Abstract: 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.

Key words: allografts, tendon, collagen cross-linking, peracetic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, morphological observation