Nam Kwangwoo, Kimura Tsuyoshi, Kishida Akio
Division of Biofunctional Molecules, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo, Japan.
Macromol Biosci. 2008 Jan 9;8(1):32-7. doi: 10.1002/mabi.200700206.
To control the crosslinking rate of the collagen gel, ethanol/water co-solvent was adopted for the reaction solvent for the collagen microfibril crosslinking. Collagen gel was prepared by using EDC and NHS as coupling agents. Ethanol did not denaturate the helical structure of the collagen and prevented the hydrolysis of EDC, but showed the protonation of carboxylate anions. In order to control the intra- and interhelical crosslink of the collagen triple helix, variations of the mole ratio of carboxyl group/EDC/NHS, and of the ethanol mole concentration were investigated. Increase in the EDC ratio against the carboxyl group increased the crosslinking rate. Furthermore, an increase in the ethanol mole concentration resulted in an increase of the crosslinking rate until ethanol mole concentration was 0.12, but showed gradual decrease as the ethanol mole concentration was further increased. This is because the adsorption of solvent by the collagen gel, protonation of carboxylate anion, and hydrolysis of EDC is at its most optimum condition for the coupling reaction when the ethanol mole concentration is 0.12. The re-crosslinking of the collagen gel showed an increase in the crosslinking rate, but did not show further increase when the coupling reaction was executed for the third time. This implied that the highest possible crosslinking rate for the intra- and interhelical is approximately 60% when EDC/NHS is used.
为了控制胶原蛋白凝胶的交联速率，采用乙醇/水共溶剂作为胶原蛋白微纤维交联反应的溶剂。使用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）作为偶联剂制备胶原蛋白凝胶。乙醇不会使胶原蛋白的螺旋结构变性，还能防止EDC水解，但会使羧酸根阴离子质子化。为了控制胶原蛋白三螺旋的螺旋内和螺旋间交联，研究了羧基/EDC/NHS摩尔比以及乙醇摩尔浓度的变化。相对于羧基，EDC比例的增加会提高交联速率。此外，乙醇摩尔浓度的增加会导致交联速率增加，直到乙醇摩尔浓度达到0.12，但随着乙醇摩尔浓度进一步增加，交联速率会逐渐降低。这是因为当乙醇摩尔浓度为0.12时，胶原蛋白凝胶对溶剂的吸附、羧酸根阴离子的质子化以及EDC的水解处于偶联反应的最适宜条件。胶原蛋白凝胶的再交联显示交联速率增加，但当进行第三次偶联反应时，交联速率没有进一步增加。这意味着当使用EDC/NHS时，螺旋内和螺旋间的最高可能交联速率约为60%。
