collagen and its preparation method, collagen composite material and its application, and a preparation method of biological materials.
背景技术Background technique
胶原蛋白是哺乳动物中含量最丰富、分布最广的功能性蛋白质，其具有低免疫原性、优良的生物活性、生物降解性和凝固性。同时作为细胞外基质的主要结构蛋白，它可以为细胞增殖和生长提供合适的微环境，提供机械强度以形成结构组织，并充当细胞粘附和信号分子，是一种非常理想的3D打印生物材料。然而，由于极性基团和非极性基团之间的强分子间和分子内氢键、离子键、范德华力和疏水键，胶原蛋白极难溶解，这极大限制了胶原蛋白在许多领域的应用。尤其是在3D打印中，由中性胶原蛋白难以控制热驱动自组装的凝胶化导致其出丝不顺。同时，胶原蛋白凝胶在缓慢的凝胶化过程中容易收缩，导致印刷结构坍塌。因此，很难使用天然未改性胶原蛋白进行复杂支架的3D生物打印。Collagen is the most abundant and widely distributed functional protein in mammals. It has low immunogenicity, excellent biological activity, biodegradability and coagulation properties. At the same time, as the main structural protein of the extracellular matrix, it can provide a suitable microenvironment for cell proliferation and growth, provide mechanical strength to form structural tissues, and act as cell adhesion and signaling molecules. It is a very ideal 3D printing biomaterial. . However, collagen is extremely difficult to dissolve due to strong intermolecular and intramolecular hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic bonds between polar and nonpolar groups, which greatly limits the use of collagen in many fields. Applications. Especially in 3D printing, it is difficult to control the gelation of heat-driven self-assembly of neutral collagen, resulting in its smooth silk production. At the same time, collagen gels tend to shrink during the slow gelation process, causing the printed structure to collapse. Therefore, it is difficult to use natural unmodified collagen for 3D bioprinting of complex scaffolds.
胶原蛋白在生物3D打印中的独特优势和潜力为实现胶原蛋白3D打印开发了对应的策略，但都是基于在酸性和低温下溶解胶原蛋白而进行3D生物打印。这种缓慢、不均匀且不利于环境的处理方法难以快速稳定地获得胶原基3D打印生物墨水。因此，开发一种快速、稳定的胶原溶解后改性方法实现胶原基生物墨水的3D生物打印具有重要意义。The unique advantages and potential of collagen in bio-3D printing have led to the development of corresponding strategies to achieve collagen 3D printing, but they are all based on dissolving collagen in acidic and low temperature for 3D bioprinting. This slow, uneven and environmentally unfriendly processing method makes it difficult to quickly and stably obtain collagen-based 3D printing bioinks. Therefore, it is of great significance to develop a fast and stable post-dissolution collagen modification method to achieve 3D bioprinting of collagen-based bioinks.
离子液体(IL)是一种在相对较低的温度下以液态存在的盐，被认为是一种绿色溶剂，具有许多优异的性能。因此，离子液体有望成为胶原溶解和改性的理想溶剂。虽然以往的研究已经证明，许多离子液体都能很好地溶解胶原蛋白，但用于经离子液体溶解的胶原蛋白保持胶原蛋白的特征结构并用于3D生物打印的研究仍属空白。因此，在胶原3D生物打印中，通过筛选离子液体的种类和胶原的溶解温度来维持胶原的结构特征，获得一种新型的胶原溶解改性策略具有重要意义。Ionic liquid (IL) is a salt that exists in a liquid state at relatively low temperatures and is considered a green solvent with many excellent properties. Therefore, ionic liquids are expected to become ideal solvents for collagen dissolution and modification. Although previous studies have proven that many ionic liquids can dissolve collagen well, research on how collagen dissolved by ionic liquids can maintain the characteristic structure of collagen and be used for 3D bioprinting is still blank. Therefore, in collagen 3D bioprinting, it is of great significance to obtain a new collagen dissolution modification strategy by screening the type of ionic liquid and the dissolution temperature of collagen to maintain the structural characteristics of collagen.
发明内容Contents of the invention
本发明的目的是为了克服现有技术存在的胶原蛋白在3D生物打印中难以维持其结构和功能的问题，提供一种改性胶原蛋白及其制备方法、胶原蛋白复合材料及其应用、以及生物材料的制备方法，该改性胶原蛋白在生物打印后也能维持胶原蛋白的结构和功能。The purpose of the present invention is to overcome the problem in the prior art that collagen is difficult to maintain its structure and function in 3D bioprinting, and to provide a modified collagen and its preparation method, collagen composite materials and their applications, and biological According to the preparation method of the material, the modified collagen can also maintain the structure and function of collagen after bioprinting.
针对上述技术问题，本发明的发明人经过深入研究发现，通过离子液体对胶原蛋白进行光交联基团的功能化修饰，可获得修饰率可控，结构保持完好的改性胶原蛋白，将该改性固化胶原蛋白有效分散在PBS中，可以获得3D打印性质优异，生物活性好的胶原蛋白生物打印材料，可用于打印具有正常功能的组织模型，例如具有正常分泌白蛋白和药物代谢功能的血管化肝组织。In view of the above technical problems, the inventor of the present invention found through in-depth research that functional modification of collagen with photo-crosslinking groups through ionic liquids can obtain modified collagen with controllable modification rate and intact structure. Modified solidified collagen is effectively dispersed in PBS to obtain 3D printing collagen bioprinting materials with excellent properties and good bioactivity, which can be used to print tissue models with normal functions, such as blood vessels with normal albumin secretion and drug metabolism functions. Transform liver tissue.
为了实现上述目的，本发明一方面提供一种改性胶原蛋白，所述改性胶原蛋白具有三股螺旋结构，并且在所述改性胶原蛋白的至少部分氨基上连接有功能基团；In order to achieve the above object, on one hand, the present invention provides a modified collagen, the modified collagen has a triple helix structure, and functional groups are connected to at least part of the amino groups of the modified collagen;
其中，所述功能基团选自如下所示的一价残基中的一种或多种，Wherein, the functional group is selected from one or more of the monovalent residues shown below,
式中，R1、R2、R3和R4各自独立地为H、甲基、乙基或丙基，优选地，R1、R2、R3和R4各自独立地为H或甲基；In the formula, R 1 , R 2 , R 3 and R 4 are each independently H, methyl, ethyl or propyl. Preferably, R 1 , R 2 , R 3 and R 4 are each independently H or methane. base;
优选地，相对于1g所述改性胶原蛋白，所述功能基团的含量为0.006-0.025μmol。Preferably, the content of the functional group is 0.006-0.025 μmol relative to 1 g of the modified collagen.
优选地，所述功能基团通过在咪唑型离子液体的存在下使胶原蛋白上的氨基与酰胺化试剂反应而形成。Preferably, the functional group is formed by reacting an amino group on collagen with an amidation reagent in the presence of an imidazole-type ionic liquid.
优选地，所述酰胺化试剂为甲基丙烯酸酐、降冰片烯二酸酐、具有式(4)所示结构的香豆素-3-活性酯中的一种或多种。Preferably, the amidation reagent is one or more of methacrylic anhydride, norbornenedioic anhydride, and coumarin-3-active ester having the structure shown in formula (4).
式(4)中，X为 In formula (4), X is
优选地，所述咪唑型离子液体为1-甲基-3-正辛基咪唑溴化物、1-已基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑硝酸盐、1-丁基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑啉双(三氟甲基磺酰基)亚胺、1-丁基-3-甲基咪唑氯盐、1-丁基-3-甲基咪唑六氟磷酸盐、1-丁基-3-甲基咪唑氢溴酸盐、1-乙基-3-甲基氯化咪唑鎓、1-烯丙基-3-甲基氯化咪唑中的一种或多种。Preferably, the imidazole ionic liquid is 1-methyl-3-n-octylimidazole bromide, 1-hexyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazole Nitrate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, 1-butyl-3-methyl 1-Butyl-3-methylimidazole chloride, 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hydrobromide, 1-ethyl-3-methylimidazolium chloride, 1 -One or more types of allyl-3-methylimidazole chloride.
本发明第二方面提供一种改性胶原蛋白的制备方法，该方法包括：在咪唑型离子液体的存在下，将胶原蛋白与酰胺化试剂接触，使得胶原蛋白的至少部分氨基进行酰胺化反应从而形成连接在氨基上的功能基团；A second aspect of the present invention provides a method for preparing modified collagen. The method includes: contacting collagen with an amidation reagent in the presence of an imidazole ionic liquid, so that at least part of the amino groups of the collagen undergo an amidation reaction. Form a functional group attached to the amino group;
其中，所述功能基团选自如下所示的一价残基中的一种或多种，Wherein, the functional group is selected from one or more of the monovalent residues shown below,
式中，R1、R2、R3和R4各自独立地为H、甲基、乙基或丙基，优选地，R1、R2、R3和R4各自独立地为H或甲基。In the formula, R 1 , R 2 , R 3 and R 4 are each independently H, methyl, ethyl or propyl. Preferably, R 1 , R 2 , R 3 and R 4 are each independently H or methane. base.
优选地，所述酰胺化反应的温度为40-120℃，优选地，所述酰胺化反应的时间为0.5-6h。Preferably, the temperature of the amidation reaction is 40-120°C, and preferably, the time of the amidation reaction is 0.5-6 h.
优选地，所述胶原蛋白与所述酰胺化试剂的重量比为1：0.01-0.5，优选为1：0.05-0.4。Preferably, the weight ratio of the collagen to the amidation reagent is 1:0.01-0.5, preferably 1:0.05-0.4.
优选地，所述胶原蛋白与所述咪唑型离子液体的重量比为1：32-1600。Preferably, the weight ratio of the collagen to the imidazole ionic liquid is 1:32-1600.
优选地，所述咪唑型离子液体为1-甲基-3-正辛基咪唑溴化物、1-已基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑硝酸盐、1-丁基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑啉双(三氟甲基磺酰基)亚胺、1-丁基-3-甲基咪唑氯盐、1-丁基-3-甲基咪唑六氟磷酸盐、1-丁基-3-甲基咪唑氢溴酸盐、1-乙基-3-甲基氯化咪唑鎓、1-烯丙基-3-甲基氯化咪唑中的一种或多种。Preferably, the imidazole ionic liquid is 1-methyl-3-n-octylimidazole bromide, 1-hexyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazole Nitrate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, 1-butyl-3-methyl 1-Butyl-3-methylimidazole chloride, 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hydrobromide, 1-ethyl-3-methylimidazolium chloride, 1 -One or more types of allyl-3-methylimidazole chloride.
