Human OLF tissues (3–5 g) were obtained during surgery from a patient admitted to the Shanghai Tenth People's Hospital, Shanghai, China (aged 53 years; male; stage IV lumbar degenerative disease, during March 2015) that was diagnosed with lumbar degenerative disease. Written informed consent was provided by the patient for the use of their tissue samples in the present study. The tissues were cut into 1–2-mm³sections and digested with 2% type-I collagenase (Sunshine Biotechnology Co., Ltd., Nanjing, China) at 37°C for 30 min. Nucleated cells (10,000 cells/cm²) were recovered and plated as mononuclear cells to obtain a higher yield. Cells were cultured in α-minimum essential medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 15% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in 5% CO₂; the medium was refreshed every 2 days. Following 1 week of culture, the cells were transferred to culture flasks coated with fibronectin (5 µg/cm², Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). A specially-designed four-point bending apparatus with flexible silicon-bottomed chambers was generated as described previously (14), and used to induce the mechanical stress model. The cells were then subjected to uniaxial tensile strain (0.5 Hz, 2,000 min, three times/day).

Cells were distributed into control, unstretched and stretched groups.

In the control group, cells were treated with PBS; in unstretched group, cells were subjected to uniaxial tensile strain without voltage; in stretched group, cells were subjected to uniaxial tensile strain (0.5 Hz, 2,000 min, three times/day).

Following 3 weeks of mechanical stress, cells (1–2×10³) were cultured in a 96-well plate for 24 h. A total of 20 µl MTT assay reagent (5 mg/ml; Beyotime Institute of Biotechnology, Haimen, China) was then added to each well, and the plates were incubated at 37°C and 5% CO₂ for 4 h. Following incubation, the culture medium was removed and 150 µl dimethyl sulfoxide (DMSO) was added to each well and allowed to dissolve the formazan crystals for 20 min at 37°C. Cell viability was measured using a microplate reader (Omega Bio-Tek, Inc., Norcross, GA, USA) at a wavelength of 490 nm.

Cells (1×10⁵) from the different groups were fixed in 95% ethanol overnight at 4°C, and stained with Oil Red O (0.25%) for 15 min at room temperature. The cells were then washed three times with 70% ethanol for 5 min. Osteogenic differentiation was observed using a microscope in three field of view (Axio Imager 2; Zeiss GmbH, Jena, Germany).

After 3 weeks mechanical stress, cells (1×10⁶) from the different groups were cultured on a 6-well plate for 24 h. Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. A total of 2 ng RNA was reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. For qPCR analysis, 1 ng cDNA template was used for each reaction and sequences were amplified using a StepOne™ Real-Time PCR system and Power SYBR^® Green PCR Master Mix (both from Applied Biosystems; Thermo Fisher Scientific, Inc.). Amplification reactions were conducted with an initial step at 95°C for 1 min, followed by 40 cycles of 55°C for 30 sec and 60°C for 30 sec. The primers used for qPCR analysis are listed in Table I and gene expression was normalized to the levels of β-actin and analyzed using the 2^−ΔΔCq method (15).

Cells (1×10⁶) after 3 weeks mechanical stress from the different groups were lysed with ice-cold lysis buffer containing a 10 µg/ml of protein inhibitor mixture (1:100; Roche Diagnostics, Indianapolis, IN, USA). Protein concentrations were then determined using a DC Protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 50 µg protein was separated by 8–12% SDS-PAGE and electrophoretically transferred onto 0.22 µm polyvinylidenedifluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then washed with5% non-fat dried milk in Tris-buffered solution plus 0.05% Tween-20 (TBST) for 2 h, prior to incubation with anti-Src (catalog no. sc-8995, dilution, 1:200), anti-bone morphogenetic protein (catalog no. sc-9003, BMP; dilution, 1:200) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-phosphorylated (p)-mothers against decapentaplegic homolog-1 (catalog no. 13820, p-Smad-1; dilution, 1:400; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-p38-mitogen-activated protein kinases (catalog no. sc-7975-R, p38MAPK; dilution, 1:300) and anti-β-actin (catalog no. sc-7210, dilution, 1:400) (both from Santa Cruz Biotechnology, Inc.) at 4°C overnight. The membranes were washed with TBST for 5 min three times and were subsequently incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (catalog no. A0208, dilution, 1:4,000; Beyotime Institute of Biotechnology) at room temperature for 1 h, and detected with electrochemiluminescence western blot detection reagents (Beyotime Institute of Biotechnology). Protein expression was analyzed using Quantity One software version 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

