Institutional Review Board approval and informed consent for sample collection were obtained prior to the collection of all samples. All of the procedures specified below were approved by the Ethical Committee of Xinqiao Hospital (the Third Military Medical University, Chongqing, China) and were in accordance with the Helsinki Declaration.

Human intervertebral disc cartilage endplate cells and CESCs were obtained from 6 patients (3 men, 3 women; 39–50 years old), following the protocol of a previous study (15). Cells were derived from surgically resected intervertebral disc cartilage endplate. CESCs at passage 3 were used in the present study. The expression profile of cell surface antigens was determined by flow cytometry. In brief, the cells were seeded in a 6-well culture plate and grown until they reached 90% confluence. After washing with PBS, the cells were stained with the following fluorescein isothiocyanate (FITC), phycoerythrin (PE) or peridinin chlorophyll protein (PerCP)-conjugated antibodies: CD73-FITC (cat. no. 11-0739-41), CD14-FITC (cat. no. 11-0149-41), CD19-FITC (cat. no. 11-0199-41), CD90-FITC (cat. no. 11-0909-41), CD34-FITC (cat. no. 11-0349-41), CD45-FITC (cat. no. 11-9459-41), CD105-PE (cat. no. 12-1057-41) or human leukocyte antigen – antigen D related (HLA-DR)-PerCP(cat. no. 9043-4724-025) (1:500 dilution, all from eBioscience; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Immunoglobulin G (eBioscience; Thermo Fisher Scientific, Inc.) was used as an isotype control. After incubation for 30 min at 37°C, cells were washed 3 times with PBS and resuspended in 200 µl PBS. Finally, labeled cells were subjected to single-channel flow cytometric analysis (BD Biosciences, Franklin Lakes, NJ, USA). The percentage of stained cells was calculated relative to the isotype control.

For cyclic tension loading, CESCs were placed on an elastic silicone membrane coated with collagen I at a density of 2×10⁵ cells per well. After reaching 80–90% confluence, cells were serum-starved in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco; Thermo Fisher Scientific, Inc.) containing 1% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) for 24 h for synchronization, followed by stretching using a Flexercell^™ Tension Plus system (FX-4000T; Flexcell International Corp., Burlington, NC, USA) at 37°C in a 5% CO₂ incubator in DMEM/F12 with 10% FBS. According to the experimental protocol, a 20% stretch elongation at a frequency of 1 Hz was applied for 12, 24, 36 or 48 h.

After stretching, cells were seeded into 96-well plates at a density of 5×10³ cells per well. After 24 h, the cell viability was assessed using a Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. In brief, 10 µl CCK-8 solution was added to each well and the plate was then incubated at 37°C for 4 h. The optical density was measured at a wavelength of 450 nm with a microplate reader. In another experiment prior to stretching, caspase-3 inhibitor Z-VAD-FMK (20 µM; Beyotime Institute of Biotechnology, Haimen, China) was added to the cell culture medium and the plate was incubated at 37°C for 20 min.

The apoptotic rate was measured with an Annexin V-FITC apoptosis detection kit I (BD Pharmingen, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. In brief, cells were washed twice with cold PBS and then resuspended in 300 µl binding buffer at a concentration of 1×10⁶ cells per ml. Annexin V-FITC solution (5 µl) and propidium iodide (PI; 5 µl of a 1 µg/ml solution) were then added to these cells, followed by incubation for 30 min at 37°C. The apoptotic incidence was detected by flow cytometry within 1 h.

Caspase-3 activity was detected with a caspase assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In brief, 2×10⁶ cells were lysed with 100 µl lysis buffer on ice for 15 min and the cell lysates were then centrifuged at 16,000 × g for 10 min at 4°C. Reaction buffer (80 µl) and acetyl-DEVD-p-nitroanilin (pNA; 10 µl) was mixed with 10 µl lysate and the mixture was then incubated for 2 h at 37°C. The optical density of free pNA was measured at an absorption wavelength of 405 nm using a microplate spectrophotometer (Dynex Technologies, Chantilly, VA, USA). Caspase-3 activity was expressed as the relative absorbance ratio of samples vs. control group.