优选地，所述酰胺化试剂为甲基丙烯酸酐、降冰片烯二酸酐、具有式(4)所示结构的香豆素-3-活性酯中的一种或多种，Preferably, the amidation reagent is one or more of methacrylic anhydride, norbornenedioic anhydride, and coumarin-3-active ester having the structure shown in formula (4),
式(4)中，X为 In formula (4), X is
优选地，该方法包括：先将胶原蛋白溶于咪唑型离子液体，然后再加入酰胺化试剂进行酰胺化反应。Preferably, the method includes: first dissolving collagen in an imidazole ionic liquid, and then adding an amidation reagent to perform an amidation reaction.
本发明第三方面提供一种胶原蛋白复合材料，该胶原蛋白复合材料包括：改性胶原蛋白、光引发剂和可选的光交联剂，该改性胶原蛋白为上述本发明的改性胶原蛋白或者由上述本发明的制备方法得到的改性胶原蛋白。The third aspect of the present invention provides a collagen composite material. The collagen composite material includes: modified collagen, a photoinitiator and an optional photocrosslinking agent. The modified collagen is the above-mentioned modified collagen of the present invention. Protein or modified collagen obtained by the above preparation method of the present invention.
本发明第四方面提供一种胶原蛋白复合材料，该胶原蛋白复合材料包括：改性胶原蛋白、光引发剂、细胞、缓冲液和可选的光交联剂，该改性胶原蛋白为上述本发明的改性胶原蛋白或者由上述本发明的制备方法得到的改性胶原蛋白。The fourth aspect of the present invention provides a collagen composite material. The collagen composite material includes: modified collagen, a photoinitiator, cells, a buffer and an optional photo-crosslinking agent. The modified collagen is the above-mentioned present invention. The modified collagen of the invention or the modified collagen obtained by the above-mentioned preparation method of the invention.
优选地，所述细胞为内皮细胞、间充质干细胞、胚胎干细胞、多能诱导干细胞、肝细胞、成纤维细胞、肝癌细胞、脑胶质瘤细胞、肾癌细胞中的一种或多种。Preferably, the cells are one or more of endothelial cells, mesenchymal stem cells, embryonic stem cells, pluripotent induced stem cells, hepatocytes, fibroblasts, liver cancer cells, glioma cells, and renal cancer cells.
在上述本发明第三或者第四方面的胶原蛋白复合材料中，优选地，所述胶原蛋白复合材料中的胶原蛋白的质量分数为1-5％。In the above-mentioned collagen composite material of the third or fourth aspect of the present invention, preferably, the mass fraction of collagen in the collagen composite material is 1-5%.
优选地，所述光引发剂为苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐、2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮、2-甲基-2-(4-吗啉基)-1-[4-(甲硫基)苯基]-1-丙酮、双(2,4,6-三甲基苯甲酰基)苯基氧化膦中的一种或多种。Preferably, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy )phenyl]-1-propanone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, bis(2,4,6- One or more of trimethylbenzoyl)phenylphosphine oxide.
优选地，所述胶原蛋白复合材料中的所述光引发剂的质量分数为0.02-0.5％，优选为0.04-0.2％。Preferably, the mass fraction of the photoinitiator in the collagen composite material is 0.02-0.5%, preferably 0.04-0.2%.
优选地，所述光交联剂为含有至少两个巯基的光交联剂，更优选为二硫苏糖醇、双巯基聚乙二醇、巯基化透明质酸、四(3-巯基丙酸)季戊四醇酯、内消旋-2,3-二巯基丁二酸、二(巯基乙酸)-1,4-丁二酯中的一种或多种。Preferably, the photo-cross-linking agent is a photo-cross-linking agent containing at least two mercapto groups, more preferably dithiothreitol, bis-mercapto polyethylene glycol, mercapto hyaluronic acid, tetrakis(3-mercaptopropionic acid) ) Pentaerythritol ester, meso-2,3-dimercaptosuccinic acid, or one or more of di(mercaptoacetic acid)-1,4-butanedioic acid.
优选地，所述胶原蛋白复合材料中的所述光交联剂的质量分数为0.1-1％。Preferably, the mass fraction of the photo-crosslinking agent in the collagen composite material is 0.1-1%.
本发明第五方面提供上述本发明的改性胶原蛋白、由上述本发明的制备方法得到的改性胶原蛋白、或者上述本发明的胶原蛋白复合材料在生物打印中的应用。The fifth aspect of the present invention provides the application of the above-mentioned modified collagen of the present invention, the modified collagen obtained by the above-mentioned preparation method of the present invention, or the above-mentioned collagen composite material of the present invention in bioprinting.
优选地，所述生物打印的方式为挤出式3D生物打印、静电纺丝、灌注水凝胶、旋涂水凝胶、注射水凝胶或者喷墨打印。Preferably, the bioprinting method is extrusion 3D bioprinting, electrospinning, infusion hydrogel, spin coating hydrogel, injection hydrogel or inkjet printing.
优选地，所述生物打印为如下的任意一种或多种：Preferably, the bioprinting is any one or more of the following:
(a)构建3D打印的胶原蛋白功能性敷料；(a) Construction of 3D printed collagen functional dressing;
(b)制备3D打印或可注射的负载细胞或功能分子的水凝胶；(b) Prepare 3D printed or injectable hydrogels loaded with cells or functional molecules;
(c)制备用于静电纺丝的产品；(c) Preparing products for electrospinning;
(d)制备用于修复神经元或促进神经干细胞分化的产品；(d) Preparing products for repairing neurons or promoting differentiation of neural stem cells;
(e)体外构建功能性的组织或器官。(e) Construct functional tissues or organs in vitro.
本发明第六方面提供一种生物材料的制备方法，该方法包括：利用上述本发明的改性胶原蛋白、由上述本发明的制备方法得到的改性胶原蛋白、或者上述本发明的胶原蛋白复合材料进行生物打印。A sixth aspect of the present invention provides a method for preparing biomaterials. The method includes: using the modified collagen of the present invention, the modified collagen obtained by the preparation method of the present invention, or the collagen composite of the present invention. Materials for bioprinting.
优选地，所述生物材料为3D细胞模型、球状体、组织模型、类器官、人体器官芯片中的一种或多种。Preferably, the biological material is one or more of 3D cell models, spheroids, tissue models, organoids, and human organ chips.
优选地，该方法还包括利用光照对改性胶原蛋白进行固化，更优选地，光照的条件包括：光照的时间为30-500s，光照的波长为350-450nm，光照功率密度为1-10mW/cm2。Preferably, the method also includes using light to solidify the modified collagen. More preferably, the light conditions include: the time of light is 30-500s, the wavelength of light is 350-450nm, and the power density of light is 1-10mW/ cm 2 .
本发明第七方面提供一种水凝胶，其包含上述本发明的改性胶原蛋白或者由上述本发明的制备方法得到的改性胶原蛋白，并且所述改性胶原蛋白具有通过功能基团之间连接而形成的交联结构。The seventh aspect of the present invention provides a hydrogel, which contains the modified collagen of the present invention or the modified collagen obtained by the preparation method of the present invention, and the modified collagen has the function of functional groups. A cross-linked structure formed by the connections between them.
优选地，所述水凝胶中还含有细胞，所述细胞为内皮细胞、间充质干细胞、胚胎干细胞、多能诱导干细胞、肝细胞、成纤维细胞、肝癌细胞、脑胶质瘤细胞、肾癌细胞中的一种或多种。Preferably, the hydrogel also contains cells, and the cells are endothelial cells, mesenchymal stem cells, embryonic stem cells, pluripotent induced stem cells, hepatocytes, fibroblasts, liver cancer cells, brain glioma cells, kidney One or more types of cancer cells.
通过上述技术方案，与现有技术相比，本发明具有以下优点：Through the above technical solution, compared with the existing technology, the present invention has the following advantages:
(1)本发明提供了一种用离子液体溶解与修饰的改性胶原蛋白的制备方法。与传统的酸溶方式相比，本发明的改性胶原蛋白可在温和条件溶解，溶解过程快，溶解度有了很大提升，至少可达20mg/ml；并且易于对胶原蛋白修饰反应活性基团，修饰率可根据需求人为调控，最高可达86％。(1) The present invention provides a method for preparing modified collagen dissolved and modified by ionic liquid. Compared with the traditional acid-soluble method, the modified collagen of the present invention can be dissolved under mild conditions, the dissolution process is fast, and the solubility is greatly improved, at least up to 20 mg/ml; and it is easy to modify reactive groups on collagen , the modification rate can be manually adjusted according to needs, up to 86%.
(2)本发明提供了一种改性胶原蛋白生物打印材料及其制备方法，包括光交联基团修饰的胶原蛋白、光引发剂、光交联剂和缓冲液，通过紫外照射形成水凝胶，具有较好的弹性和剪切变稀特性，可用于3D打印。(2) The present invention provides a modified collagen bioprinting material and a preparation method thereof, which includes collagen modified with photo-crosslinking groups, a photoinitiator, a photo-crosslinking agent and a buffer, and forms hydrogels through ultraviolet irradiation. Glue, which has good elasticity and shear thinning properties, can be used for 3D printing.
(3)本发明提供可利用光引发巯烯反应进行化学交联的胶原蛋白生物打印材料，引入生物相容性好的交联剂(如SH-PEG-SH)，适用于制备利用生物正交反应进行交联的含细胞水凝胶。以快速和正交的巯烯反应为基础的胶原蛋白生物打印材料具有潜在的生物活性和生物相容性。(3) The present invention provides collagen bioprinting materials that can utilize light-initiated thiene reactions for chemical cross-linking, introduce cross-linking agents with good biocompatibility (such as SH-PEG-SH), and are suitable for the preparation and utilization of bio-orthogonal Reaction cross-linking of cell-containing hydrogels. Collagen bioprinting materials based on fast and orthogonal thiene reactions have potential bioactivity and biocompatibility.
(4)本发明提供的胶原蛋白复合材料，原料易得，制备方法简单，具有较好的打印性质且可保持一定的三股螺旋结构，有潜力成为一种用于组织和器官生物打印的生物材料。(4) The collagen composite material provided by the present invention has easily available raw materials, a simple preparation method, good printing properties and can maintain a certain triple helix structure, and has the potential to become a biomaterial for bioprinting of tissues and organs. .
