Apoptosis Occurs in the Anterior Talofibular Ligament of Patients With Chronic Lateral Ankle Instability: An In Vitro Study : Clinical Orthopaedics and Related Research®

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Apoptosis Occurs in the Anterior Talofibular Ligament of Patients With Chronic Lateral Ankle Instability: An In Vitro Study

Choi, Youn-Ho MD1; Cho, Sung-Hyun MD1; Seo, JeongYong MD1; Ahn, Jae Hoon MD, PhD2; Kim, Yoon-Chung MD, PhD1

Author Information
Clinical Orthopaedics and Related Research: December 2022 - Volume 480 - Issue 12 - p 2420-2429
doi: 10.1097/CORR.0000000000002337

Abstract

Introduction

Ankle sprain is one of the most common musculoskeletal injuries [35, 40], accounting for 40% of all sports-related injuries [9, 45]. Ten percent of emergency department visits are for acute ankle injuries [6, 48]. Most acute sprains affect the lateral ankle, and anterior talofibular ligament injury is common [17, 32]. Anterior talofibular ligament injury causes anterolateral ankle instability; 33% of patients with an acute anterior talofibular ligament injury experience chronic lateral ankle instability [3, 46]. If untreated, chronic lateral ankle instability can lead to ankle arthritis [13]. Nonoperative management is the treatment of choice for acute anterior talofibular ligament injury [6, 35]. Studies have recommended early functional rehabilitation after initial nonoperative treatment as the standard for Grade I and II anterior talofibular ligament injuries [4, 8, 19, 22]. There is no consensus about the standard treatment for Grade III anterior talofibular ligament injuries, although they can also be treated nonoperatively. Surgery should be considered based on individual circumstances [7, 21, 45]. For patients with chronic lateral ankle instability, functional rehabilitation after initial treatment is also recommended, but if symptoms persist despite nonoperative treatment, ligament repair surgery may be recommended [6, 40]. Such widely accepted opinions have been validated using clinical scoring systems, physical examinations, stress radiography, ultrasonography, and MRI [6, 35, 45]. However, to the best of our knowledge, no attempt has been made to investigate biological evidence regarding the difference between chronic and acutely injured anterior talofibular ligaments.

We suspected that biological differences between chronic and acutely injured ligaments could influence differences in treatment strategies. Apoptosis refers to planned cell death and is critical for body homeostasis [42]. Apoptosis eliminates harmful cells and regulates cell populations [31, 33]. However, uncontrolled apoptosis can cause irreversible degenerative diseases such as osteoarthritis and neurodegeneration [5, 20, 23]. To our knowledge, Yuan et al. [50] were the first to report that apoptosis is involved in tendon degeneration after rotator cuff tear. Uysal et al. [44] found that excessive apoptosis was associated with degeneration of the human meniscus. We theorized that apoptosis might be involved in progressive anterior talofibular ligament degeneration after acute anterior talofibular ligament injury but more so after chronic lateral ankle instability. If it is revealed that apoptosis occurs more in the anterior talofibular ligament tissues of patients with chronic lateral ankle instability than in patients with acute anterior talofibular ligament injury, biochemical and histological differences between the two diseases can be demonstrated. This may provide additional evidence for different treatment strategies between chronic lateral ankle instability and acute anterior talofibular ligament injury.

The purpose of this study was to investigate whether apoptosis occurs in the anterior talofibular ligament of patients with chronic lateral ankle instability. We sought to (1) elucidate the difference in the extent of apoptosis between patients with chronic lateral ankle instability and those with acute anterior talofibular ligament injury. In addition, we asked: (2) What is the expression level of apoptotic enzymes such as caspases 3, 7, 8, and 9 and cytochrome c in each patient group? (3) Is there a correlation between apoptotic activities and the symptom duration period of chronic lateral ankle instability?

