The study was reviewed and approved by the University Ethics Committee. Written informed consent was obtained from all subjects.
This study included 19 men and 11 women with idiopathic ONFH (Arco stage II) and a mean age of 30.6 years. All patients received the following evaluations before surgery: Harris hip score (HHS), visual analogue scale (VAS) of pain, routine blood tests, liver and kidney function tests, comprehensive coagulation tests, erythrocyte sedimentation rate (ESR), C-reactive protein level, electrocardiogram (ECG), chest anteroposterior radiography, bilateral hip anteroposterior radiography, and bilateral hip plain magnetic resonance imaging (MRI).
Harvest and isolation of autologous stem cells
Prior to surgery, patients received recombinant granulocyte colony-stimulating factor (GCSF, 30 IU IM qd ×5 days) to induce stem cell mobilization in the bone marrow . The patient was placed in the supine position under anesthesia. Bone marrow aspiration from the iliac crest was performed, and 100 ml of bone marrow and 100 ml of peripheral blood were harvested. The bone marrow and blood were placed in heparin-coated centrifuge tubes and centrifuged twice at 4000 r/min for 10 min. A 30-ml cell suspension was obtained, and 15 ml of this suspension was added to a collagen sponge to generate a gel-like cell-material composite for surgery. The remaining 15 ml was used to assess cell number and viability.
Mononuclear cell (MNC) count
MNCs were isolated by density gradient centrifugation at 3000 rpm for 30 min with a Percoll cell separator. After centrifugation, the solution was divided into three layers; the middle layer contained the MNCs, which were isolated and counted with a microscope eyepiece reticle.
Isolation and culture of human bone marrow-derived MSCs (hBMSCs)
The cells were resuspended at a 1:1 ratio in the culture medium (Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin), placed in cell culture flasks at a density of 3×106 cells/mL, and cultured in an incubator at 37℃ and 5% CO2. The cells were passaged at 80% confluence using a 0.2% trypsin solution. Cells at passage three (P3) were used for the subsequent experiments.
Immunophenotypic characterization of hBMSCs
Cells at P3 were collected after digestion. A 100-μl suspension containing 1×106 cells was immunostained for cell surface markers and analyzed using an Aria SE flow cytometer and Cell Quest Pro software. hBMSCs were identified as cells positive for CD105, CD73, CD44, and CD90 and negative for CD34, CD45, and HLA-DR. The cells were incubated with CD44-APC, CD90-FITC, CD105-CY5.5, CD73-PE, CD34-PE, CD45-PE, and HLA-DR-PE at the suppliers' recommended dilutions for 45 min at room temperature in the dark. Flow cytometry was performed after two washes with phosphate-buffered saline (PBS).
The cell ultrastructure was observed using transmission electron microscopy (TEM, Tecnai 10, FEI, Hillsboro, OR, USA). Specimen preparation for TEM was as follows: P3 cells were concentrated by low-speed centrifugation (2000 rpm). The cell pellets were prefixed in 2.5% glutaraldehyde and then rinsed three times with PBS. Subsequently, the cell pellets were postfixed with 1% osmium tetroxide, rinsed three times with PBS, and dehydrated in a series of acetone in distilled water (30%, 50%, 70%, 90%, 95%, and 100%). Finally, the dehydrated cell pellets were embedded and sectioned with a diamond knife. The ultrathin specimen sections were stained with uranyl acetate and lead citrate for 30 min each and then observed by TEM.
Quantitation of multilineage differentiation
Cells at P3 were trypsinized and plated onto 100 mm2 tissue culture plates at 105 cells per plate. After the cells were incubated in the culture medium for 1 day, the medium was replaced with either osteogenic medium containing DMEM, 10 mM β-glycerophosphate, 0.1 M dexamethasone, 50 g/ml L-ascorbic acid 2-phosphate, and 10 g/ml insulin or chondrogenic medium containing DMEM, 1% (v/v) FBS, 10 ng/ml rh-TGFβ1, 50 mg/L ascorbic acid, 6.25 mg/ml insulin, 10-7 M dexamethasone, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Culture media were replaced every 3 days. Cells were assessed at 14 days after inducing differentiation. The quantitative analysis of osteogenic differentiation was performed by measuring alkaline phosphatase (ALP) activity with an Alkaline Phosphatase Assay Kit (Abcam, Cambridge, MA) per the manufacturer's protocol and by quantifying alizarin red S staining of calcified tissues according to a standard protocol. Toluidine blue (D8857, NobleRyder, China) staining was performed to evaluate chondrogenic differentiation.
hBMSCs were stained with alizarin red S after 14 days of osteogenic induction and with toluidine blue after 14 days of chondrogenic induction. The area percentage of staining was determined individually by ImageJ version 1.50d. Images were first converted to a grayscale stack by selecting the RGB values, activating thresholding, and adjusting the region of interest based on the original colored image. This highlights the region of interest within the grayscale in red, and the area percentages were measured.
Core decompression and stem cell composite implantation
Upon achieving successful anesthesia, a 3 cm incision was made below the greater trochanter. Under C-arm fluoroscopy guidance, a 2.5 mm Kirschner wire (K-wire) was drilled into the lesion site of the femoral head (2-3 mm beyond the subchondral level) via the femoral neck. A 6.5 mm drill was drilled into the same site over the K-wire. A customized long-handled curette was used to completely remove the lesion tissue beneath the cartilage. Fluoroscopy was performed to ensure complete lesion removal. Next, the collagen sponge-cell composites were injected to fill this site. A piece of muscle membrane was used to cover the filled site to prevent cell leakage. All procedures were performed by the same surgical team (Fig. 1a-f).
Postoperative management and follow-up
The patients were instructed to walk with a supportive device for 9 months after surgery. The HHS and imaging studies of the treated hip were used to assess the clinical efficacy during follow-up. Patients' pain assessments were scored on a VAS from 0 cm (no pain) to 100 cm (severe pain).
HHS of the hip: The delta HHS was calculated as the difference between the HHS before surgery and the HHS at 9 months after surgery. A greater difference was believed to represent more significant functional improvement.
Necrotic area evaluation by MRI: A GE Signa 1.5T superconducting MR (USA) was used for the hip examination. The coronal T1-weighted images were selected to measure the necrotic area angle α and the central angle β of the femoral head (Fig. 2a, b). The necrotic area ratio (before vs after treatment) of each hip was determined according to FengChao Zhao's method . The repair ratio was calculated according to the following formula, and a higher repair ratio indicates more significant lesion repair. (see formula 1 in the Supplementary Files)
The mean repair ratio was set as the threshold to divide the patients into group A (ratio above the mean) and group B (ratio below the mean). The ultrastructure, proliferative capacity, and multidirectional differentiation ability were compared between the groups.
SPSS version 21.0 was used for statistical analyses. The paired t test, nonpaired t test, and Spearman correlation analysis were used. All tests were two-tailed at the 5% significance level.