附图说明Description of the drawings
图1为本发明的离子液体中用甲基丙烯酸酐和降冰片烯二酸酐修饰胶原蛋白方法的流程示意图。Figure 1 is a schematic flow chart of the method of modifying collagen with methacrylic anhydride and norbornenedioic anhydride in the ionic liquid of the present invention.
图2为牛跟腱I型胶原蛋白经不同咪唑型离子液体和水处理后的溶解状态。Figure 2 shows the dissolution state of bovine Achilles tendon type I collagen after treatment with different imidazole ionic liquids and water.
图3为牛跟腱I型胶原蛋白及其经1-乙基-3-甲基咪唑硝酸盐溶解后再生的X射线衍射图谱。Figure 3 shows the X-ray diffraction pattern of bovine Achilles tendon type I collagen and its regeneration after dissolution with 1-ethyl-3-methylimidazole nitrate.
图4中的(A)为明胶(Gelatin)、牛跟腱I型胶原蛋白原料(Col)、经离子液体处理后再生的胶原蛋白(IL-Col)、以及不同比例的甲基丙烯酸酐修饰的胶原蛋白(Col-Me)的聚丙烯酰胺凝胶电泳图谱(SDS-PAGE)；(B)为明胶(Gelatin)、牛跟腱I型胶原蛋白原料(Col)、经离子液体处理后的胶原蛋白(IL-Col)、以及不同比例的降冰片烯二酸酐修饰的胶原蛋白(Col-Nor)的SDS-PAGE电泳图谱；(C)为明胶(Gelatin)、牛跟腱I型胶原蛋白原料(Col)、经离子液体处理后的胶原蛋白(IL-Col)、以及不同比例的甲基丙烯酸酐修饰的胶原蛋白(Col-Me)的圆二色图谱；(D)为明胶(Gelatin)、牛跟腱I型胶原蛋白原料(Col)、经离子液体处理后的胶原蛋白(IL-Col)、以及不同比例的降冰片烯二酸酐修饰的胶原蛋白(Col-Nor)的圆二色图谱。(A) in Figure 4 is gelatin (Gelatin), bovine Achilles tendon type I collagen raw material (Col), regenerated collagen after ionic liquid treatment (IL-Col), and different proportions of methacrylic anhydride modified Polyacrylamide gel electrophoresis pattern (SDS-PAGE) of collagen (Col-Me); (B) is gelatin (Gelatin), bovine Achilles tendon type I collagen raw material (Col), and collagen after ionic liquid treatment (IL-Col), and SDS-PAGE electrophoresis patterns of norbornedioic anhydride-modified collagen (Col-Nor) in different proportions; (C) is gelatin (Gelatin), bovine Achilles tendon type I collagen raw material (Col ), circular dichroism spectra of collagen treated with ionic liquids (IL-Col), and collagen modified with different proportions of methacrylic anhydride (Col-Me); (D) is gelatin, beef heel Circular dichroism spectra of tendon type I collagen raw material (Col), ionic liquid-treated collagen (IL-Col), and collagen modified with different proportions of norbornedioic anhydride (Col-Nor).
图5为本发明所述经光交联基团甲基丙烯酸酐修饰的牛跟腱I型胶原蛋白的核磁共振氢谱。Figure 5 is the hydrogen nuclear magnetic resonance spectrum of bovine Achilles tendon type I collagen modified by the photo-crosslinking group methacrylic anhydride according to the present invention.
图6为本发明所述经光交联基团降冰片烯二酸酐修饰的牛跟腱I型胶原蛋白的核磁共振氢谱。Figure 6 is a hydrogen nuclear magnetic resonance spectrum of bovine Achilles tendon type I collagen modified by the photo-crosslinking group norbornene dianhydride according to the present invention.
图7为本发明所述Col-Nor配制的胶原蛋白、甲基丙烯酸酐修饰的明胶(GelMA)和牛跟腱I型胶原蛋白原料(Col)用于3D打印所得打印体的照片。Figure 7 is a photo of the printed body obtained by 3D printing using the collagen prepared by Col-Nor, methacrylic anhydride-modified gelatin (GelMA) and bovine Achilles tendon type I collagen raw material (Col) according to the present invention.
图8中的(A)为酶联免疫吸附剂测定(ELISA)法测定通过Col-Me打印的血管化肝组织的人白蛋白分泌水平；(B)为ELISA法测定通过Col-Nor打印的血管化肝组织的人白蛋白分泌水平。(A) in Figure 8 is an enzyme-linked immunosorbent assay (ELISA) method for measuring the human albumin secretion level of vascularized liver tissue printed by Col-Me; (B) is an ELISA method for measuring blood vessels printed by Col-Nor. Human albumin secretion levels in liver tissue.
图9中的(A)为用β-萘黄酮处理Col-Nor包裹的血管化肝组织的CYP1A1酶活性；(B)为用β-萘黄酮处理Col-Nor包裹的血管化肝组织的CYP1A2酶活性；(C)为用利福平处理Col-Nor包裹的血管化肝组织的CYP3A4酶活性。(A) in Figure 9 shows the CYP1A1 enzyme activity of Col-Nor-coated vascularized liver tissue treated with β-naphthoflavone; (B) shows the CYP1A2 enzyme activity of Col-Nor-coated vascularized liver tissue treated with β-naphthoflavone. Activity; (C) is the CYP3A4 enzyme activity of Col-Nor-coated vascularized liver tissue treated with rifampicin.
具体实施方式Detailed ways
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。The endpoints of ranges and any values disclosed herein are not limited to the precise range or value, but these ranges or values are to be understood to include values approaching such ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges. These values The scope shall be deemed to be specifically disclosed herein.
本发明第一方面提供一种改性胶原蛋白，所述改性胶原蛋白具有三股螺旋结构，并且在所述改性胶原蛋白的至少部分氨基上连接有功能基团；A first aspect of the present invention provides a modified collagen, the modified collagen has a triple helix structure, and functional groups are connected to at least part of the amino groups of the modified collagen;
其中，所述功能基团选自如下所示的一价残基中的一种或多种，Wherein, the functional group is selected from one or more of the monovalent residues shown below,
式中，R1、R2、R3和R4各自独立地为H、甲基、乙基或丙基。In the formula, R 1 , R 2 , R 3 and R 4 are each independently H, methyl, ethyl or propyl.
上式中，表示与胶原蛋白上的游离氨基(-NH2)形成键/>从而连接在胶原蛋白上。本发明图1中示出了功能基团为式(1)且R1为甲基的改性胶原蛋白(Col-Me)、以及功能基团为式(2)的改性胶原蛋白(Col-Nor)的结构。In the above formula, Indicates the formation of a bond with the free amino group (-NH 2 ) on collagen/> thereby connecting to collagen. Figure 1 of the present invention shows modified collagen (Col-Me) whose functional group is formula (1) and R 1 is methyl, and modified collagen (Col-Me) whose functional group is formula (2). Nor) structure.
根据本发明，优选的情况下，R1、R2、R3和R4各自独立地为H或甲基，更优选地，R1为甲基，R2、R3和R4均为H。According to the present invention, preferably, R 1 , R 2 , R 3 and R 4 are each independently H or methyl. More preferably, R 1 is methyl, and R 2 , R 3 and R 4 are all H. .
在本发明中，“三股螺旋结构”是指活性胶原蛋白(例如天然胶原蛋白)所具有的三股螺旋结构。也就是说，本发明的改性胶原蛋白的改性过程不显著影响胶原蛋白的四级结构，本发明的改性胶原蛋白具有改性前胶原蛋白(例如天然胶原蛋白)的活性。用于制备本发明改性胶原蛋白的胶原蛋白可以为天然提取胶原蛋白或者人工手段合成的胶原蛋白。本发明的胶原蛋白可以为I型、II型、III型或IV型，优选为I型胶原蛋白。In the present invention, the "triple helix structure" refers to the triple helix structure of active collagen (eg, natural collagen). That is to say, the modification process of the modified collagen of the present invention does not significantly affect the quaternary structure of collagen, and the modified collagen of the present invention has the activity of modifying procollagen (such as natural collagen). The collagen used to prepare the modified collagen of the present invention can be naturally extracted collagen or collagen synthesized by artificial means. The collagen of the present invention can be type I, type II, type III or type IV, and is preferably type I collagen.
优选的情况下，上述功能基团可以为光交联基团、离子交联基团、或者热诱导交联基团等，优选为光交联基团。Preferably, the above-mentioned functional group can be a photo-crosslinking group, an ion cross-linking group, a heat-induced cross-linking group, etc., and is preferably a photo-crosslinking group.
作为上述功能基团的形成方法，没有特别的限定，只要在保持胶原蛋白三股螺旋结构的情况下，完成改性即可。例如，所述功能基团可以通过在咪唑型离子液体的存在下使胶原蛋白上的氨基与酰胺化试剂反应而形成。所述酰胺化试剂根据需要形成的功能基团进行选择，例如可以为甲基丙烯酸酐、降冰片烯二酸酐、具有式(4)所示结构的香豆素-3-活性酯中的一种或多种。There is no particular limitation on the formation method of the above-mentioned functional groups, as long as the modification is completed while maintaining the triple helix structure of collagen. For example, the functional group can be formed by reacting an amino group on collagen with an amidation reagent in the presence of an imidazole-type ionic liquid. The amidation reagent is selected according to the functional group that needs to be formed. For example, it can be one of methacrylic anhydride, norbornenedioic anhydride, and coumarin-3-active ester with the structure shown in formula (4). or more.
式(4)中，X为 In formula (4), X is
上述香豆素-3-活性酯的制备例如可以参考ACS Appl.Mater.Interfaces2019,11,22973-22978由香豆素-3-羧酸为原料合成。The preparation of the above-mentioned coumarin-3-active ester can be synthesized from coumarin-3-carboxylic acid as raw material with reference to ACS Appl.Mater.Interfaces2019, 11, 22973-22978.