Patients and Methods

Study Design and Tissue Collection

We prospectively enrolled 50 patients between March 2019 and February 2021 from a single center (Fig. 1). The mean age of all patients was 42 ± 16 years. There were 26 males and 24 females. We treated 25 patients with chronic lateral ankle instability who reported persistent pain and mechanical instability for at least 3 months after their most recent lateral ankle sprain. All of them were considered potentially eligible based on the inclusion criteria that 6-week functional rehabilitation did not lead to an improvement in the patient’s pain and ankle instability compared with the first visit to the clinic. The chronic lateral ankle instability group was treated using an open modified Broström operation. During the procedure, the proximal portion of diseased anterior talofibular ligament tissue was collected (Fig. 2A). Three patients were excluded because the remaining ligament was so severely degenerated that biopsies were impossible, and another patient was excluded because the amount of tissue collected was not sufficient to make a sample for laboratory analysis, leaving 21 tissue samples from patients for analysis. We treated 25 patients with acute anterior talofibular ligament injury (control group) with confirmed concomitant anterior talofibular ligament injury during ankle fracture surgery. The ankle fractures were all unstable, including bimalleolar fractures, trimalleolar fractures, and ankle fracture-dislocations. Those were highly likely to involve anterior talofibular ligament injury. During open reduction and internal fixation, the condition of the anterior talofibular ligament was visually checked. After confirming its rupture or laxity by intraoperative anterior drawer test, the proximal portion of injured anterior talofibular ligament tissue was collected (Fig. 2B). We designated patients with acute anterior talofibular ligament injury with ankle fractures as a control group instead of those with isolated anterior talofibular ligament injury by acute ankle sprains for the following reasons. Given that acute ankle sprains are usually well treated with nonoperative treatment rather than operative treatment, it is not easy to collect injured anterior talofibular ligament tissue by surgical biopsy from patients with isolated acute ankle sprain in an actual clinical setting. Five patients were excluded because their anterior talofibular ligaments were deemed stable with the intraoperative anterior drawer test, and another three patients were excluded because the injured ligaments were severely damaged and the quality of collected tissue was insufficient to make a sample for laboratory analysis, leaving 17 tissue specimens from patients for analysis.

F1
Fig. 1:
This flowchart details the patient inclusion and exclusion criteria for this study.
F2
Fig. 2:
A-B The proximal portion of anterior talofibular ligament tissue (white arrows) near the distal fibula (white asterisks) was harvested during (A) the modified Broström operation in patients from the chronic lateral ankle instability group and (B) open reduction and internal fixation of distal fibular fractures in the setting of acute anterior talofibular ligament injury in the acute anterior talofibular ligament injury group.

All operations were performed by the same surgeon (Y-CK). All procedures were performed through a lateral approach using a J-shaped incision to investigate the anterior talofibular ligament. Ultimately, tissues were collected from 21 patients (11 males and 10 females) in the chronic lateral ankle instability group with a mean age of 37 ± 14 years and from 17 patients (6 males and 11 females) in the acute anterior talofibular ligament injury group with a mean age of 49 ± 17 years. There was no difference in mean age or sex distribution between the two groups.

Ligamentous tissues from both groups were stored at -70°C by flash freezing. A portion of each of the ligamentous tissues was also immediately fixed in 10% formalin for 4 to 6 hours and embedded in paraffin for in situ detection of apoptotic cells and immunohistochemistry.

TUNEL Assay

Apoptotic cells were detected in situ using a commercially available terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL) apoptosis kit (ab206386, Abcam). The TUNEL assay is a method for detecting DNA fragments caused by apoptosis using paraffin-embedded human ligament tissues sections (4-μm thick). Terminal deoxynucleotidyl transferase is an enzyme that recognizes the exposed 3'-OH ends of DNA fragments and catalyzes the attachment of biotinylated nucleotides to the ends of 3'-OH [10]. Biotin labeling uses streptavidin-conjugated peroxidase and the colorimetric substrate diaminobenzidine. Labeled DNA fragments are brown in color.

The sample sections were deparaffinized and rehydrated in graded alcohol solutions. After incubating the sections in proteinase K (20 μm/mL) for 15 minutes at room temperature and washing them in deionized water, we inactivated endogenous peroxidase by immersing the sections in 3% hydrogen peroxide solution for 5 minutes at room temperature. Then, the sections were incubated in an equilibration buffer and terminal deoxynucleotidyl transferase at 37°C for 1 hour. After washing the sections in phosphate-buffered saline and incubating them in streptavidin-conjugated peroxidase for 30 minutes at room temperature, we immersed the sections in 3,3'-diaminobenzidine hydrochloride. Subsequently, the sections were counterstained with methyl green, and normal cell nuclei were observed (in green). Apoptotic cells were identified as cells with brown-stained nuclei under optical microscopy, and the number of cells was counted. Eight slides from the chronic lateral ankle instability group and nine slides from the acute anterior talofibular ligament injury group were evaluated for each patient ligament. Three of 10 separate fields were combined for reporting purposes for each cohort [10, 25, 30].

