Barnes Catherine P, Pemble Charles W, Brand David D, Simpson David G, Bowlin Gary L
Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia 23284-3067, USA.
Tissue Eng. 2007 Jul;13(7):1593-605. doi: 10.1089/ten.2006.0292.
In trying to assess the structural integrity of electrospun type II collagen scaffolds, a modified but new technique for cross-linking collagen has been developed. Carbodiimides have been previously used to cross-link collagen in gels and in lyophilized native tissue specimens but had not been used for electrospun mats until recently. This cross-linking agent, and in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), is of extreme interest, especially for tissue-engineered scaffolds composed specifically of native polymers (e.g., collagen), because it is a zero-length cross-linking agent that has not been shown to cause any cytotoxic reactions. The unique aspect of the cross-linking protocol in this study involves the use of ethanol as the solvent for the cross-linking agent, because the pure collagen electrospun mats immediately disintegrate when placed in an aqueous solution. This study examines 2 concentrations of EDC with and without the addition of N-hydroxysuccinimide to the reaction (which has been shown to result in higher cross-linking yields in aqueous solutions) to test the hypothesis that the use of EDC in a nonaqueous solution will cross-link electrospun type II collagen fibrous matrices in a comparable manner to typical glutaraldehyde fixation protocols. The use of EDC is compared with the cross-linking effects of glutaraldehyde via mechanical testing (uniaxial tensile testing) and biochemical testing (analysis of the percentage of free amino groups). The stress-strain curves of the cross-linked samples demonstrated uniaxial tensile behavior more characteristic of native tissue than do the dry, untreated samples. The heated, 50% glutaraldehyde cross-linking protocol resulted in a mean peak stress of 0.76 MPa, a mean strain at break of 127.30%, and a mean tangential modulus of 0.89 MPa; mean values for the samples treated with the EDC protocols ranged from 0.35 to 0.60 MPa for peak stress, from 111.83 to 159.23% for strain at break, and from 0.57 to 0.92 MPa for tangential modulus. Low and high concentrations (20 mM and 200 mM, respectively) of EDC alone were comparable in extent of cross-linking (29% and 29%, respectively) to the heated 50% glutaraldehyde cross-linking protocol (30% cross-linked).
在试图评估静电纺丝II型胶原蛋白支架的结构完整性时，已开发出一种改良的新型胶原蛋白交联技术。碳二亚胺此前已用于在凝胶和冻干的天然组织标本中交联胶原蛋白，但直到最近才用于静电纺丝垫。这种交联剂，特别是1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐（EDC），备受关注，尤其是对于由天然聚合物（如胶原蛋白）组成的组织工程支架，因为它是一种零长度交联剂，尚未显示出会引起任何细胞毒性反应。本研究中交联方案的独特之处在于使用乙醇作为交联剂的溶剂，因为纯胶原蛋白静电纺丝垫放入水溶液中会立即分解。本研究考察了2种浓度的EDC，反应中添加和不添加N-羟基琥珀酰亚胺（已证明在水溶液中可产生更高的交联产率），以检验在非水溶液中使用EDC将以与典型戊二醛固定方案相当的方式交联静电纺丝II型胶原蛋白纤维基质的假设。通过机械测试（单轴拉伸测试）和生化测试（游离氨基百分比分析），将EDC的使用与戊二醛的交联效果进行比较。交联样品的应力-应变曲线显示出比干燥、未处理样品更具天然组织特征的单轴拉伸行为。加热的50%戊二醛交联方案产生的平均峰值应力为0.76 MPa，平均断裂应变率为127.30%，平均切线模量为0.89 MPa；用EDC方案处理的样品的峰值应力平均值范围为0.35至0.60 MPa，断裂应变率范围为111.83至159.23%，切线模量范围为0.57至0.92 MPa。单独使用低浓度和高浓度（分别为20 mM和200 mM）的EDC的交联程度（分别为29%和29%）与加热的50%戊二醛交联方案（30%交联）相当。
