Apoptosis Occurs in Anterior Talofibular Ligament of Chronic Lateral Ankle Instability: Biochemical Evidence from Bench to Bed

Abstract

Category: Ankle; Basic Sciences/Biologics; Sports Introduction/Purpose: Chronic lateral ankle instability (CLAI) is a common entity that can result in degenerative arthritis if left untreated. CLAI is treated operatively, while acute ligament injury, so-called 'ankle sprain' is primarily treated nonoperatively with good clinical outcomes. This widely accepted opinion has been proved by clinical scoring system, physical examination, stress radiographs, ultrasonography, or magnetic resonance imaging. However, some surgeons often rush to perform the operative repair for acute ankle ligament injury. Apoptosis has been demonstrated as a considerable cause of ligament degeneration. Up to date, there are few reports of biomolecular and histochemical evidence focused on the 'apoptosis' that occurred in the torn anterior talofibular ligament (ATFL). The aim of this study is to elucidate the apoptosis that occurs within the ATFL of CLAI. Methods: Ligamentous tissues were collected from 15 patients undergoing modified Broström operation (group C) and 10 patients undergoing distal fibular fracture surgery as controls (group A). We allocated distal fibular fracture patients as an alternative to acute lateral ankle ligament injury to harvest acutely injured ATFL tissue. In both groups, the tissue samples were harvested at ATFL. Apoptotic cells were determined by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay. Caspase 3/7, 8, 9, and cytochrome c activities were measured to determine the intracellular apoptosis pathway using Western blot. Immunohistochemistry staining was also performed to detect the expression of caspases and cytochrome c. Results: The TUNEL assay revealed a large number of positive staining cells in group C that included ATFL of CLAI. The apoptotic activities of ligaments from group C were significantly higher than those of group A (P < 0.05). Immunohistochemistry also revealed an increase in expression of caspase 3/7, 8, 9, and cytochrome c more in group C than in group A. Conclusion: This study demonstrates that the degenerated ATFL following CLAI showed higher apoptotic activity than acutely sprained ligament. Based on our biochemical findings, we could remind previous understanding about surgical indication and timing of lateral ankle ligament injury. We suggest that a clinical relevance between our laboratory research and previous knowledge Further study should be conducted to focus on biological augmentation to reverse or prevent further apoptosis within the lateral ankle ligament complex including ATFL.