Caspases and Cytochrome c Activity

The expression levels of caspase 3, 7, 8, and 9 and cytochrome c, which are proteins contributing to apoptosis, were quantitated via Western blotting. There are two pathways of apoptosis: intrinsic and extrinsic. Caspase 8 is involved in the latter pathway, and caspase 9 and cytochrome c are involved in the former [24, 28]. Caspases 3 and 7 are then activated to complete apoptosis [12, 29, 47].

We added 100 μL of radioimmunoprecipitation assay buffer (Thermo Scientific) along with a protease cocktail inhibitor and phosphatase inhibitor to each sample followed by homogenization on ice and centrifugation at 14,000 rpm for 15 minutes at 4°C. Then, we transferred each supernatant to a new tube. We measured protein concentrations using the standard Bradford assay. The samples were loaded onto 10% (w/v) sodium dodecyl sulfate-polyacrylamide gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene fluoride membranes, and the membranes were blocked in blocking buffer (5% [w/v] bovine serum albumin in tris-buffered saline with 0.1% [v/v] tween 20) for 1 hour. The membranes were then incubated with monoclonal antibodies against human caspases 3, 7, 8, and 9 (Lab Vision Corp) and cytochrome c (Cell Signaling Technology) at 1:1000 dilution overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). The protein bands were imaged using an enhanced chemiluminescence kit (GE Healthcare Bio-Sciences Corp) and quantitated with ImageJ software (National Institutes of Health). In the chronic lateral ankle instability group, eight samples for caspase 3 and nine samples for caspases 7, 8, 9 and cytochrome c were evaluated for each patient. In the acute anterior talofibular ligament injury group, nine samples for caspases 3 and 9 and cytochrome c and 10 samples for caspases 7 and 8 were evaluated for each patient. Three samples were combined for reporting purposes for each cohort. The method used is similar to that performed by Ha et al. [11] to evaluate apoptosis in the nucleus pulposus.

Using immunohistochemistry, caspases 3, 7, 8, and 9 and cytochrome c were detected in situ. After overnight fixation in 10% (v/v) buffered formalin, the samples were washed and dehydrated through a graded series of alcohol baths. Paraffin-embedded samples were blocked in 5% (v/v) horse serum for 20 minutes, and 4-μm-thick sections were prepared, deparaffinized, and rehydrated in a graded series of alcohol baths. Then, the samples were subjected to heat-induced epitope retrieval with a citrate buffer with a pH of 6.0. Endogenous peroxidase was inactivated via immersion in 0.3% (v/v) hydrogen peroxide. The monoclonal antibodies listed above were added, and incubation proceeded overnight at 4°C. After washing the samples with phosphate-buffered saline, we added a biotinylated secondary antibody (Vectastain Elite ABC Kit, Vector Laboratories) for 20 minutes, which resulted in the development of streptavidin-conjugated peroxidase and chromogen (3,3ʹ-diaminobenzidine tetrachloride). Protein distributions were observed via optical microscopy after counterstaining with hematoxylin. In the chronic lateral ankle instability group, eight samples for caspase 3 and nine samples for caspases 7, 8, and 9 and cytochrome c were evaluated for each patient. In the acute anterior talofibular ligament injury group, nine samples for caspases 3 and 9 and cytochrome c and 10 samples for caspases 7 and 8 were evaluated for each patient. Three samples were combined for reporting purposes for each cohort. The method used is similar to that performed by Lee et al. [30] to evaluate apoptosis in diseased rotator cuffs.