为了保持胶原蛋白三股螺旋结构，优选使用的咪唑型离子液体可以为1-甲基-3-正辛基咪唑溴化物、1-已基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑硝酸盐、1-丁基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑啉双(三氟甲基磺酰基)亚胺、1-丁基-3-甲基咪唑氯盐、1-丁基-3-甲基咪唑六氟磷酸盐、1-丁基-3-甲基咪唑氢溴酸盐、1-乙基-3-甲基氯化咪唑鎓、1-烯丙基-3-甲基氯化咪唑中的一种或多种，更优选为1-乙基-3-甲基咪唑硝酸盐和/或1-甲基-3-正辛基咪唑溴化物。In order to maintain the triple helix structure of collagen, the preferred imidazole ionic liquids can be 1-methyl-3-n-octylimidazole bromide, 1-hexyl-3-methylimidazole tetrafluoroborate, 1-ethyl 1-ethyl-3-methylimidazole nitrate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazole bis(trifluoromethylsulfonyl)imide, 1 -Butyl-3-methylimidazole chloride, 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hydrobromide, 1-ethyl-3-methyl One or more of 1-allyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazole chloride, more preferably 1-ethyl-3-methylimidazole nitrate and/or 1-methyl- 3-n-Octylimidazole bromide.
作为改性反应的程度，优选地，相对于1g所述改性胶原蛋白，所述功能基团的含量为0.006-0.025μmol，优选为0.01-0.02μmol，例如0.02μmol。As the degree of modification reaction, preferably, the content of the functional group is 0.006-0.025 μmol, preferably 0.01-0.02 μmol, such as 0.02 μmol, relative to 1 g of the modified collagen.
本发明第二方面提供一种改性胶原蛋白的制备方法，该方法包括：在咪唑型离子液体的存在下，将胶原蛋白与酰胺化试剂接触，使得胶原蛋白的至少部分氨基进行酰胺化反应从而形成连接在氨基上的功能基团；A second aspect of the present invention provides a method for preparing modified collagen. The method includes: contacting collagen with an amidation reagent in the presence of an imidazole ionic liquid, so that at least part of the amino groups of the collagen undergo an amidation reaction. Form a functional group attached to the amino group;
其中，所述功能基团选自如下所示的一价残基中的一种或多种，Wherein, the functional group is selected from one or more of the monovalent residues shown below,
式中，R1、R2、R3和R4各自独立地为H、甲基、乙基或丙基。In the formula, R 1 , R 2 , R 3 and R 4 are each independently H, methyl, ethyl or propyl.
在本发明中，作为原料的胶原蛋白可以为天然胶原蛋白或者人工手段合成的胶原蛋白。In the present invention, the collagen as the raw material can be natural collagen or collagen synthesized by artificial means.
根据本发明，优选地，所述酰胺化试剂为甲基丙烯酸酐、降冰片烯二酸酐、具有式(4)所示结构的香豆素-3-活性酯中的一种或多种。According to the present invention, preferably, the amidation reagent is one or more of methacrylic anhydride, norbornenedioic anhydride, and coumarin-3-active ester having the structure shown in formula (4).
式(4)中，X为 In formula (4), X is
为了适当地进行改性，胶原蛋白与酰胺化试剂的重量比可以为1：0.01-0.5，优选为1：0.05-0.4，例如1：0.2。In order to carry out modification appropriately, the weight ratio of collagen to amidation reagent can be 1:0.01-0.5, preferably 1:0.05-0.4, such as 1:0.2.
为了更好地保持胶原蛋白三股螺旋结构并进行改性，优选地，所述咪唑型离子液体为1-甲基-3-正辛基咪唑溴化物、1-已基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑硝酸盐、1-丁基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑啉双(三氟甲基磺酰基)亚胺、1-丁基-3-甲基咪唑氯盐、1-丁基-3-甲基咪唑六氟磷酸盐、1-丁基-3-甲基咪唑氢溴酸盐、1-乙基-3-甲基氯化咪唑鎓、1-烯丙基-3-甲基氯化咪唑中的一种或多种，更优选为1-乙基-3-甲基咪唑硝酸盐和/或1-甲基-3-正辛基咪唑溴化物。为了更好地保持胶原蛋白三股螺旋结构并进行改性，胶原蛋白与咪唑型离子液体的重量比可以为1：32-1600，例如1：64，具体可以为1：32、1：64、1：160、1：320、1：640、1：1600等。In order to better maintain the triple helical structure of collagen and modify it, preferably, the imidazole ionic liquid is 1-methyl-3-n-octylimidazole bromide, 1-hexyl-3-methylimidazole tetrazole Fluoroborate, 1-ethyl-3-methylimidazole nitrate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazole bis(trifluoromethyl Sulfonyl)imine, 1-butyl-3-methylimidazole chloride, 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole hydrobromide, One or more of 1-ethyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazole chloride, more preferably 1-ethyl-3-methylimidazole nitrate and/or 1-methyl-3-n-octylimidazole bromide. In order to better maintain the triple helix structure of collagen and modify it, the weight ratio of collagen to imidazole ionic liquid can be 1:32-1600, such as 1:64, specifically it can be 1:32, 1:64, 1 :160, 1:320, 1:640, 1:1600, etc.
根据本发明，所述酰胺化反应的温度可以为40-120℃，优选为50-80℃，例如50℃。优选地，所述酰胺化反应的时间可以为0.5-6h，优选为4h。According to the present invention, the temperature of the amidation reaction may be 40-120°C, preferably 50-80°C, such as 50°C. Preferably, the amidation reaction time can be 0.5-6h, preferably 4h.
作为进行酰胺化反应的方式，没有特别的限定，只要能够得到本发明的改性胶原蛋白即可。例如该方法可以包括：先将胶原蛋白溶于咪唑型离子液体，然后再加入酰胺化试剂进行酰胺化反应。图1示出了本发明制备方法的具体例子，胶原蛋白(Collagen)经过离子液体(Ionic Liquid)溶解后得到溶解的胶原蛋白(IL-COL，Solubilized Collagen)，然后再经过酰胺化反应修饰改性基团从而得到改性胶原蛋白(Col-Me或者Col-Nor)。There is no particular limitation on the method of carrying out the amidation reaction, as long as the modified collagen of the present invention can be obtained. For example, the method may include: first dissolving collagen in an imidazole ionic liquid, and then adding an amidation reagent to perform an amidation reaction. Figure 1 shows a specific example of the preparation method of the present invention. Collagen (Collagen) is dissolved by ionic liquid (Ionic Liquid) to obtain dissolved collagen (IL-COL, Solubilized Collagen), which is then modified through amidation reaction. groups to obtain modified collagen (Col-Me or Col-Nor).
另外，酰胺化反应完成后，需要从反应体系中去除咪唑型离子液体，从而得到所需的改性胶原蛋白。作为去除咪唑型离子液体的方法，例如可以采用透析(截留分子量例如可以为5-10kDa)。In addition, after the amidation reaction is completed, the imidazole ionic liquid needs to be removed from the reaction system to obtain the required modified collagen. As a method for removing imidazole-type ionic liquids, for example, dialysis can be used (the molecular weight cutoff can be, for example, 5-10 kDa).
本发明第三方面提供一种胶原蛋白复合材料，该胶原蛋白复合材料包括：改性胶原蛋白、光引发剂和可选的光交联剂，该改性胶原蛋白为上述本发明的改性胶原蛋白或者由上述本发明的制备方法得到的改性胶原蛋白。The third aspect of the present invention provides a collagen composite material. The collagen composite material includes: modified collagen, a photoinitiator and an optional photocrosslinking agent. The modified collagen is the above-mentioned modified collagen of the present invention. Protein or modified collagen obtained by the above preparation method of the present invention.
为了提供良好的生物打印效果，优选地，所述胶原蛋白复合材料中的胶原蛋白的质量分数为1-5％，优选为1.5-3％，例如2-2.5％。In order to provide good bioprinting effect, preferably, the mass fraction of collagen in the collagen composite material is 1-5%, preferably 1.5-3%, such as 2-2.5%.
本发明的胶原蛋白复合材料中的光引发剂只要能够完成胶原蛋白的打印即可。优选地，所述光引发剂为苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐(LAP)、2-羟基-2-甲基-1-[4-(2-羟基乙氧基)苯基]-1-丙酮、2-甲基-2-(4-吗啉基)-1-[4-(甲硫基)苯基]-1-丙酮、双(2,4,6-三甲基苯甲酰基)苯基氧化膦中的一种或多种。优选地，所述胶原蛋白复合材料中的所述光引发剂的含量为0.02-0.5％，优选为0.04-0.2％，例如0.04％、0.05％或0.1％。The photoinitiator in the collagen composite material of the present invention only needs to be able to complete collagen printing. Preferably, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl)lithium phosphate (LAP), 2-hydroxy-2-methyl-1-[4-(2-hydroxy Ethoxy)phenyl]-1-propanone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, bis(2,4 , one or more of 6-trimethylbenzoyl)phenylphosphine oxide. Preferably, the content of the photoinitiator in the collagen composite material is 0.02-0.5%, preferably 0.04-0.2%, such as 0.04%, 0.05% or 0.1%.
本发明的胶原蛋白复合材料中的光交联剂可以根据需要添加，例如在功能基团为上述式(2)时，优选含有光交联剂，并且优选使用含有至少两个巯基的光交联剂，更优选为二硫苏糖醇、双巯基聚乙二醇(SH-PEG-SH，例如分子量500-5000g/mol)、巯基化透明质酸(分子量例如可以为70000-150000g/mol)、四(3-巯基丙酸)季戊四醇酯、内消旋-2,3-二巯基丁二酸、二(巯基乙酸)-1,4-丁二酯中的一种或多种。优选地，所述胶原蛋白复合材料中的所述光交联剂的质量分数为0.1-1％，优选为0.2-0.8％，更优选为0.3-0.5％，例如0.4％。The photocrosslinking agent in the collagen composite material of the present invention can be added as needed. For example, when the functional group is the above formula (2), it is preferable to contain a photocrosslinking agent, and it is preferable to use a photocrosslinking agent containing at least two thiol groups. Agents, more preferably dithiothreitol, dithiol polyethylene glycol (SH-PEG-SH, for example, molecular weight 500-5000g/mol), thiolated hyaluronic acid (molecular weight, for example, 70000-150000g/mol), One or more of pentaerythritol tetrakis(3-mercaptopropionate), meso-2,3-dimercaptosuccinic acid, and di(mercaptoacetic acid)-1,4-butanediester. Preferably, the mass fraction of the photo-crosslinking agent in the collagen composite material is 0.1-1%, preferably 0.2-0.8%, more preferably 0.3-0.5%, such as 0.4%.
