Repair of tendons treated with peracetic acid-ethanol and gamma irradiation by EDC combined with NHS: a morphological, biochemical and biomechanical study in vitro

Abstract

The purpose of this study was to investigate whether 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) combined with n-hydroxysuccinimide (NHS) can repair tendon damage caused by peracetic acid-ethanol and gamma irradiation sterilization. The semitendinosus tendons of 15 New Zealand white rabbits were selected as experimental materials, and the tendons were sterilized in a solution containing 1% (v/w) peracetic acid and 24% (v/w) ethanol. After 15 kGy gamma irradiation sterilization, the tendons were randomly divided into three groups (n = 10). The tendons were repaired with EDCs of 0, 2.5 and 5 mM combined with 5 mM NHS for 6 h, the tendons were temporarily stored at - 80 ± °C. The arrangement and spatial structure of collagen fibers were observed by light microscopy and scanning electron microscopy, the collagen type and collagen crimp period were observed under a polarizing microscope, and the collagen fibril diameter and its distribution were measured by transmission electron microscopy, from which the collagen fibril index and mass average diameter were calculated. The resistance of collagen to enzymolysis was detected by the free hydroxyproline test, and tensile fracture and cyclic loading tests of each group of tendons were carried out, from which the elastic modulus, maximum stress, maximum strain, strain energy density and cyclic creep strain were calculated. The obtained results showed that the gap between loose collagen fibers in the 0 mM control group was wider, the parallel arrangement of tendons in the 2.5 and 5 mM groups was more uniform and regular and the fiber space decreased, the crimp period in the 5 mM group was lower than that in the 0 mM group (P < 0.05), and the concentration of hydroxyproline in the 5 mM group (711.64 ± 77.95 μg/g) was better than that in the control group (1150.57 ± 158.75 μg/g). The elastic modulus of the 5 mM group (424.73 ± 150.96 MPa) was better than that of the 0 mM group (179.09 ± 37.14 MPa). Our results show that EDC combined with NHS can repair damaged tendons after peracetic acid-ethanol and gamma radiation treatment, and 5 mM EDC has better morphological performance, anti-enzymolysis ability and biomechanical properties than 2.5 mM EDC.

Keywords: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; Allografts; Collagen cross-linking; Peracetic acid; Tendon.