Relationship Between Apoptotic Activities and the Duration Period of Chronic Lateral Ankle Instability Symptoms

The relationship between the relative optical densities of each enzyme was confirmed by Western blot analysis, and the duration of the patient’s symptoms was analyzed by correlation analysis using a Pearson correlation coefficient. Symptom duration was defined as the period from the first onset of any symptoms identified in the patient’s medical history to the time of surgery. Of the 21 patients in the chronic lateral ankle instability group, 19 patients were finally evaluated; two patients who had an ambiguous response about the onset of symptoms were excluded.

Primary and Secondary Study Outcomes

Our primary study goal was to identify the different extent of apoptosis between patients with chronic lateral ankle instability and those with acute anterior talofibular ligament injury. To achieve this, we investigated apoptotic cells in injured anterior talofibular ligaments by TUNEL assay. We quantified the expression level of apoptotic enzymes by Western blot and detected apoptotic enzymes by immunohistochemistry. We compared results and data between the two groups.

Our secondary study goal was to determine whether there was a correlation between apoptotic activities and the symptom duration of chronic lateral ankle instability. To achieve this, we performed statistical analyses for each apoptotic enzyme activity and the duration of the patient’s symptom.

Ethical Approval

We obtained ethical review board approval to perform this study.

Statistical Analysis

We used a nonpaired t-test with continuity correction for the comparison of protein expression levels between the two groups. The relationship between the expression of apoptotic enzyme and the duration of chronic lateral ankle instability symptoms was analyzed using the Pearson correlation coefficient. All analyses were performed with R software (version 3.5.3; R Development Core Team, R Foundation for Statistical Computing). The level of statistical significance was set at p < 0.05.

Results

Cell Apoptosis

We identified more apoptosis in the chronic lateral ankle instability group than in the acute anterior talofibular ligament injury group. The TUNEL assay qualitatively demonstrated more brown (apoptotic) cells in the chronic lateral ankle instability group than in the acute anterior talofibular ligament injury group (Fig. 3).

F3
Fig. 3:
A-B Representative images of anterior talofibular ligament cross sections of TUNEL staining. TUNEL-positive (apoptotic) cells were stained brown and normal cells were stained blue. (A) In the chronic lateral ankle instability group, more TUNEL-positive cells (black arrows) were evident than in (B) the acute anterior talofibular ligament injury group (counterstain, methyl green; original magnification, x200 for Fig. 3A and x400 for Fig. 3B).

Caspases and Cytochrome c Activity

The expression level of five apoptotic enzymes was higher in the chronic lateral ankle instability group than in the acute anterior talofibular ligament injury group (Fig. 4). The relative optical density of caspase 3 was 117 in the chronic lateral ankle instability group and 59 in the acute anterior talofibular ligament injury group (mean difference 58 [95% confidence interval (CI) 31 to 86]; p < 0.001). The relative optical density for caspase 7 was 138 in the chronic lateral ankle instability group and 45 in the acute anterior talofibular ligament injury group (mean difference 93 [95% CI 58 to 128]; p < 0.001), whereas for caspase 8, it was 126 in the chronic lateral ankle instability group and 68 in the acute anterior talofibular ligament injury group (mean difference 58 [95% CI 29 to 89]; p < 0.001), and for caspase 9, it was 128 in the chronic lateral ankle instability group and 54 in the acute anterior talofibular ligament injury group (mean difference 74 [95% CI 44 to 104]; p < 0.001). The relative optical density of cytochrome c was 139 in the chronic lateral ankle instability group and 51 in the acute anterior talofibular ligament injury group (mean difference 88 [95% CI 46 to 129]; p < 0.001). Immunohistochemistry qualitatively demonstrated that caspases 3, 7, 8, and 9 and cytochrome c were detected more in the vascular endothelial cells of the chronic lateral ankle instability group ligaments than in those of the acute anterior talofibular ligament injury group (Fig. 5).

F4
Fig. 4:
Western blotting data are shown in this graph. The apoptotic activities of ligaments from the chronic lateral ankle instability group were higher than those of ligaments in the acute anterior talofibular ligament injury group (p < 0.001 for all comparisons). aVisualization of each mean difference in expression levels of five apoptotic enzymes between the two groups.
F5
Fig. 5:
Cross section of immunohistochemical staining of caspases 3, 7, 8, and 9 and cytochrome c is shown. The proteins were stained brown; more stained cells were observed in the chronic lateral ankle instability group than in the acute anterior talofibular ligament injury group (counterstain, hematoxylin; original magnification, x200).

