Ligamentum flavum tissues were harvested aseptically during surgery, which was performed to decompress the spinal cord for myeloradiculopathy, from four thoracic OLF patients who were documented by magnetic resonance imaging and computed tomography. As a control, the ligamentum flavum was obtained from four non-OLF patients during surgery for thoracic spine trauma. OLF or non-OLF was preoperatively diagnosed by X-ray, computerized tomography, and magnetic resonance imaging, and further confirmed by histological examination. Fig. 1 shows histological characterization of the ossified ligamentum flavum. Details of these patients are shown in Table 1. Informed consent was obtained from each patient, and this study was approved by the Ethics Committee of the Southern Medical University.

Cells were isolated from the ligamentum flavum tissues as described previously18 with some modifications. Briefly, the specimens were washed with phosphate-buffered saline (PBS), after which any surrounding tissues attached to the specimen were carefully removed, including bone, fat, and connective tissue. The collected ligaments were minced into pieces of approximately 0.5 mm³ and washed twice with PBS. The minced tissue was digested at 37℃ for 60 min with 0.2% collagenase type I (Sigma-Aldrich Corp., St. Louis, MO, USA) in Dulbecco's Modified Eagle's medium (DMEM, Gibco BRL, Melbourne, Victoria, Australia) containing no serum. Collagenase-treated ligament chips were washed with DMEM and then placed in 6-well plates in DMEM supplemented with 10% fetal bovine serum (Gibco BRL.), 100 U/mL penicillin and 100 pg/mL streptomycin, and incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37℃. The medium was changed at two-day intervals, and the explants were daily checked for cell outgrowth using an inverted light microscope. The outgrown cells were harvested before confluence and subcultured after trypsinization with 0.2% trypsin/ 0.02% ethylenediamine tetraacetic acid (EDTA). Cells at the third passage were used for experiments. Cell lines obtained from OLF and non-OLF patients were named as OLF cells and non-OLF cells, respectively.

For immunofluorescence analysis, monolayers of cells were grown on glass coverslips inserted in 6-well plates. After rinsing with PBS, the cells were fixed in 4% paraformaldehyde in PBS for 10 min and permeabilized with PBS containing 0.5% TritonX-100 for 5 min at room temperature. Blocking was performed with 5% bovine serum albumin (BSA) in PBS for 30 min, and the cells were subsequently incubated overnight at 4℃ with primary antibodies. The antibodies were used at the following dilutions: goat polyclonal anti-collagen type I antibody 1 : 50 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and goat polyclonal anti-Collagen type III antibody 1 : 50 (Santa Cruz Biotech-nology.); goat polyclonal anti-Osteocalcin antibody 1 : 25 (Santa Cruz Biotechnology.); mouse monoclonal anti-Collagen type II antibody 1 : 25 (Thermo Fisher Scientific Inc, Fremont, CA, USA). Following three washes in PBS (5 mins each), the cells were incubated for 60 min at room temperature with a 1 : 100 dilution of the secondary anti-bodies: fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat IgG (ZSGB-BIO Inc., Beijing, China) and FITC-conjugated goat anti-mouse IgG (ZSGB-BIO Inc.) respec-tively. After three additional washes in PBS, the cell nuclei were counterstained with 10 µg/mL propidium iodide (PI) (Sigma-Aldrich Corp.) for 10 min at room temperature and rinsed again in PBS. Finally, the coverslips were mounted onto microscopic slides using 50% glycerol in PBS. Negative controls were performed in parallel, except that cells were treated with 5% BSA in PBS without primary antibody. Images were obtained and analyzed using the LSM-510 laser confocal microscope system (Carl Zeiss, Göttingen, Germany).

Total RNA was extracted from cultured cells using a TRIZOL® Reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. First-strand cDNA was synthesized by reverse transcription of 5 µg of total RNA using SuperScript™ III First-Strand Synthesis System (Invitrogen.). PCR amplification was performed with GoTaq® Green Master Mix (Promega, Madison, WI, USA) using the human specific primers, which are shown in Table 2. The house-keeping gene beta-actin was used as an internal control. Cycling conditions for PCR were as follows: for osteocalcin, collagen type I, collagen type II and collagen type III, 35 cycles of 45 s at 94℃, 45 s at 60℃, 1 min at 72℃, and 5 min at 72℃; for beta-actin, 35 cycles of 30 s at 94℃, 30 s at 55℃, 30s at 72℃, and 5 min at 72℃. PCR products were subjected to 2% agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light exposure.