Study population
36 patients diagnosed with SIONFH were admitted to the Third Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine between May and December 2021. Additionally, 36 healthy individuals served as control subjects. Diagnosis of SIONFH was based on medical history, physical examination, and X-ray and MRI findings. Patients had been receiving 2 g of prednisone or an equivalent steroid for a minimum of 3 months, with diagnosis occurring within the preceding 2 years (20). All patients were classified according to the ARCO staging system (6). Inclusion criteria comprised: a) Patients classified as stage II, III, or IV according to the ARCO staging; b) Adult patients aged between 18 and 65 years. Exclusion criteria included: a) Patients with concurrent large joint pathologies; b) Patients with chronic comorbidities such as hypertension, diabetes mellitus, coronary heart disease, etc. Healthy control subjects had no history of hip pain or steroid use and presented normal anteroposterior pelvic and frog leg radiographs.
Ethical approval
The study adhered to the Declaration of Helsinki, and patients provided informed consent and received approval from the hospital ethics committee (GYH202101-04) before participation.
Sample collection
Approximately 5 ml of peripheral venous blood was collected from fasting subjects in both groups, centrifuged at 1,000 r/min for 5 minutes after natural coagulation at 4℃, and serum was extracted and stored at -80℃. Femoral head specimens were obtained post-total hip arthroplasty (THA). Samples were collected from the subchondral necrotic area or healthy bone, approximately 1-3 mm below the cartilage, for analysis.
H&E staining of bone tissue
Bone specimens from necrotic areas of both groups were fixed in 4% paraformaldehyde solution at room temperature for 24 hours. Sequential decalcification, dehydration, and paraffin embedding were performed. 5-μm sections were stained with hematoxylin and eosin (H&E) and examined under a microscope (BX53, Olympus Corporation, Japan). Five randomly chosen high-magnification fields were used to count bone gaps, with the percentage of voids indicating the total apoptotic cell rate. Percentage of voids = (number of voids/number of bone gaps) × 100%.
Immunohistochemistry of p62
Paraffin sections (5 um thick) were deparaffinized, hydrated, antigen-repaired, and immunohistochemically stained for p62 expression using the En Vision two-step staining technique. Staining results were observed and captured under a light microscope (3DHISTEC, Pannoramic MIDI). Image J software was used for quantitative analysis, with the percentage of positive areas serving as a quantitative index of p62 and comparison.
Immunofluorescence of p62
Following dewaxing, antigen repair, and serum closure of 5 um thick sections, primary antibodies were incubated overnight at 4℃. After washing three times, sections were incubated with corresponding secondary antibodies for 2 hours at room temperature and stained with DAPI nuclear stain. Image J software was utilized to calculate the p62 positive cell rate. Positive cell rate = (number of positive cells/total number of cells) × 100%.
Enzyme-linked immunosorbent assay for p62
Serum p62 protein concentration was determined via enzyme-linked immunosorbent assay (ELISA) using a kit from Cusabio, Wuhan, Hubei Province, China, following kit instructions.
Micro CT Scan
Bone specimens were scanned using NEMO Micro CT. Avatar 3.0 software was used to observe changes in femoral head shape, quantitative analysis of bone microstructure, and 3D reconstruction.
Statistical analysis
SPSS 23.0 statistical software was employed for data analysis. Measurement data were expressed as mean ± standard deviation (mean ± SD). Independent samples t-test and X2 test were used for continuous and categorical data, respectively, with P < 0.05 considered statistically significant.