Correlation Between Apoptotic Activities and the Symptom Duration of Chronic Lateral Ankle Instability

Apoptotic activity was not correlated with the symptom duration of chronic lateral ankle instability for any of the enzymes examined [39]. The Pearson correlation coefficient was 0.22 (95% CI -0.25 to 0.69; p = 0.36) for caspase 3, 0.29 (95% CI -0.16 to 0.74; p = 0.23) for caspase 7, and 0.29 (95% CI -0.16 to 0.74; p = 0.23) for caspase 9.

Discussion

An anterior talofibular ligament injury can progress to chronic lateral ankle instability [3, 46]. Acute anterior talofibular ligament injury is usually treated nonoperatively, whereas chronic lateral ankle instability often requires ligament repair surgery. We wondered why the clinical course and treatment strategies between the two diseases were different. We sought to answer that question by investigating the biological evidence because previous studies had provided only clinical or radiological evidence [6, 35, 45]. Based on previous studies that apoptosis is associated with musculoskeletal tissue degeneration [26, 30, 50], we sought to identify the biological differences between chronic lateral ankle instability and acute anterior talofibular ligament injury by focusing on apoptosis associated with degeneration of the anterior talofibular ligament. The first key finding of our study was that qualitatively more apoptosis occurred in anterior talofibular ligament tissues in the chronic lateral ankle instability group than in the acute anterior talofibular ligament injury group. The second important result was that more apoptotic enzymes were expressed in the chronic lateral ankle instability group than in the acute anterior talofibular ligament injury group. These results demonstrated biochemical and histological differences between chronic lateral ankle instability and acute anterior talofibular ligament injury. More apoptosis in the ligament suggests that ligament degeneration may also progress further. In this study, we have provided biological evidence as to why acute ankle anterior talofibular ligament injury is better treated with nonoperative treatment than chronic lateral ankle instability and why surgical ligament repair is more required for chronic lateral ankle instability than acute anterior talofibular ligament injury.

Limitations

Our study has several limitations. First, the number of included samples was reduced from 50 to 38. Although the final sample size was small, we do not believe this to be a disqualifying shortcoming because it was sufficient to demonstrate differences between the chronic lateral ankle instability group and the acute anterior talofibular ligament injury group. However, we were not able to demonstrate a correlation between the level of apoptotic enzyme expression and the symptom duration of chronic lateral ankle instability. The Pearson correlation coefficient between these two factors was not statistically significant. Given our findings related to this topic, this might have been achieved with a larger sample size. Second, it is possible that the group of patients in this study might differ from patients of other races. However, we do not believe this is likely because reports have been made that the rate of ACL rupture in the knee differs by patient anatomy rather than race [41, 43]. Third, the results according to sex were not analyzed. We analyzed only demographic data to minimize selection bias between males and females. We found no difference in the initial patient enrollment. Examining possible differences between the two groups by sex requires designing a new prospective study on the subject. Fourth, in the acute anterior talofibular ligament injury group, anterior talofibular ligament tissue could only be collected from patients presenting with acute anterior talofibular ligament injury and distal fibular fracture, for which surgery was required. It cannot be assumed that the anterior talofibular ligament in patients presenting with an acute anterior talofibular ligament injury without bone fracture would demonstrate the same findings as in the acute anterior talofibular ligament injury group of our study. However, since acute anterior talofibular ligament injury is usually treated well with nonoperative treatment, it is not easy to perform a surgical biopsy of acutely injured ligament tissue from patients with isolated acute ankle sprain in an actual clinical setting. Fifth, tissue samples in our study were collected once at the time of surgery, so it was difficult to identify how ligament tissue changes histologically over time. Patients with chronic lateral ankle instability often have a long history of symptoms, and the duration of symptoms can only be assessed by relying on the patient’s memory, which contains unavoidable recall biases.