优选的情况下，本发明中利用甲基丙烯酸酐修饰的改性胶原蛋白，其交联是无规链增长光聚合的结果，会产生高的初始自由基浓度并在凝胶化后产生异质疏水动力学链。虽无需外加光交联剂，但交联模式对于某些容易受到自由基介导的损伤的细胞类型可能并不理想。因此本发明还提供光引发的巯烯反应化学交联的胶原蛋白生物打印材料，引入生物相容性好的SH-PEG-SH等作为交联剂，适用于制备带有正交交联的充满细胞的水凝胶。同时巯烯反应不受氧的抑制，较低的自由基浓度即可引发反应。因此，硫醇-烯水凝胶的交联非常快，并产生具有理想化和正交结构的水凝胶网络。Preferably, the cross-linking of the modified collagen modified with methacrylic anhydride in the present invention is the result of random chain growth photopolymerization, which will produce a high initial free radical concentration and produce heterogeneity after gelation. Hydrophobic kinetic chain. Although no external photo-cross-linking agent is required, the cross-linking pattern may not be ideal for certain cell types that are susceptible to free radical-mediated damage. Therefore, the present invention also provides a photo-initiated thiene reaction chemically cross-linked collagen bioprinting material, which introduces SH-PEG-SH and the like with good biocompatibility as a cross-linking agent, and is suitable for preparing full-fill membranes with orthogonal cross-linking. Cellular hydrogel. At the same time, the thiene reaction is not inhibited by oxygen, and the reaction can be initiated at a lower free radical concentration. Therefore, cross-linking of thiol-ene hydrogels is very rapid and results in hydrogel networks with idealized and orthogonal structures.
本发明第四方面提供一种胶原蛋白复合材料，该胶原蛋白复合材料包括：改性胶原蛋白、光引发剂、细胞、缓冲液和可选的光交联剂，该改性胶原蛋白为上述本发明的改性胶原蛋白或者由上述本发明的制备方法得到的改性胶原蛋白。The fourth aspect of the present invention provides a collagen composite material. The collagen composite material includes: modified collagen, a photoinitiator, cells, a buffer and an optional photo-crosslinking agent. The modified collagen is the above-mentioned present invention. The modified collagen of the invention or the modified collagen obtained by the above-mentioned preparation method of the invention.
在上述本发明第四方面的改性胶原蛋白、光引发剂和光交联剂的具体种类和含量等与上述第三方面相同，在此不在赘述。The specific types and contents of the modified collagen, photoinitiator and photocrosslinking agent in the fourth aspect of the present invention are the same as those in the third aspect and will not be described again here.
根据本发明，所述细胞例如可以是需要进行生物打印的细胞，具体可以为内皮细胞、间充质干细胞、肝细胞、成纤维细胞、肝癌细胞、脑胶质瘤细胞、肾癌细胞中的一种或多种。上述细胞可以是人源的，也可以是非人源的(例如小鼠、大鼠、兔、牛、猪等)。具体地，内皮细胞例如为静脉内皮细胞(如RFP-HUVEC)，间充质干细胞例如为人源间充质干细胞(hMSCs)，肝细胞例如为原代肝细胞(PHHs)。According to the present invention, the cells can be, for example, cells that need to be bioprinted, specifically, they can be one of endothelial cells, mesenchymal stem cells, hepatocytes, fibroblasts, liver cancer cells, brain glioma cells, and renal cancer cells. Kind or variety. The above-mentioned cells may be of human origin or non-human origin (such as mouse, rat, rabbit, cow, pig, etc.). Specifically, endothelial cells are, for example, intravenous endothelial cells (such as RFP-HUVEC), mesenchymal stem cells are, for example, human mesenchymal stem cells (hMSCs), and liver cells are, for example, primary hepatocytes (PHHs).
根据本发明的一个优选的实施方式，作为用于制备血管化肝组织的胶原蛋白复合材料，其中采用的细胞包括静脉内皮细胞、间充质干细胞和原代肝细胞。According to a preferred embodiment of the present invention, as a collagen composite material for preparing vascularized liver tissue, the cells used include venous endothelial cells, mesenchymal stem cells and primary hepatocytes.
为了保持上述细胞的活性，该胶原蛋白复合材料中还含有缓冲液。这里的缓冲液例如可以为磷酸盐缓冲液PBS、或者用于细胞培养的培养基等。作为上述培养基，例如肝细胞培养基(hepatocyte medium，HM培养基)和内皮细胞培养基(endothelial cell growthmedium-2，EGM-2)等。In order to maintain the activity of the above-mentioned cells, the collagen composite material also contains a buffer. The buffer here may be, for example, phosphate buffered saline (PBS) or a medium used for cell culture. Examples of the above-mentioned culture medium include hepatocyte medium (HM medium), endothelial cell growth medium (EGM-2), and the like.
本发明第五方面提供上述本发明的改性胶原蛋白、由上述本发明的制备方法得到的改性胶原蛋白、或者上述本发明的胶原蛋白复合材料在生物打印中的应用。The fifth aspect of the present invention provides the application of the above-mentioned modified collagen of the present invention, the modified collagen obtained by the above-mentioned preparation method of the present invention, or the above-mentioned collagen composite material of the present invention in bioprinting.
通过采用本发明的改性胶原蛋白或者胶原蛋白复合材料，可以打印得到三维的胶原蛋白或者含有胶原蛋白的复合结构。例如可以为3D细胞模型、球状体、组织模型、类器官、人体器官芯片中的一种或多种。By using the modified collagen or collagen composite material of the present invention, three-dimensional collagen or a composite structure containing collagen can be printed. For example, it can be one or more of 3D cell models, spheroids, tissue models, organoids, and human organ chips.
所述生物打印的方式没有特别的限定，只要能适当地打印改性胶原蛋白即可，例如可以为挤出式3D生物打印、静电纺丝、灌注水凝胶、旋涂水凝胶、注射水凝胶或者喷墨打印等。优选地，所述生物打印为如下的任意一种或多种：(a)构建3D打印的胶原蛋白功能性敷料；(b)制备3D打印或可注射的负载细胞或功能分子的水凝胶；(c)制备用于静电纺丝的产品；(d)制备用于修复神经元或促进神经干细胞分化的产品；(e)体外构建功能性的组织或器官。The method of bioprinting is not particularly limited, as long as the modified collagen can be properly printed. For example, it can be extrusion 3D bioprinting, electrospinning, infusion hydrogel, spin coating hydrogel, or water injection. Gel or inkjet printing, etc. Preferably, the bioprinting is any one or more of the following: (a) constructing a 3D printed collagen functional dressing; (b) preparing a 3D printed or injectable hydrogel loaded with cells or functional molecules; (c) Preparing products for electrospinning; (d) Preparing products for repairing neurons or promoting differentiation of neural stem cells; (e) Constructing functional tissues or organs in vitro.
根据本发明的一个优选的实施方式，作为打印的方法，可以通过如下进行，将上述本发明的改性胶原蛋白和细胞、或者上述本发明的胶原蛋白复合材料作为生物打印墨水载入生物3D打印机，通过3D打印方法按设计形状制造含细胞的立体水凝胶，在体外培养条件下获得组织模型(如血管化组织模型)。According to a preferred embodiment of the present invention, as a printing method, the above-mentioned modified collagen and cells of the present invention, or the above-mentioned collagen composite material of the present invention can be loaded into a bio-3D printer as a bioprinting ink. , create a three-dimensional hydrogel containing cells according to the designed shape through 3D printing, and obtain a tissue model (such as a vascularized tissue model) under in vitro culture conditions.
进一步地，本发明的改性胶原蛋白可以在打印完成后，通过交联固化成型。交联方式可以采用光照。优选地，光照的条件可以包括：光照的时间为30-500s，光照的波长为350-450nm，光照功率密度为1-10mW/cm2。在本发明的一个特别优选的实施方式中，光照的条件包括：光照的时间为2min，光照的波长为365nm，光照功率密度为5mW/cm2。Furthermore, the modified collagen of the present invention can be formed by cross-linking and solidification after printing is completed. The cross-linking method can use light. Preferably, the illumination conditions may include: the illumination time is 30-500s, the illumination wavelength is 350-450nm, and the illumination power density is 1-10mW/cm 2 . In a particularly preferred embodiment of the present invention, the illumination conditions include: the illumination time is 2 minutes, the illumination wavelength is 365nm, and the illumination power density is 5mW/cm 2 .
根据本发明，所述光照使用的光源可以为可见光光源，例如能够提供波长为400-700nm的光源。作为所述光源例如可以为氙灯光源等。According to the present invention, the light source used for the illumination may be a visible light source, for example, a light source capable of providing a wavelength of 400-700 nm. The light source may be, for example, a xenon lamp source.
本发明第六方面提供一种生物材料的制备方法，该方法包括：利用上述本发明的改性胶原蛋白、由上述本发明的制备方法得到的改性胶原蛋白、或者上述本发明的胶原蛋白复合材料进行生物打印。A sixth aspect of the present invention provides a method for preparing biomaterials. The method includes: using the modified collagen of the present invention, the modified collagen obtained by the preparation method of the present invention, or the collagen composite of the present invention. Materials for bioprinting.
进一步地，为了得到更好的三维结构，优选地，该方法还包括利用光照对改性胶原蛋白进行固化。改性胶原蛋白(水凝胶)通过3D打印成型后，可方便的通过光照固化形成稳定水凝胶。Furthermore, in order to obtain a better three-dimensional structure, preferably, the method also includes using light to solidify the modified collagen. After the modified collagen (hydrogel) is formed by 3D printing, it can be easily cured by light to form a stable hydrogel.
优选地，光照的条件包括：光照的时间为30-500s，光照的波长为350-450nm，光照功率密度为1-10mW/cm2。在本发明的一个特别优选的实施方式中，光照的条件包括：光照的时间为2min，光照的波长为365nm，光照功率密度为5mW/cm2。Preferably, the illumination conditions include: the illumination time is 30-500s, the illumination wavelength is 350-450nm, and the illumination power density is 1-10mW/cm 2 . In a particularly preferred embodiment of the present invention, the illumination conditions include: the illumination time is 2 minutes, the illumination wavelength is 365nm, and the illumination power density is 5mW/cm 2 .
