a Human Genetics, Sri Ramachandra University , Porur, Chennai , India.
b Cell and Tissue Engineering Laboratory, Sri Ramachandra University , Porur, Chennai , India.
Artif Cells Nanomed Biotechnol. 2016 Aug;44(5):1318-25. doi: 10.3109/21691401.2015.1029629. Epub 2015 May 9.
Electrospinning is a well-established technique that uses a high electric field to fabricate ultrafine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers, due to its widespread occurrence in nature and its biocompatibility. Electrospinning of collagen alone has been reported, with fluoroalcohols such as hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE), but the resultant collagen lost its characteristic ultrastructural integrity of D-periodicity 67 nm banding, confirmed by transmission electron microscopy (TEM), and the fluoroalcohols used were toxic to the environment. In this study, we describe the use of glacial acetic acid and DMSO to dissolve collagen and generate electrospun nanofibers of collagen type 1, which is non-toxic and economical. TEM analysis revealed the characteristic feature of native collagen triple helical repeats, showing 67 nm D-periodicity banding pattern and confirming that the ultrastructural integrity of the collagen was maintained. Analysis by scanning electron microscopy (SEM) showed fiber diameters in the range of 200-1100 nm. Biocompatibility of the three-dimensional (3D) scaffolds was established by MTT assays using rat skeletal myoblasts (L6 cell line) and confocal microscopic analysis of immunofluorescent-stained sections of collagen scaffolds for muscle-specific markers such as desmin and actin. Primary neonatal rat ventricular cardiomyocytes (NRVCM) seeded onto the collagen scaffolds were able to maintain their contractile function for a period of 17 days and also expressed higher levels of desmin when compared with 2D cultures. We report for the first time that collagen type 1 can be electrospun without blending with copolymers using the novel benign solvent combination, and the method can be potentially explored for applications in tissue engineering.
静电纺丝是一种成熟的技术，它利用高电场从天然和合成聚合物中制造超精细纤维支架，以模拟细胞微环境。胶原蛋白是最受欢迎的生物聚合物之一，因为它广泛存在于自然界中，并且具有生物相容性。已经有报道称，胶原蛋白可以单独进行静电纺丝，使用氟醇，如六氟异丙醇（HFIP）和三氟乙醇（TFE），但通过透射电子显微镜（TEM）证实，所得的胶原蛋白失去了其特征性的超微结构完整性，即 67nm 的 D 周期带状结构，并且使用的氟醇对环境有毒。在这项研究中，我们描述了使用冰醋酸和 DMSO 溶解胶原蛋白并生成胶原蛋白 1 型的电纺纳米纤维，这种方法既无毒又经济。TEM 分析显示了天然胶原蛋白三螺旋重复的特征，显示出 67nm 的 D 周期带状结构，证实了胶原蛋白的超微结构完整性得以保持。扫描电子显微镜（SEM）分析显示纤维直径在 200-1100nm 范围内。通过使用大鼠骨骼肌母细胞（L6 细胞系）进行 MTT 分析以及对胶原蛋白支架进行免疫荧光染色的共聚焦显微镜分析，研究了三维（3D）支架的生物相容性，以评估其对肌肉特异性标志物（如结蛋白和肌动蛋白）的相容性。原代新生大鼠心室心肌细胞（NRVCM）接种到胶原蛋白支架上后，能够维持其收缩功能长达 17 天，并且与 2D 培养相比，表达更高水平的结蛋白。我们首次报道，胶原蛋白 1 型可以使用新型良性溶剂组合进行静电纺丝，而无需与共聚物共混，并且该方法可能在组织工程中得到探索。
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