碳化二亚胺联合N-羟基琥珀酰亚胺交联过氧乙酸-乙醇处理后的肌腱:体外形态学特征

Peracetic acid-ethanol treated tendons crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide combined with N-hydroxysuccinimide:morphological features in vitro

背景:过氧乙酸已被证明能有效灭活异体肌腱中的潜在微生物,但会使胶原分子内、分子间交联受到破坏,因此如何减弱或者修复这种损伤,灭菌的同时改善肌腱形态学特性是一个待解决的问题。目的:探讨碳化二亚胺/N-羟基琥珀酰亚胺交联是否对过氧乙酸-乙醇处理后的肌腱起保护作用。方法:选取18只新西兰大白兔,切取半腱肌肌腱(36条)作为实验材料。将肌腱分3组处理,每组12条:对照组肌腱脱细胞后置入过氧乙酸-乙醇溶液中进行灭菌处理;实验1组在对照组处理的基础上,置入MES缓冲液中浸泡24 h,置入含50 mmol/L MES、2.5 mmol/L碳化二亚胺、5 mmol/L N-羟基琥珀酰亚胺的乙醇交联液中浸泡6 h;实验2组在对照组处理的基础上,置入MES缓冲液中浸泡24 h,置入含50 mmol/L MES、5 mmol/L碳化二亚胺、5 mmol/L N-羟基琥珀酰亚胺的乙醇交联液中浸泡6 h;3组处理后均进行伽马辐照灭菌,分别进行光学显微镜、扫描电镜、透射电镜与偏光显微镜观察。结果与结论:①光学显微镜、扫描电镜:空白组胶原纤维无序性较强,肌腱细胞散在分布,腱束间连接松散纤维间隙较宽,纤维缠绕在一起,波形紊乱;两实验组肌腱平行排列且均匀规则,呈波浪形,纤维间隙小,以实验2组表现更为显著;②透射电镜:实验2组胶原原纤维直径、胶原原纤维指数大于对照组、实验1组(P<0.05),实验1组、实验2组胶原纤维质量平均直径大于对照组(P<0.05),对照组胶原纤维密度大于实验1组、实验2组(P<0.05);③偏光显微镜:3组均为Ⅰ型胶原纤维占比较大、Ⅲ型胶原纤维占比较小,对照组纤维密度较小,间隙较宽松;实验1组纤维密度有所增加,实验2组纤维间隙最小、密度最为大,对照组胶原卷曲周期大于实验1组、实验2组(P<0.05);④结果表明,碳化二亚胺联合N-羟基琥珀酰亚胺交联处理能在微观角度改善肌腱的受损程度,对过氧乙酸-乙醇处理后肌腱起保护作用,且碳化二亚胺浓度为5 mmol/L较2.5 mmol/L处理可获得更好的肌腱形态学特征。

BACKGROUND:Peracetic acid has been proven to effectively inactivate potential microorganisms in allograft tendons,but disrupts intramolecular and intermolecular cross-links of collagen.Therefore,how to weaken or repair this kind of damage and improve the morphological properties of tendon while sterilizing is a problem to be solved.OBJECTIVE:To explore whether 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide can protect tendons treated peracetic acid-ethanol.METHODS:Eighteen New Zealand white rabbits were selected as model animals,and the semitendinosus tendons were cut as experimental materials(n=36).Tendons were divided into three groups(n=12).The tendons in the control group were decellularized and placed in peracetic acid-ethanol solution for sterilization.On the basis of the treatment of the control group,the tendons in the experimental group 1 were placed in MES buffer solution for 24 hours,and then placed in the ethanol cross-linking solution containing 50 mmol/L MES,2.5 mmol/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,and 5 mmol/L N-hydroxysuccinimide for 6 hours.On the basis of the treatment of the control group,the tendons in the experimental group 2 were placed in MES buffer solution for 24 hours,and then placed in the ethanol cross-linking solution containing 50 mmol/L MES,5 mmol/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,and 5 mmol/L N-hydroxysuccinimide for 6 hours.After treatment,the tendons were sterilized by gamma irradiation and observed by optical microscope,scanning electron microscope,transmission electron microscope,and polarizing microscope.RESULTS AND CONCLUSION:(1)Optical microscope and scanning electron microscope:In the blank group,the collagen fibers were more disordered;tenocytes were scattered;the connection between tendon bundles was loose and the fiber gap was wider;the fibers were entangled together;the waveform was disordered.The parallel arrangement of tendons in the two experimental groups was more uniform,regular and wavy,and the interfiber gap became smaller and the performance in the experimental group 2 was more significant.(2)Transmission electron microscope:Collagen fibril diameter and collagen fibril index in experimental group 2 were greater than those in control group and experimental group 1(P<0.05).The average diameter of collagen fibers in experimental group 1 and experimental group 2 was greater than that in the control group(P<0.05).The density of collagen fibers in the control group was greater than that in the experimental group 1 and the experimental group 2(P<0.05).(3)Polarizing microscope:In the three groups,the proportion of type Ⅰ collagen fibers was relatively large,and the proportion of type Ⅲ collagen fibers was relatively small.The control group had less fiber density and loose gaps.In the experimental group 1,fiber density increased.The fiber gap of the experimental group 2 was the smallest and the density was the largest.The collagen crimp cycle in the control group was greater than that in the experimental group 1 and the experimental group 2(P<0.05).(4)The results showed that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide combined with N-hydroxysuccinimide could lessen the degree of tendon damage at the microscopic level,and could protect the tendon after peracetic acid-ethanol treatment.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide concentration of 5 mmol/L had better morphological characteristics than 2.5 mmol/L.