本发明第七方面提供一种水凝胶，其包含上述本发明的改性胶原蛋白或者由上述本发明的制备方法得到的改性胶原蛋白，并且所述改性胶原蛋白具有通过功能基团之间连接而形成的交联结构。The seventh aspect of the present invention provides a hydrogel, which contains the modified collagen of the present invention or the modified collagen obtained by the preparation method of the present invention, and the modified collagen has the function of functional groups. A cross-linked structure formed by the connections between them.
上述交联结构可以通过利用光照对改性胶原蛋白进行固化而得到。The above cross-linked structure can be obtained by solidifying modified collagen using light.
优选的情况下，所述水凝胶中还含有细胞。并且本发明的改性胶原蛋白的生物打印以及交联过程可以保留细胞的活性。所述细胞为内皮细胞、间充质干细胞、胚胎干细胞、多能诱导干细胞、肝细胞、成纤维细胞、肝癌细胞、脑胶质瘤细胞、肾癌细胞中的一种或多种。Preferably, the hydrogel also contains cells. Moreover, the bioprinting and cross-linking process of modified collagen of the present invention can retain the activity of cells. The cells are one or more of endothelial cells, mesenchymal stem cells, embryonic stem cells, pluripotent induced stem cells, liver cells, fibroblasts, liver cancer cells, brain glioma cells, and renal cancer cells.
以下将通过实施例对本发明进行详细描述。实施例中未注明具体条件者，按照本领域常规实验条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。The present invention will be described in detail below through examples. If no specific conditions are specified in the examples, the experiments shall be carried out in accordance with the conventional experimental conditions in this field. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
实施例1：牛跟腱I型胶原蛋白(Col)在不同咪唑型离子液体中的溶解Example 1: Dissolution of bovine Achilles tendon type I collagen (Col) in different imidazole ionic liquids
将牛跟腱I型胶原蛋白按质量分数为2.5％分别在水和不同咪唑型离子液体中溶解，其中咪唑型离子液体分别为1-甲基-3-正辛基咪唑溴化物、1-已基-3-甲基咪唑四氟硼酸盐、1-乙基-3-甲基咪唑硝酸盐和1-丁基-3-甲基咪唑四氟硼酸盐。在90℃下溶解12h后，对未溶解的离子液体组继续加热至120℃。The bovine Achilles tendon type I collagen was dissolved in water and different imidazole ionic liquids with a mass fraction of 2.5%. The imidazole ionic liquids were 1-methyl-3-n-octyl imidazole bromide and 1-hexyl ionic liquid. 1-ethyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazole nitrate and 1-butyl-3-methylimidazole tetrafluoroborate. After dissolving at 90°C for 12 hours, the undissolved ionic liquid group was continued to be heated to 120°C.
所得的溶解结果如图2所示，牛跟腱I型胶原蛋白仅在在1-甲基-3-正辛基咪唑溴化物(120℃)和1-乙基-3-甲基咪唑硝酸盐(90℃)中溶解均匀。进一步对1-乙基-3-甲基咪唑硝酸盐(90℃)中牛跟腱I型胶原蛋白分别在35℃、40℃、45℃和50℃溶解，发现50℃即可对质量分数为2.5％的牛跟腱I型胶原蛋白实现均匀溶解。The dissolution results obtained are shown in Figure 2. Bovine Achilles tendon type I collagen was only dissolved in 1-methyl-3-n-octylimidazole bromide (120°C) and 1-ethyl-3-methylimidazole nitrate. (90℃) dissolves evenly. Further, bovine Achilles tendon type I collagen in 1-ethyl-3-methylimidazole nitrate (90℃) was dissolved at 35℃, 40℃, 45℃ and 50℃ respectively. It was found that 50℃ can dissolve the mass fraction of 2.5% bovine Achilles tendon type I collagen achieves uniform dissolution.
另外，将牛跟腱I型胶原蛋白按质量分数为2.5％在0.08mM乙酸水溶液中溶解，在4℃下最终实现均匀溶解，而提高温度则导致溶解度降低。In addition, bovine Achilles tendon type I collagen was dissolved in a 0.08mM acetic acid aqueous solution with a mass fraction of 2.5%, and uniform dissolution was finally achieved at 4°C. Increasing the temperature resulted in a decrease in solubility.
实施例2：牛跟腱I型胶原蛋白及其经1-乙基-3-甲基咪唑硝酸盐溶解再生产物的X射线衍射表征Example 2: X-ray diffraction characterization of bovine Achilles tendon type I collagen and its regenerated products dissolved by 1-ethyl-3-methylimidazole nitrate
将3.2g 1-乙基-3-甲基咪唑硝酸盐加热到在50℃，等待该离子液体全部融化后。加入50mg牛跟腱I型胶原蛋白，在50℃加热搅拌使牛跟腱I型胶原蛋白经2h溶解均匀。同时作为对比酸溶胶原，将质量体积分数为0.3％同批次胶原蛋白加入0.08mM乙酸中，4℃下经12h溶解均匀。将体系通过截留分子量为6-8kDa透析袋(美国Spectra/Por透析膜，132650)透析三天，期间换水9次。透析完成后，通过冻干获得经1-乙基-3-甲基咪唑硝酸盐溶解再生的牛跟腱I型胶原蛋白(IL-Col)和经0.08mM乙酸溶解再生的牛跟腱I型胶原蛋白(H+-Col)。Heat 3.2g of 1-ethyl-3-methylimidazole nitrate to 50°C and wait until the ionic liquid is completely melted. Add 50 mg of bovine Achilles tendon type I collagen, heat and stir at 50°C to dissolve the bovine Achilles tendon type I collagen evenly over 2 hours. At the same time, as a comparison of acid-solubilized collagen, 0.3% mass volume fraction of collagen from the same batch was added to 0.08mM acetic acid and dissolved evenly at 4°C for 12 hours. The system was dialyzed for three days through a dialysis bag with a molecular weight cutoff of 6-8 kDa (American Spectra/Por dialysis membrane, 132650), during which the water was changed 9 times. After dialysis, bovine Achilles tendon type I collagen (IL-Col) dissolved and regenerated with 1-ethyl-3-methylimidazole nitrate and bovine Achilles tendon type I collagen dissolved and regenerated with 0.08mM acetic acid were obtained by freeze-drying. Protein (H + -Col).
将牛跟腱I型胶原蛋白和上述得到的IL-Col和H+-Col用于X射线衍射表征，所得结果如图3所示。图谱结果表明IL-Col和H+-Col均保留了牛跟腱I型胶原蛋白的无序结构和特有的三股螺旋结构。Bovine Achilles tendon type I collagen and the IL-Col and H + -Col obtained above were used for X-ray diffraction characterization, and the results are shown in Figure 3. The chromatogram results showed that both IL-Col and H + -Col retained the disordered structure and unique triple helical structure of bovine Achilles tendon type I collagen.
实施例3：不同比例甲基丙烯酸酸酐修饰牛跟腱I型胶原蛋白的合成Example 3: Synthesis of bovine Achilles tendon type I collagen modified with different proportions of methacrylic anhydride
将3.2g 1-乙基-3-甲基咪唑硝酸盐加热到在50℃，等待该离子液体全部融化后。加入50mg牛跟腱I型胶原蛋白，在50℃加热搅拌溶解4小时。溶解完毕后，分别加入2.5μL、5μL、10μL的甲基丙烯酸酐(依次记为Col-Me-1、Col-Me-2和Col-Me-4)，继续搅拌加热4h。反应完毕后，再将体系通过截留分子量为6-8kDa透析袋透析三天，期间换水9次。透析完成后，通过冻干获得不同比例甲基丙烯酸酐修饰胶原蛋白(Col-Me)。Heat 3.2g of 1-ethyl-3-methylimidazole nitrate to 50°C and wait until the ionic liquid is completely melted. Add 50 mg of bovine Achilles tendon type I collagen, heat, stir and dissolve at 50°C for 4 hours. After the dissolution is completed, add 2.5 μL, 5 μL, and 10 μL of methacrylic anhydride (recorded as Col-Me-1, Col-Me-2, and Col-Me-4 in sequence), and continue stirring and heating for 4 h. After the reaction is completed, the system is dialyzed through a dialysis bag with a molecular weight cutoff of 6-8kDa for three days, during which the water is changed 9 times. After dialysis, methacrylic anhydride-modified collagen (Col-Me) with different proportions was obtained by freeze-drying.
将10μL质量分数为0.3％的明胶(Gelatin，Gel)、Col、IL-Col和不同比例修饰的Col-Me分别与10μL 2ⅹloading buffer混合均匀。再将混合物离心5分钟，然后在沸水中加热10分钟。将加热的混合物上样至SurePAGE预成型凝胶(Genscript，M00652，梯度浓度为4-12％)上。电泳结束后，凝胶用考马斯蓝快速染色溶液(Beyotime，P0017)染色，并用Bio-RadUniversal Hood III成像，所得结果如图4中的(A)所示，其中1、2、4分别表示Col-Me-1、Col-Me-2和Col-Me-4。并对其中的Gel、Col、IL-Col和Col-Me用于圆二色光谱表征，所得结果如图4中的(C)所示。同时对Col-Me-1进行核磁共振氢谱表征，所得结果如图5所示。Mix 10 μL of gelatin (Gel), Col, IL-Col and modified Col-Me in different proportions with a mass fraction of 0.3% with 10 μL of 2ⅹ loading buffer. Centrifuge the mixture for another 5 minutes and heat in boiling water for 10 minutes. The heated mixture was loaded onto a SurePAGE precast gel (Genscript, M00652, gradient concentration 4-12%). After electrophoresis, the gel was stained with Coomassie Blue Rapid Staining Solution (Beyotime, P0017) and imaged with Bio-Rad Universal Hood III. The results are shown in (A) in Figure 4, where 1, 2, and 4 represent respectively. Col-Me-1, Col-Me-2 and Col-Me-4. Gel, Col, IL-Col and Col-Me were characterized by circular dichroism spectra, and the results are shown in (C) in Figure 4. At the same time, Col-Me-1 was characterized by hydrogen nuclear magnetic resonance spectroscopy, and the results are shown in Figure 5.
