4.1 Volunteers
This study was approved by the Ethical Committee of the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University.
Inclusion criteria of cases: ① Patients diagnosed and treated from January 2018 to January 2023; ② Age ≥ 18 years, ≤ 40 years; ③ The injury of ATFL was diagnosed by medical history, physical examination and imaging examination.
Exclusion criteria of cases: ① Patients with other organ injuries or limb fractures; ② Patients with injury of other ligaments of the same side ankle joint; ③ Patients with ankle deformity, disease, or repeated ankle pain and swelling before the injury.
Publicly available single-cell RNA datasets of cartilage tissues, obtained from 5 volunteers. None of the 5 volunteers were diagnosed with ankle-related conditions.
4.2 Instruments
All 164 cases of patients with ATFL injury received MRI examination, using PhilipsIntera 1.5T NovaDual double gradient superconducting MRI scanner, ankle coil, and conventional TSE sequence axial, sagittal, coronal and sagittal SPIR sequence scanning (Fig.1 A, B and C). Matrix 256 × 256, FOV 14cm, layer thickness 3mm. The patient lay supinely, with legs naturally straightened, the transverse axis of the joint was vertical to the axis of the bed length, and the scanning range was upper covering the lower tibiofibular joint, and lower covering the margo inferior calcaneus.
The MRI data obtained were read by two senior physicians of our department, and the degree of damage of the ATFL injury and OLT was diagnosed and measured on the PACS system. In case of disagreement, it shall be decided by superior experts.
Single-cell RNA datasets of healthy talus cartilage obtained from 5 volunteers were processed and integrated by Seurat and Harmony R packages. Cell-Chat was used for analysis of cell-cell communication. Compute-Commun-Prob was used to calculate the communication probability between various cells and infer the communication network between cells. Compute-Commun-Prob-Pathway was used to infer intercellular communication at the signaling pathway level.
4.3 Observation target
4.3.1 The classification of ATFL injury and OLT
The classification of MRI findings of ATFL injury was: 0 normal; Grade 1: slight thickening or thinning; Grade 2: partial tear, with increased signal; Grade 3: complete tear, ligament discontinuity or defect, irregular ligament shape and high signal; Grade 4: ambiguous.
The MRI manifestations of OLT were recorded by Hepple classification as follows: stage I: only the surface of articular cartilage was damaged; stage IIa: not only articular cartilage injuries, but also subchondral fractures and peripheral bone marrow edema; Stage IIb: articular cartilage injury and subchondral fracture, but without peripheral bone marrow edema; stage III: osteochondral fragments were separated, but there was no displacement; stage IV: bone fragments were separated, and displaced; stage V: subchondral cysts were formed.
4.3.2 Lesion location (the nine equal surface area zones)
Lesion location was determined using a 9-zone anatomic localization scheme on MRI.9
4.3.3 Lesion size (cm2)
The size of lesion was evaluated by elliptical area calculation formula.
4.3.4 Thickness index
Thickness index refers to the measurement method in Tao’s article.10
4.3.5 AOFAS
The function of foot was evaluated using the American Orthopaedic Foot and Ankle Association (AOFAS) scoring scale. The result is obtained by simply adding up the scores of each section. The total score is 100 points.
4.3.6 VAS
The pain of foot was assessed using Visual Analog Scoring (VAS). Patients were asked to draw a mark on the 10 cm horizontal line based on their own feelings to indicate how pain they suffered.The number from 0 to 10 indicating no pain to severe pain.
4.4 Statistical Analysis
All statistical analysis was conducted using SPSS Version 26.0 (IBM, Armonk, NY, USA) with any necessary extensions. The correlation analysis between ATFL injury grade and the Hepple stage of OLT determined by MRI imaging was performed. The chi-square test was used to determine the risk factors associated with OLT by taking gender, age, course of disease, injured side, injured area, and thickness index as independent variables while taking the incidence rate and the severity of OLT as the dependent variable. Multivariate logistic regression analysis was used to determine the independent risk factors, the corresponding odds ratio (OR) and the 95% confidence interval (CI). Respectively, the correlation between ATFL injury grade and the Hepple stage of OLT and AOFAS and VAS were analyzed.
The parameters were expressed as the mean ± standard deviation (SD). All hypothesis tests were performed at a significance level of 5%, with P-values of 0.05 or less considered statistically significant.
4.5 Data availability statements
The single-cell RNA sequence datasets collected in the study are presented publicly in the GEO repository, accession number GSE216578.