关键词: 前交叉韧带, 动物模型, 免疫组化, 透射电镜, 超微结构, 韧带化

Abstract: BACKGROUND: As a dominant breed pig in southwest China, the southern Yunnan small-ear pig has been widely used as an experimental animal in the basic research of other disciplines, but there are still no reports on its application in anterior cruciate ligament reconstruction.
OBJECTIVE: To establish a large animal model of the southern Yunnan small-ear pig with anterior cruciate ligament with autologous Achilles tendon was established.
METHODS: Twenty adult female Yunnan small-ear pigs were equally randomized into two groups. In the autologous Achilles tendon group, the right knee anterior cruciate ligament was reconstructed with autologous Achilles tendon as a graft, while in the sham-operated group, a similar operation was performed on the right knee without any treatment of the anterior cruciate ligament. General conditions of each pig were observed and recorded before and 12 months after surgery. Ligaments and grafts were taken for gross observation and MAS scoring. Hematoxylin-eosin staining was performed to observe morphological characteristics of ligaments. The staining and arrangement of type I and type III collagen were evaluated by immunohistochemistry. Transmission electron microscopy was used to observe the type, size, diameter, ratio, and distribution of collagen fibers in ligaments. 
RESULTS AND CONCLUSION: All animals had normal diet and activity, good wound healing, no obvious inflammatory reaction, no local purulent infection, and no significant changes in mental and urinary conditions compared with those before surgery. The reconstructed cruciate ligament of the knee was intact, with no stiffness and normal range of motion. Both the anterior drawer and Lachman tests were negative. Gross observation of the graft: 12 months after surgery, the grafts was in good position, with good integrity, obvious tension, ligament color close to the original anterior cruciate ligament, and complete surface synovial coverage. Most of the intraarticular ligaments in the autologous Achilles tendon group were defined as MAS I type and a few were defined as MAS II type. In the sham-operated group, the intraarticular ligament was defined as MAS I type. Hematoxylin-eosin staining indicated that, 12 months after surgery, collagen fibers in the autologous Achilles tendon group began to appear bundled, isotropic, and uniformly arranged, with more obvious isotropic corrugations, and the nuclei were mainly linear or spindle-shaped, which were similar to those in normal anterior cruciate ligament tissue of the sham-operated group. Immunohistochemistry results indicated that, 12 months after surgery, there was a higher expression of type I collagen and significantly less expression of type III collagen in the reconstructed anterior cruciate ligament in the autologous Achilles tendon group. The degree of type I and type III staining was similar in the two groups. Under the transmission electron microscope, the diameter, arrangement and density of collagen fibers in the reconstructed anterior cruciate ligament of the autologous Achilles tendon group were similar to those of the original anterior cruciate ligament at 12 months after surgery, indicating that the ligament remodeling process had been basically completed in the autologous Achilles tendon group at 12 months after surgery. Through a comprehensive evaluation of animal general conditions, ligament general view, MAS score, hematoxylin-eosin staining, immunohistochemistry, and transmission electron microscopy observation, we successfully established a large animal model of anterior cruciate ligament reconstruction using autogenous Achilles tendon in southern Yunnan small-ear pigs, with good morphological, histological and ultrastructural results. 

Key words: anterior cruciate ligament, animal model, immunohistochemistry, transmission electron microscopy, ultrastructure, ligamentization