图4中的(A)凝胶电泳结果中IL-Col、Col-Me-1和Col-Me-2保留有I型胶原蛋白的β、α1和α2的特征条带，符合I型胶原蛋白的特征。而Col-Me-4则没有对应的特征条带，表明Col-Me-4失去I型胶原蛋白的特征。图5的核磁共振氢谱中Col-Me-1表现出I型胶原蛋白特征氢谱，同时表现出甲基丙烯酸酸酐修饰后的特定氢谱峰。以上结果表明通过1-乙基-3-甲基咪唑硝酸盐溶解后的牛跟腱I型胶原蛋白可成功修饰上甲基丙烯酸酸酐，并且Col-Me-1和Col-Me-2保留I型胶原蛋白特征。In the gel electrophoresis result of (A) in Figure 4, IL-Col, Col-Me-1 and Col-Me-2 retain the characteristic bands of β, α1 and α2 of type I collagen, which is consistent with the characteristics of type I collagen. feature. However, Col-Me-4 has no corresponding characteristic band, indicating that Col-Me-4 has lost the characteristics of type I collagen. In the hydrogen nuclear magnetic resonance spectrum of Figure 5, Col-Me-1 exhibits a characteristic hydrogen spectrum of type I collagen, and also exhibits a specific hydrogen spectrum peak after modification with methacrylic anhydride. The above results indicate that bovine Achilles tendon type I collagen dissolved by 1-ethyl-3-methylimidazole nitrate can be successfully modified with methacrylic anhydride, and Col-Me-1 and Col-Me-2 retain type I collagen. Collagen characteristics.
图4中的(C)圆二色光谱结果中，相比于明胶，Col、IL-Col、Col-Me-1和Col-Me-2均分别在196nm和221nm附近存在明显的负峰和正峰，这表明IL-Col、Col-Me-1和Col-Me-2均保持了Col的三股螺旋结构。而明胶与Col-Me-4在221nm附近没有正峰，表明Col-Me-4未保留三股螺旋结构，其结构接近于明胶。In the circular dichroism spectrum result (C) in Figure 4, compared to gelatin, Col, IL-Col, Col-Me-1 and Col-Me-2 all have obvious negative and positive peaks near 196nm and 221nm respectively. , which shows that IL-Col, Col-Me-1 and Col-Me-2 all maintain the triple helix structure of Col. However, gelatin and Col-Me-4 have no positive peaks near 221 nm, indicating that Col-Me-4 does not retain the triple helix structure and its structure is close to gelatin.
实施例4：不同比例降冰片烯二酸酐修饰牛跟腱I型胶原蛋白的合成Example 4: Synthesis of bovine Achilles tendon type I collagen modified with different proportions of norbornenedioic anhydride
将3.2g 1-乙基-3-甲基咪唑硝酸盐加热到在50℃，等待该离子液体全部融化后。加入50mg胶原蛋白，在50℃加热搅拌溶解4小时。溶解完毕后，分别加入2.5mg、5mg、10mg降冰片烯二酸酐(依次记为Col-Nor-1、Col-Nor-2和Col-Nor-4)，继续搅拌加热4小时。反应完毕后，再将体系通过截留分子量为6-8kDa透析袋透析三天，期间换水9次。透析完成后，通过冻干获得不同比例降冰片烯二酸酐修饰胶原蛋白(Col-Nor)。Heat 3.2g of 1-ethyl-3-methylimidazole nitrate to 50°C and wait until the ionic liquid is completely melted. Add 50 mg of collagen, heat, stir and dissolve at 50°C for 4 hours. After the dissolution is completed, add 2.5 mg, 5 mg, and 10 mg of norbornenedioic anhydride (recorded as Col-Nor-1, Col-Nor-2, and Col-Nor-4 in sequence), and continue stirring and heating for 4 hours. After the reaction is completed, the system is dialyzed through a dialysis bag with a molecular weight cutoff of 6-8kDa for three days, during which the water is changed 9 times. After dialysis, norbornenedic anhydride-modified collagen (Col-Nor) with different proportions was obtained by freeze-drying.
将10μL的明胶(Gel)、Col、IL-Col和不同比例修饰的Col-Nor(3mg/mL)分别与10μL2ⅹloading buffer混合均匀。再将混合物离心5分钟，然后在沸水中加热10分钟。将加热的混合物上样至SurePAGE预成型凝胶(Genscript，M00652，梯度浓度为4-12％)上。电泳结束后，凝胶用考马斯蓝快速染色溶液(Beyotime，P0017)染色，并用Bio-Rad Universal HoodIII成像，所得结果如图4中的(B)所示，其中1、2、4分别表示Col-Nor-1、Col-Nor-2和Col-Nor-4。并对其中的Gel、Col、IL-Col和Col-Nor用于圆二色光谱表征，所得结果如图4中的(D)所示。同时对修饰所得胶原蛋白进行核磁共振氢谱表征，对应的Col-Nor-4结果如图6所示。Mix 10 μL of gelatin (Gel), Col, IL-Col, and Col-Nor (3 mg/mL) modified in different proportions with 10 μL of 2ⅹ loading buffer. Centrifuge the mixture for another 5 minutes and heat in boiling water for 10 minutes. The heated mixture was loaded onto a SurePAGE precast gel (Genscript, M00652, gradient concentration 4-12%). After electrophoresis, the gel was stained with Coomassie blue rapid staining solution (Beyotime, P0017) and imaged with Bio-Rad Universal HoodIII. The results are shown in (B) in Figure 4, where 1, 2, and 4 represent respectively. Col-Nor-1, Col-Nor-2 and Col-Nor-4. Gel, Col, IL-Col and Col-Nor were used for circular dichroism spectral characterization, and the results are shown in (D) in Figure 4. At the same time, the modified collagen was characterized by hydrogen nuclear magnetic resonance spectroscopy, and the corresponding Col-Nor-4 results are shown in Figure 6.
图4中的(B)凝胶电泳结果中IL-Col、Col-Nor-1、Col-Nor-2和Col-Nor-4均保留有I型胶原蛋白的β、α1和α2的特征条带。图6中的核磁共振氢谱中Col-Nor-4表现出I型胶原蛋白特征氢谱，同时表现出降冰片烯二酸酐修饰后的特定氢谱峰。以上结果表明通过1-乙基-3-甲基咪唑硝酸盐溶解后的牛跟腱I型胶原蛋白可成功修饰降冰片烯基团，并具有胶原蛋白的特征结构。In the gel electrophoresis results of (B) in Figure 4, IL-Col, Col-Nor-1, Col-Nor-2 and Col-Nor-4 all retain the characteristic bands of β, α1 and α2 of type I collagen. . In the hydrogen nuclear magnetic resonance spectrum in Figure 6, Col-Nor-4 exhibits a characteristic hydrogen spectrum of type I collagen, and also exhibits a specific hydrogen spectrum peak after modification by norbornene dianhydride. The above results show that bovine Achilles tendon type I collagen dissolved by 1-ethyl-3-methylimidazole nitrate can be successfully modified with norbornene groups and has the characteristic structure of collagen.
图4中的(D)圆二色光谱结果中，相比于明胶，Col、IL-Col、Col-Nor-1、Col-Nor-2和Col-Nor-4均分别在196nm和221nm附近存在明显的负峰和正峰，这表明IL-Col和Col-Nor均保持了Col的三股螺旋结构。In the circular dichroism spectrum result (D) in Figure 4, compared to gelatin, Col, IL-Col, Col-Nor-1, Col-Nor-2 and Col-Nor-4 all exist near 196nm and 221nm respectively. There are obvious negative and positive peaks, which indicates that both IL-Col and Col-Nor maintain the triple helical structure of Col.
实施例5：Col-Nor生物打印复合材料的挤出式打印Example 5: Extrusion printing of Col-Nor bioprinting composites
将所述修饰所得Col-Nor-4、LAP、光交联剂(SH-PEG2000-SH，2000g/mol)和PBS(重量比为1：0.02：0.16：40)在37℃下溶解完全，获得Col-Nor水凝胶。同时将同浓度的商用GelMA、LAP和PBS(重量比为1：0.02：40)在37℃下溶解完全获得GelMA水凝胶，并将胶原蛋白(Col)在0.08mM的乙酸水溶液中按在常用的可溶浓度(重量比1：8，超过该浓度将难以保证充分溶解)在4℃溶解12h再调至中性后得Col水凝胶。The modified Col-Nor-4, LAP, photo-crosslinking agent (SH-PEG2000-SH, 2000g/mol) and PBS (weight ratio 1:0.02:0.16:40) were completely dissolved at 37°C to obtain Col-Nor hydrogel. At the same time, commercial GelMA, LAP and PBS of the same concentration (weight ratio 1:0.02:40) were dissolved at 37°C to completely obtain GelMA hydrogel, and collagen (Col) was added to the commonly used solution in 0.08mM acetic acid aqueous solution. The soluble concentration (weight ratio 1:8, exceeding this concentration will be difficult to ensure full dissolution) was dissolved at 4°C for 12 hours and then adjusted to neutrality to obtain Col hydrogel.
将上述生物打印复合材料吸入3D打印所用注射器。Col-Nor-4水凝胶和GelMA水凝胶在3D打印机中选定待打印的模型文件，设定打印机料仓温度为25℃，底板温度为10℃，进行挤出式打印，并进一步通过光照强度为5mW/cm2，365nm光源光交联固化2分钟。而Col水凝胶则在4℃环境下进行3D打印，打印完成后置于37℃中固化30min。水凝胶固化完成后加入足量磷酸盐缓冲溶液浸泡五分钟，使整个物体浸没在溶液中，得到光固化成型具有弹性的水凝胶，所得结果如图7所示。Inhale the above bioprinting composite material into the syringe used for 3D printing. Col-Nor-4 hydrogel and GelMA hydrogel select the model file to be printed in the 3D printer, set the printer bin temperature to 25°C and the bottom plate temperature to 10°C, perform extrusion printing, and further pass The light intensity is 5mW/cm 2 and the 365nm light source is used for photo-crosslinking and curing for 2 minutes. The Col hydrogel is 3D printed at 4°C, and after printing is completed, it is cured at 37°C for 30 minutes. After the hydrogel is cured, add a sufficient amount of phosphate buffer solution and soak it for five minutes so that the entire object is immersed in the solution, and a light-cured elastic hydrogel is obtained. The results are shown in Figure 7.