Discussion of Key Findings

We found that more apoptosis occurred in anterior talofibular ligament tissues in chronic lateral ankle instability than in acute anterior talofibular ligament injury. This suggests that ligament degeneration may have progressed more in chronic lateral ankle instability than in acute anterior talofibular ligament injury. However, the exact role of apoptosis in anterior talofibular ligament degeneration remains unclear. Although chondrocyte apoptosis in patients with osteoarthritis has been extensively studied by previous researchers, the relationship between apoptosis and cartilage degeneration is not fully understood because it is difficult to determine how apoptosis contributes to degeneration. It is unclear whether chondrocyte apoptosis causes osteoarthritis or whether apoptosis is merely a byproduct of osteoarthritis as well [1, 14, 15, 18]. However, it is possible to infer how apoptosis might contribute to anterior talofibular ligament degeneration. Yuan et al. [50] found that rotator cuff degeneration was accompanied by apoptosis of fibroblasts or fibroblast-like cells, which are the major cells of tendons. These cells produce and maintain the extracellular matrix collagen and noncollagenous components [2]. Therefore, apoptosis may impair the synthesis of the extracellular matrix and thus cause tendon degeneration [37, 50]. We suggest that anterior talofibular ligament degeneration has a similar etiology. In the immunohistochemistry analyses of our study, apoptotic enzymes were found in vascular endothelial cells of the ligament. Apoptosis of vascular endothelial cells is known to cause vascular damage, vascular leak, coagulation, and inflammation and has the potential to further accelerate tissue degeneration [49].

We found that the expression of all proteins involved in apoptotic activities was higher in the chronic lateral ankle instability group than in the acute anterior talofibular ligament injury group. With this key finding, we can discuss the causes of apoptosis. There may be various causes of apoptosis in damaged anterior talofibular ligaments. Apoptosis involves intrinsic (mitochondrial) and extrinsic pathways [27, 38]. The intrinsic pathway is activated by problems in the cell, such as DNA damage or intracellular oxidative stress [16, 36]. On the other hand, extrinsic pathway activity commences with binding of the Fas ligand (an extrinsic factor) to the Fas receptor (CD95) on the cell membrane [34]. Caspase 9 and cytochrome c are involved in the intrinsic pathway and caspase 8 in the extrinsic pathway [51]. Caspases 3 and 7 receive signals from these proteins in both pathways and execute apoptosis [16]. In our study, since expression of all five apoptotic enzymes was increased substantially, it can be assumed that both intrinsic and extrinsic pathways were activated. DNA damage, intracellular oxidative stress, and Fas ligand, which are the causes of apoptosis as suggested above, may be involved together. However, a gap in knowledge remains as to which of these factors dominates. Future investigations into which factors dominate ligament apoptosis and knowing how to prevent these factors will enable the development of novel therapies for the treatment of chronic lateral ankle instability.

We found no correlation between increased expression of caspases 3, 7, and 9 and symptom duration in the chronic lateral ankle instability group. Although patients were recruited prospectively in this study, it was difficult to identify histological changes in the ligament over time because tissue samples were collected once at the time of surgery. However, a positive correlation between enzyme expression level and symptom duration could be useful to evaluate apoptotic activity with prolonged symptom duration. To further clarify whether such a correlation exists, differently designed studies with larger samples should be conducted in the future.

Conclusion

In our study of chronic lateral ankle instability, the apoptotic activity of the anterior talofibular ligament was higher than in acute anterior talofibular ligament injury. The anterior talofibular ligament in chronic lateral ankle instability is more associated with degenerative changes than the ligament in acute anterior talofibular ligament injury. Our results revealed biological differences in the anterior talofibular ligament between the two diseases. This may support the difference in treatment strategies for chronic lateral ankle instability that requires surgical repair and acute anterior talofibular ligament injury that does not require urgent surgical repair. Further research should focus on apoptotic timing as well as biological augmentation to reverse or prevent apoptosis within anterior talofibular ligament. If further studies of methods to downregulate the effects of DNA damage, intracellular oxidative stress, and Fas ligand that causes apoptosis are investigated, a new treatment strategy for chronic lateral ankle instability might be possible.

Acknowledgments

We thank Senior Research Engineer Seung June Noh BSc for his assistance with the experiments and laboratory analyses. We thank Sei Won Kim MD, PhD for his help with the statistical analysis. Also, we thank Textcheck (http://www.textcheck.com/certificate/jL9gdM) for English-language editing.

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