All data are expressed as the mean ± standard deviation, and represent the mean values of at least three independent experiments using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). Comparisons among groups were analyzed using one-way analysis of variance, followed by the Tukey Kramer post hoc test. P<0.05 was considered to indicate a statistically significant difference.

OLF cells were extracted using a fibronectin differential-adhesion assay, and OLF cell morphology was observed following 1 week of culture. As shown in Fig. 1, OLF cells exhibited a uniform morphology and were well-distributed in the culture flasks. In addition, the cells exhibited a shuttle shape until the end of week 1 (Fig. 1).

When comparing the viability of OLF cells among the different experimental groups, the control and unstretched groups demonstrated similar levels of cell viability at 1, 2 and 3 weeks (Fig. 2). However, mechanical stress significantly increased OLF cell viability following 2 (P<0.05) and 3 weeks (P<0.01) of mechanical stimulation when compared with the controls (Fig. 2).

Oil Red O staining was performed to analyze the effect of 3 weeks of mechanical stress on the osteogenic differentiation of OLF cells. The osteogenic differentiation rate of the control group appeared to be similar to that of the unstretched group (Fig. 3). By contrast, mechanical stress effectively promoted the osteogenic differentiation rate and increased the size of OLF cells, when compared with the control group (Fig. 3).

RT-qPCR analysis was performed to investigate OC, ALP and RUNX-2 mRNA expression levels following mechanical stress stimulation at 3 weeks. As shown in Fig. 4, the mRNA expression levels of OC, ALP and RUNX-2 were comparable when comparing the control group and unstretched groups. By contrast, mechanical stress significantly increased the expression of OC, ALP and RUNX-2 mRNA in OLF cells when compared with the control group (P<0.01; Fig. 4).

In order to investigate the mechanisms underlying the effects of mechanical stress on osteogenic OLF cell differentiation, Ets-1 and SOX-2 mRNA expression was determined by RT-qPCR analysis. Following quantitative analysis, no significant difference in Ets-1 and SOX-2 miRNA expression levels were observed between the control and unstretched groups (Fig. 5). However, mechanical stress significantly inhibited Ets-1 and SOX-2 mRNA expression in OLF cells when compared with the control cells (P<0.01; Fig. 5).

Western blot analysis revealed that the control and unstretched groups exhibited similar levels of Src protein expression (Fig. 6). By contrast, mechanical stress significantly inhibited Src protein expression in OLF cells when compared with the control group (P<0.01; Fig. 6).

To further elucidate the underlying mechanisms of mechanical stress on osteogenic OLF cell differentiation, p-p38MAPK protein was detected using western blot analysis. No significant differences inp-p38MAPK protein expression were observed between the control and unstretched groups (Fig. 7). However, mechanical stress significantly increased p-p38MAPK protein expression, when compared with the control group (P<0.01; Fig. 7).

BMP protein expression was determined by western blot analysis. As shown in Fig. 8, no significant difference in BMP protein expression was observed between the control and unstretched groups. However, mechanical stress significantly increased BMP protein expression in OLF cells when compared with the control group (P<0.01; Fig. 8).

p-Smad-1 protein expression in OLF cells was analyzed using western blot analysis. As shown in Fig. 9, no significant difference in the protein expression of p-Smad-1 was observed between the control and unstretched groups. However, mechanical stress significantly increased p-Smad-1 protein expression in OLF cells, when compared with the control group (P<0.01; Fig. 9).