图7中打印立体结构的形状保真度表明Col-Nor-4胶原蛋白水凝胶成型效果优异，可完成二维及三维的高精度打印，而同浓度的GelMA在3D打印中成型效果差，容易塌陷。此外，胶原蛋白原料则难以打印。The shape fidelity of the printed three-dimensional structure in Figure 7 shows that Col-Nor-4 collagen hydrogel has excellent molding effect and can complete two-dimensional and three-dimensional high-precision printing, while GelMA with the same concentration has poor molding effect in 3D printing. Collapse easily. In addition, collagen raw materials are difficult to print.
实施例6：ELISA法测定Col-Me和Col-Nor打印的血管化肝组织中的白蛋白分泌Example 6: Determination of albumin secretion in vascularized liver tissue printed by Col-Me and Col-Nor by ELISA
将Col-Nor-4、LAP、光交联剂SH-PEG2000-SH与细胞混合均匀配制胶原蛋白复合材料，其中含改性胶原蛋白25mg/mL、红色荧光标记的静脉内皮细胞(RFP-HUVEC)5×106细胞/mL、人源间充质干细胞(hMSCs)5×105细胞/mL和原代肝细胞(PHHs)聚集体1×104细胞/mL，并且Col-Nor-4、LAP、光交联剂的重量比同实施例5。将上述体系转入3D打印所用注射器中，打印成型后，通过实施例5中交联方式光交联固化成型。光交联完成后，打印组织在含有20ng/mL血管内皮生长因子(VEGF，R&D)和10ng/mL碱性成纤维细胞生长因子(bFGF，R&D)的50％(v/v)肝细胞培养基(其组成为含有1％N-2培养基添加剂(Gibco，17502-048)、1％B27(Gibco，12587010)、1mM N-乙酰-L-半胱氨酸(Sigma-Aldrich)、10mM烟酰胺(Solarbio)、2ng/mL重组人FGF10(Peprotech)、50ng/mL重组人EGF(Peprotech)、25ng/mL重组人HGF(Peprotech)、10nm人胃泌素I[Leu15](Sigma-Aldrich)、5μM A-83-01(TocrisBioscience)，10μM rho激酶抑制剂Y-27632(Selleck)、50ng/mL Wnt3a蛋白(StemimuneLLC)、1％胎牛血清(Ausbian)和1％青霉素链霉素(Solarbio)的DMEM/F12(Gibco，12634-010)培养基和50％(v/v)含有20ng/mL血管内皮生长因子(VEGF，R&D)和10ng/mL碱性成纤维细胞生长因子(bFGF，R&D)的EGM-2培养基(Lonza，CC-3162)的培养基中培养。其中将等量的人肝癌细胞(HepG2)聚集体代替PHHs聚集体作为对照组。通过人白蛋白ELISA试剂盒(Abcam，ab179887)在第1天、第3天、第5天和第7天收集细胞上清液确定打印组织的分泌的人白蛋白水平，所得结果如图8中的(B)所示。Mix Col-Nor-4, LAP, photo-crosslinking agent SH-PEG2000-SH and cells evenly to prepare a collagen composite material, which contains 25 mg/mL modified collagen and red fluorescently labeled venous endothelial cells (RFP-HUVEC). 5×10 6 cells/mL, human mesenchymal stem cells (hMSCs) 5×10 5 cells/mL and primary hepatocyte (PHHs) aggregates 1×10 4 cells/mL, and Col-Nor-4, LAP , the weight ratio of the photo-crosslinking agent is the same as in Example 5. Transfer the above system into a syringe used for 3D printing. After printing and molding, the system is photo-crosslinked and solidified using the crosslinking method in Example 5. After photo-cross-linking is completed, the printed tissue is cultured in 50% (v/v) hepatocyte culture medium containing 20 ng/mL vascular endothelial growth factor (VEGF, R&D) and 10 ng/mL basic fibroblast growth factor (bFGF, R&D). (Its composition contains 1% N-2 medium additive (Gibco, 17502-048), 1% B27 (Gibco, 12587010), 1mM N-acetyl-L-cysteine (Sigma-Aldrich), 10mM nicotinamide (Solarbio), 2ng/mL recombinant human FGF10 (Peprotech), 50ng/mL recombinant human EGF (Peprotech), 25ng/mL recombinant human HGF (Peprotech), 10nm human gastrin I [Leu15] (Sigma-Aldrich), 5μM A-83-01 (TocrisBioscience), 10 μM rho kinase inhibitor Y-27632 (Selleck), 50 ng/mL Wnt3a protein (Stemimune LLC), 1% fetal calf serum (Ausbian), and 1% penicillin-streptomycin (Solarbio) in DMEM /F12 (Gibco, 12634-010) medium and 50% (v/v) EGM containing 20 ng/mL vascular endothelial growth factor (VEGF, R&D) and 10 ng/mL basic fibroblast growth factor (bFGF, R&D) -2 medium (Lonza, CC-3162) medium. An equal amount of human hepatoma cell (HepG2) aggregates were used instead of PHHs aggregates as a control group. Human albumin ELISA kit (Abcam, ab179887) Cell supernatants were collected on days 1, 3, 5, and 7 to determine the secreted human albumin levels of the printed tissues, and the results are shown in Figure 8 (B).
利用上述方法，同样制备和测定利用含Col-Me-1的胶原蛋白复合材料进行生物打印所得PHHs聚集体的人白蛋白水平，所得结果如图8中的(A)所示。Using the above method, the human albumin levels of PHHs aggregates obtained by bioprinting using collagen composite materials containing Col-Me-1 were also prepared and measured. The results are shown in Figure 8 (A).
图8结果表明，通过Col-Me(A)和Col-Nor(B)3D打印的血管化肝组织均可稳定保持白蛋白分泌，这表明利用Col-Me和Col-Nor打印的血管化肝组织具有肝组织的潜在功能。Figure 8 The results show that the vascularized liver tissue 3D printed by Col-Me (A) and Col-Nor (B) can stably maintain albumin secretion, which shows that the vascularized liver tissue printed by Col-Me (A) and Col-Nor (B) can stably maintain albumin secretion. Has the potential function of liver tissue.
实施例7：用药物β-萘黄酮和利福平刺激Col-Nor打印的血管化肝组织中细胞色素P450中CYP1A1、CYP1A2和CYP3A4的酶活性。Example 7: The drugs β-naphthoflavone and rifampicin were used to stimulate the enzymatic activities of CYP1A1, CYP1A2 and CYP3A4 in cytochrome P450 in vascularized liver tissue printed by Col-Nor.
将实施例6中通过Col-Nor打印的血管化肝组织，在特定诱导剂的存在下，培养36小时。同时将原代肝细胞在培养皿中进行二维(2D)培养，培养36小时作为对照。通过P450GloTM分析试剂盒(Promega Corporation)测量不同的CYP450活性，其中，细胞色素P450家族1亚家族A成员1和2(CYP1A1和CYP1A2)的酶活性通过25μMβ-萘黄酮(Sigma-Aldrich)诱导剂存在下测定，细胞色素P450家族3亚家族A成员4(CYP3A4)的酶活性通过25μM利福平(Sigma-Aldrich)诱导剂存在下测定，同时补充等量的二甲基亚砜作为对照组，所得结果如图9所示。The vascularized liver tissue printed by Col-Nor in Example 6 was cultured in the presence of a specific inducer for 36 hours. At the same time, primary hepatocytes were cultured in two-dimensional (2D) culture dishes for 36 hours as a control. Different CYP450 activities were measured by P450GloTM Assay Kit (Promega Corporation), in which the enzymatic activities of cytochrome P450 family 1 subfamily A members 1 and 2 (CYP1A1 and CYP1A2) were present in the presence of 25 μM β-naphthoflavone (Sigma-Aldrich) inducer The enzyme activity of cytochrome P450 family 3 subfamily A member 4 (CYP3A4) was measured in the presence of 25 μM rifampicin (Sigma-Aldrich) inducer, and an equal amount of dimethyl sulfoxide was supplemented as a control group. The results are shown in Figure 9.
图9表明相比于2D培养的原代肝细胞，经Col-Nor 3D打印的血管化肝组织表现出更强的CYP1A1、CYP1A2和CYP3A4的酶活性，由此可知3D打印的血管化肝组织具有更好的代谢功能。总之，上述这些结果表明，Col-Nor水凝胶可以支持肝细胞存活，并具有潜在的肝功能。Figure 9 shows that compared with 2D cultured primary hepatocytes, vascularized liver tissue printed by Col-Nor shows stronger enzyme activities of CYP1A1, CYP1A2 and CYP3A4. It can be seen that 3D printed vascularized liver tissue has Better metabolic function. Taken together, these results indicate that Col-Nor hydrogel can support hepatocyte survival and have potential liver function.
通过上述实施例1-7可知，本发明的经1-乙基-3-甲基咪唑硝酸盐溶解后修饰的牛跟腱I型胶原蛋白具有优异的生物3D打印性能和良好的生物活性。相比于传统胶原蛋白的酸溶修饰，牛跟腱I型胶原蛋白经1-乙基-3-甲基咪唑硝酸盐溶解后可快速、简单、批量的对胶原蛋白进行各类基团修饰。同时修饰所得的胶原蛋白保持I型胶原蛋白的特征结构和生物活性，并赋予其优异的生物活性。其作为胶原蛋白复合材料可在体外实现血管化肝组织的构建，并具备肝组织潜在的功能和药物代谢能力。It can be seen from the above Examples 1-7 that the bovine Achilles tendon type I collagen modified by dissolving 1-ethyl-3-methylimidazole nitrate of the present invention has excellent bio-3D printing performance and good biological activity. Compared with the acid-soluble modification of traditional collagen, bovine Achilles tendon type I collagen can be quickly, simply and batch-modified with various groups after being dissolved by 1-ethyl-3-methylimidazole nitrate. At the same time, the modified collagen maintains the characteristic structure and biological activity of type I collagen and gives it excellent biological activity. As a collagen composite material, it can realize the construction of vascularized liver tissue in vitro, and has the potential function and drug metabolism ability of liver tissue.
本发明为发展胶原蛋白的修饰提供了全新的策略，这为实现胶原蛋白的3D生物打印的方面发挥了重要作用，并且提供了一种胶原蛋白的修饰的新方法。The present invention provides a new strategy for the development of collagen modification, which plays an important role in realizing 3D bioprinting of collagen, and provides a new method of collagen modification.
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical concept of the present invention, many simple modifications can be made to the technical solution of the present invention, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the disclosed content of the present invention. All belong to the protection scope of the present invention.
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