碳化二亚胺作为胶原蛋白交联剂的生物安全性评价

Evaluate biosecurity of I - ethyl -3 -3 - dimethyl - aminopropyl carbodiimide as crosslinking agent for collagen

目的对碳化二亚胺（EDAC）的生物安全性，探讨其作为交联剂是否有毒。方法取3只健康的大白兔，皮内注射浓度为1％和2％的EDAC生理盐水溶液200山，于2、4、24、48h和1周观察皮肤有无水肿、红斑，计算原发刺激指数，判断皮肤刺激反应类型；取20只健康的昆明小鼠，按照10μL／g腹腔注射2％的EDAC，于4、8、12、24和48h观察小鼠有无轻、中、重度毒性反应和死亡；用浓度为0．4％、1．0％、2．0％和4．0％的EDAC处理L929小鼠成纤维细胞，于6、12和24h加细胞计数试剂盒（CCK-8）培养4h，用酶标仪观察吸光度值，计算细胞相对生长率；用EDAC交联Ⅱ型胶原蛋白复合软骨微粒修复猪膝关节缺损的软骨，对照组未用EDAC交联Ⅱ型胶原蛋白修复猪膝关节软骨缺损，1个月后取标本，进行大体和组织切片观察关节内软骨情况。结果观察到1．0％和2．0％的EDAC在4h对皮肤有中度的刺激反应；2．0％的EDAC腹腔注射的小鼠出现重度毒性反应和死亡；不同浓度EDAC对L929细胞的生长均有一定抑制作用，且随着EDAC浓度的增加，其对细胞的抑制作用越强。EDAC交联Ⅱ型胶原蛋白组关节内软骨见不同程度的剥脱、软化，而对照组未见软骨破坏。结论皮肤刺激实验、急性全身毒性实验和细胞毒性实验均证明EDAC对活体组织和细胞具有毒性作用，原位成胶接种细胞或植入体内，可能需要考虑更换无毒的交联剂。

Objective To evaluate the biosecurity of 1 - ethyl - 3 - 3 - dimethyl - aminopropyl carbodiimide （EDAC） as crosslinking agent demonstrate whether EDAC are poisonous and provide theoreti- cal basis for the biosecurity for EDAC crosslink collagen. Methods Three New Zealand white rabbits were injected 1% , 2% EDAC 200 μl intradermally to observe the occurrence of swellin＇g and erythema in 2 hours, 4 hours, 24 hours, 48 hours and 1 week, then calculated the primary irritant index （PⅡ） and eval- uated the type of skin response to the EDAC. L929 mouse fibroblast cells were treated with 0.4% , 1.0% , 2. 0% , 4. 0% EDAC and cultured for 6 hours, 12 hours, 24 hours, then added cell counting kit- 8 （ CCK - 8 ） and cultured for 4 hours to observe optical density by using microplate reader and calculate the relative growth rate of cells. With EDAC crosslinked type Ⅱ collagen and composited particles of cartilage to repair cartilage defect, and the control groups used type Ⅱ collagen composited particle of cartilage. Taken specimens after one mouth, general tissue and histological section of cartilage were observed. Re- suits 1.0% and 2.0% EDAC have a moderate toxicity to skin in 4 hours. Different concentrations of EDAC can inhibit growth of L929 cells, and with the increase of concentration of EDAC, inhibition growth of cells will be stronger. EDAC crosslinking typeⅡ collagen groups was seen different degrees of softening and stripping, While the control group did not see cartilage damage. Conclusion Skin irritation test, a- cute systemic toxicity test and cell toxicity test demonstrated EDAC is toxic on living organ and cell.