Shrestha Rupendra, Palat Asha, Punnoose Alan M, Joshi Shailesh, Ponraju D, Paul Solomon F D
Department of Human Genetics, Sri Ramachandra University, Chennai, India; Cell and Tissue Engineering Laboratory, Centre for Regenerative Medicine and Stem Cell Research, Sri Ramachandra University, Chennai, India.
Cell and Tissue Engineering Laboratory, Centre for Regenerative Medicine and Stem Cell Research, Sri Ramachandra University, Chennai, India.
Tissue Cell. 2016 Dec;48(6):634-643. doi: 10.1016/j.tice.2016.07.007. Epub 2016 Jul 29.
Electrospun nanofibres have been shown to exhibit extracellular matrix (ECM)-like characteristics required for tissue engineering in terms of porosity, flexibility, fibre organization and strength. This study focuses on developing novel cellulose acetate phthalate (CAP) scaffolds by electrospinning for establishing 3-D chondrocyte and neuronal cultures. Five solvent combinations were employed in fabricating the fibres, namely, acetone/ethanol (9:1), dimethylformamide/tetrahydrofuran/acetone (3:3:4), tetrahydrofuran/acetone (1:1), tetrahydrofuran/ethanol (1:1) and chloroform/methanol (1:1). The electrospun fibres were characterized by scanning electron microscopy (SEM) analysis and confirmed to be within the nanometre range. Based on the morphology of the fibers from SEM results, two solvent combinations such as acetone/ethanol and dimethylformamide/tetrahydrofuran/acetone were selected for stabilization as CAP exhibits a pH dependent solubility. Fourier-Transform Infrared (FTIR) analysis revealed the hydrolysis of CAP which was overcome by EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] and EDC/NHS (N-hydroxysuccinimide) cross-linking resulting in its stability (pH of 7.2) for three months. MTT [3-(4, 5-dimethylthiazol-2-yl)-1, 5-diphenyltetrazolium bromide] assay performed using L6 myoblast confirmed the biocompatibility of the scaffolds. 3-D primary chondrocyte and neuronal cultures were established on the scaffolds and maintained for a period of 10 days. H&E staining and SEM analysis showed the attachment of the chondrocytes and neurons on CAP scaffolds prepared using dimethylformamide/tetrahydrofuran/acetone and acetone/ethanol respectively.
在孔隙率、柔韧性、纤维结构和强度方面,电纺纳米纤维已被证明具有组织工程所需的细胞外基质(ECM)样特性。本研究的重点是通过静电纺丝开发新型醋酸邻苯二甲酸纤维素(CAP)支架,用于建立三维软骨细胞和神经元培养体系。在制备纤维时使用了五种溶剂组合,即丙酮/乙醇(9:1)、二甲基甲酰胺/四氢呋喃/丙酮(3:3:4)、四氢呋喃/丙酮(1:1)、四氢呋喃/乙醇(1:1)和氯仿/甲醇(1:1)。通过扫描电子显微镜(SEM)分析对电纺纤维进行了表征,并确认其在纳米范围内。基于SEM结果中纤维的形态,选择了两种溶剂组合,如丙酮/乙醇和二甲基甲酰胺/四氢呋喃/丙酮,用于稳定化处理,因为CAP表现出pH依赖性溶解性。傅里叶变换红外(FTIR)分析揭示了CAP的水解,通过1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和EDC/N-羟基琥珀酰亚胺(NHS)交联克服了这一问题,使其在pH为7.2的条件下稳定三个月。使用L6成肌细胞进行的MTT [3-(4,5-二甲基噻唑-2-基)-1,5-二苯基四氮唑溴盐] 测定证实了支架的生物相容性。在支架上建立了三维原代软骨细胞和神经元培养体系,并维持10天。苏木精-伊红(H&E)染色和SEM分析表明,软骨细胞和神经元分别附着在使用二甲基甲酰胺/四氢呋喃/丙酮和丙酮/乙醇制备的CAP支架上。