Between May 2013 and April 2019, we conducted this non-randomized, controlled study at our department. Over the 6-year period, 84 patients sustained a unilateral lateral ankle instability and received arthroscopic all-inside lateral collateral ligament repair were prospectively included. This protocol was approved by the Institutional Review Board of Wenzhou Medical University, and each participant gave informed written consent before enrollment.
The diagnosis of chronic lateral ankle instability was confirmed mainly by clinical examination and magnetic resonance imaging (MRI) [12]. Exclusion criteria were cases with a follow-up less than one year, open injuries at or near the surgical site, ankle fracture, serious synovitis, peripheral vascular and neuromuscular disorders. Then 32 cases were excluded, resulting in the enrolment of 52 cases, in which 27 patients who were treated surgically with the tourniquet inflated were grouped into the Control group, and 25 cases with the tourniquet applied, but not inflated during operation were grouped into the non- tourniquet (NT) group.
All procedures were performed by one of two experienced surgeons. The normal saline in a 3-L bag without any other additional agent was used as the irrigation fluid. The arthroscopic pump (Arthrex Inc, Naples, FL, USA) for fluid management was applied with the intra-articular pressure set to 40 mmHg. A thigh tourniquet was applied in all patients. But it was inflated to 350 mmHg in Control group only. In NT group, the tourniquet was not inflated and served as a security device in case of acute massive bleeding. After adequate anaesthesia, the patient was kept in the supine position and operated arthroscopically through standard anteromedial and anterolateral portals with the help of anatomic landmarks. Diagnostic arthroscopy was performed firstly though the anteromedial portal, and associated intra- articular lesions were managed accordingly, such as the osteochondral lesions of the talus were treated by chondroplasty, and anterior tibial osteophytes were treated by cheilectomy. An accessory anterolateral portal was established approximately 1 cm proximal to the fibular tip and just anterior to it. Then the anterior talofibular ligament (ATFL) was completely detached off the fibular origin and the fibular footprint areas was observed. After preparing the bone bed, a 3.5- mm bioabsorbable suture anchor (Arthrex, Naples, FL, USA) was inserted, which was placed approximately 0.5 to 1 cm proximal to the distal tip of the anterio- inferior fibula. An 18G hollow needle with a 2 − 0 nylon thread was introduced and into distal ATFL. Then a nylon loop was made and enlarged, and retrieved through the accessory anterolateral portal, and the needle was withdrawn. One end of the sutures was passed through the loop, and the loop was pulled to introduce the mid-portion of the suture through the ATFL. After that the free end of the suture was passed through the loop and pulled to create a self-cinching stitch. The other end of anchor suture was used to draw the self-cinching stitch tightening the ATFL. Finally, additional knots (2 square knots and 2 granny knots) were applied to increase the fixation strength. No intra-articular injection of local anaesthetic or analgesia was used. A negative pressure drainage was applied, which was withdrawn on postoperative day 2. All patients were kept non- weight-bearing for 2 weeks, and subsequently started partial weight-bearing with crutch assistance. Beginning at the fourth week, patients began weight-bearing and started to access normal activity. Full return to sports or heavy labour was permitted after 3 months postoperatively.
Blood samples from the medial malleolus vein were obtained at 10 min before incision (T1),after the completion of surgery (T2), and 30 min after tourniquet deflation (T3) for measurement of MDA, IMA, TOS, TAS and OSI levels. All samples were centrifuged at 3000 rpm, and the serum and plasma samples were stored at − 80 °C until further biochemical analysis.
MDA concentration was measured using the method as described by Yagi [13]. In brief, 2.4 ml of N/12 H2SO4 and 0.3 ml of 10% phosphotungstic acid were mixed into 0.3 ml of serum. After being kept at room temperature for 5 min, the mixture was centrifuged at 1600 g for 10 min. Then the supernatant was removed, and the sediment was suspended in distilled water. After addition of 1 ml of 0.67% thiobarbituric acid, the mixture was heated at 100℃ for 60 min and subsequently centrifuged at 1600 g for 10 min. The absorbance of the organic layer was read at 532 nm. MDA levels were calculated as nmol/ml, with tetramethoxypropane used as the control.
IMA level was analyzed using the method as described Bar-Or et al [14]. 200 µl of serum was mixed with 50 µL of 0.1% cobalt chloride (CoCl2. 6H2O; Sigma, St Louis, Missouri, USA). After 10 minutes of sufficient cobalt albumin binding, 50 µl of dithiothreitol (DTT; Sigma, 1.5 mg/ml H2O) was added. Subsequently, the mixture was quenched for 2 min and 1.0 mL of 0.9% NaCl was added. A colorimetric control was prepared, in which distilled water was substituted for the same volume of 1.5 mg/ml of DTT. Sample absorbency was analyzed at 470 nm using a spectrophotometer (Thermo Scientific, Massachusetts, USA). Finally, the colour of the DTT-containing specimens were compared with that of the control ones, and the outcomes were described as absorbance units (ABSUs).
TOS and TAS levels were determined using the commercial colorimetric kits (Rel Assay Diagnostics, Shanghai, China) following the manufacturer’s instruction. Their outcomes were calculated as umol H2O2 equiv/L and mmol Trolox equiv/L, respectively. OSI level was determined as the TOS: TAS ratio and expressed as a percentage [(TOS, umol H2O2 equiv/L)/ (TAS, mmol Trolox equiv/L) × 0.1].
Demographic data including age, gender, Body Mass Index (BMI), American Society of Anesthesiologists (ASA) grade, and associated medical comorbidities (alcohol or tobacco use, hypertension, diabetes mellitus, respiratory and cardiovascular disease) were documented. And peri- operative characteristics including anaesthesia method, surgical duration, co-existing injuries, postoperative drainage volume, and length of hospital stay.
The main outcome measures were recorded as following: (1) The amount of swelling of the ankle, by measuring the circumference at the level of the malleoli (preoperative, postoperative Days 2 and 5); (2) Pain, measured by a visual analog scale (VAS), with scores ranging from 0 (no pain) to 10 points (worst imaginable pain) (preoperative, postoperative Days 2 and 5); (3) The levels of MDA, IMA,TOS, TAS and OSI measured as described above; (4) The functional outcome, as measured by American Orthopaedic Foot and Ankle Society (AOFAS) ankle hindfoot score (preoperative, postoperative Month 1 and Year 1) [15]; (5) Complication rate [wound leakage, wound infection, superficial nerve injuries, joint stiffness and deep vein thrombosis (DVT)]. A wound leakage was defined based on the consensus of the Work group of the Musculoskeletal Infection Society (WMIS), which is any leakage longer than 72 hours after wound closure [16]. Identification of wound infection was determined based on the standard Centers for Disease Control (CDC) definitions [17]. A wound culture was performed following the onset of an infectious complication.
Statistically analysis
The statistical analysis was performed using SPSS software (Version 20.0; SPSS, Chicago, IL), all statistical tests were two – sided and P-values < 0.05 were considered significant. Continuous variables were presented as means ± standard deviation (SD) and were analyzed with t test if normally distributed or Mann- Whitney U test if not normally distributed. Categorical variables were presented as frequencies and percentages were analyzed using the chi-square test or Fisher,s exact test. The sample size was calculated based on the level of ankle circumference postoperatively. Based on our previous data, a sample of 50 patients (25 in each group) was adequate to detect a 10% difference between groups at 80% power and a p-value of 0.05.