Cells were lysed using lysis buffer containing 1 mM phenylmethylsulfonylfluoride (Beyotime Institute of Biotechnology), Protein concentration was determined with the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene diflouride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, the membrane was blocked in 5% non-fat milk and 0.1% Tween-20 containing Tris-buffered saline (TBST) at room temperature for 1 h and then incubated with the respective primary antibodies overnight at 4°C. After washing with TBST, the membrane was then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Immunoblotting was detected by enhanced chemiluminescence (BeyoECLPlus, Beyotime, Beijing, China) and images of blots were captured on a ImageQuant LAS4010 (GE Healthcare, Little Chalfont, UK). Western blot bands were quantified using ImageJ software (ImageJ 1.41; National Institutes of Health, Bethesda, NJ, USA). The primary antibodies used for western blot analysis were as follows: B-cell lymphoma 2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) (cat. no. ab10433; mouse monoclonal), Bcl-2 (cat. no. ab59348; rabbit monoclonal), Bcl-2 homologous antagonist killer (Bak; cat. no. ab69404; rabbit monoclonal), Bcl-2-associated X protein (Bax; cat. no. ab53154; rabbit monoclonal) and Bcl extra large (Bcl-xl; cat. no. ab32370; rabbit monoclonal) (all used at 1:500 dilution; Abcam, Cambridge, MA, USA) and GAPDH (cat. no. TA-08, 1:2,000 dilution; Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China). The secondary antibodies used in the present study were as follows: Horse radish peroxidase (HRP)-labeled goat anti-mouse (cat. no. A0216) and anti-rabbit (cat. no. A0208) Immunoglobulin G (IgG) (H+L) (all 1:1,000 dilution; Beyotime Institute of Biotechnology).

Data were analyzed using SPSS 13.0 statistical software (SPSS, Inc., Chicago, IL, USA). Values are expressed as the mean ± standard deviation. Differences between groups were assessed using the one-way analysis of variance and the Least Significant Difference test. P<0.05 was considered to indicate a statistically significant difference.

The surface antigens of CESCs were investigated in passage 3 cells. Flow cytometric analysis revealed that the CESCs were negative for CD14, CD19, CD34, CD45 and HLA-DR, but positive for CD73, CD90 and CD105 (Fig. 1), which was similar to the result of a previous study by our group (15). Considering that the pluripotency of CESCs has already been demonstrated in a previous study (15), these results indicated that the cells used in the present study possessed the properties of stem cells.

The effects of a tensile stretch on the viability of CESCs were assessed. After 20% stretch elongation at a frequency of 1 Hz for 12, 24, 36, or 48 h, the viability of CESCs was assessed using a CCK-8. The results demonstrated that compared with static control cells (0 h), the viability of CESCs decreased with the increase of stretching time (P<0.05; Fig. 2A).

Furthermore, it was assessed whether the decrease in viability of CESCs was due to apoptosis. The apoptotic rate was detected by flow cytometry with Annexin V and PI double labeling. PI labels all dead cells, including necrotic cells and those at the final stages of apoptosis, whereas cells in early apoptosis were only stained with Annexin V. Representative graphs obtained by flow cytometric analysis of cells after Annexin V-FITC and PI double staining are presented in Fig. 2B. The apoptotic incidence in the static control group was 1.27%. The results demonstrated that the apoptotic incidence was significantly increased in CESCs subjected to cyclic stretch compared to that in the control group in a time-dependent manner (Fig. 2B). Hence, the apoptotic incidence in the CESCs was increased to 5.12, 7.06, 12.06 and 20.00% after cyclic stretch for 12, 24, 36 and 48 h, respectively (Fig. 2C). These results demonstrated that a cyclic tensile stretch decreased the viability and increased the apoptotic rate of CESCs.

Since cyclic stretch increased cell apoptosis in a time-dependent manner, the apoptotic mechanism in CESCs was then assessed. In order to understand whether apoptosis induced by cyclic stretch was caspase-dependent, the activity of caspase-3 was measured. The results indicated that the activity of caspase-3 in cells subjected to cyclic stretch increased in a time-dependent manner compared with that in static control cells (Fig. 3A), which was consistent with the apoptosis incidence (Fig. 3B and C). This finding suggested that cyclic stretch activated caspase-3 in CESCs, which finally induced cell apoptosis.

To further investigate the molecular mechanism of cyclic stretch-induced apoptosis, the expression of BNIP3 and Bcl-2 in CESCs was assessed. As presented in Fig. 3B, compared with the static control cells, the expression of pro-apoptotic protein BNIP3 increased in cells subjected to 20% stretch elongation at a frequency of 1 Hz for 12, 24, 36 and 48 h. Furthermore, the expression of anti-apoptotic protein Bcl-2 began to decrease after cells had been subjected to a 20% stretch elongation for 36 h (Fig. 3C). These results suggested that upregulation of BNIP3 and downregulation of Bcl-2 probably contributed to apoptosis induced by cyclic stretch in CESCs.

To identify the role of apoptosis in cells exposed to cyclic stretch, caspase-3 was repressed to evaluate cell viability and apoptosis. As presented in in Fig. 4A and B, pro-apoptotic protein Bak and Bax were significantly increased after cells were subjected to a 20% stretch for 36 h, while Bcl-xl was decreased, which indicated marked apoptosis in the cells. When the caspase-3 inhibitor was added to the cell culture medium, the caspase-3 activity in the stretched cells restored to near basal levels (Fig. 4C), and the cell viability in the stretched cells (Fig. 4D) was also restored comparing with stretched cells without caspase-3 inhibitor. Cell apoptosis (Fig. 4E and F) was also significantly reduced compared with those in stretched cells without caspase-3 inhibitor treatment.