All experimental animals were used in accordance with the relevant guidelines [SYXK (YU) 2012-0012] of the Ethics Committee at Southwest Hospital affiliated to the Third Military Medical University (Chongqing, China).

Twenty-five healthy New Zealand rats (weight, 250 g; age, 6–8 weeks) were obtained from the Animal Center of Third Military Medical University (Chongqing, China) and sacrificed by excessive carbon dioxide exposure. Briefly, after NP tissues were separated from the harvested thoracic and lumbar discs, NP tissue samples were sequentially digested with Gibco 0.25% trypsin (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 3–5 min at 37°C and Sigma-Aldrich type I collagenase (0.25%; Merck KGaA, Darmstadt, Germany) for 10 min. Subsequently, NP cell pellets were collected by centrifugation (500 × g at 4°C for 5 min) and cultured in Dulbecco's modified Eagle's medium/F12 (Gibco; Thermo Fisher Scientific, Inc. medium containing Gibco 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 1% (v/v) penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) under standard conditions (37°C, 20% O₂ and 5% CO₂).

NP cells were seeded in a 24-well plate and grown to 40–50% confluence. Subsequently, NP cells were incubated with 400 µl fresh culture medium containing 40 µl concentrate of recombinant lentiviral vectors (Shanghai GenePharma Co., Ltd., Shanghai, China) for 48 h to overexpress N-CDH in the NP cells (NP-N-CDH). NP cells transfected with negative vectors served as controls (NP-N-CDH-NC). Thereafter, the transfected cells were further selected via puromycin for 4–6 days. N-CDH overexpression in NP cells was verified by quantitative polymerase chain reaction (qPCR) and western blotting assays.

The transfected or un-transfected NP cells were suspended in collagen solution (1 mg/ml; Shengyou Biotechnology Co., Ltd., Hangzhou, China) and seeded into the prepared bovine decalcified bone matrix scaffold [DBM; 10×10×5 mm (1×10⁷ cells per DBM)], provided by Tissue Engineering Center of the Third Military Medical University (Chongqing, China). After NP cells seeded in the scaffold were pre-cultured under standard conditions (37°C, 20% O₂ and 5% CO₂) for 2 days, NP cells seeded in the DBM scaffolds were perfusion-cultured at 37°C in the tissue culture chambers of the self-developed bioreactor (Fig. 1) for 5 days, and simultaneously subjected to dynamic compression (20% deformation at a frequency of 1.0 Hz for 4 h once per day).

Subsequent to compression, NP cells seeded in the scaffold were collected by digestion with 0.05% trypsin and 0.1% collagenase I. The NP cells (1×10⁴ per group) were allowed to adhere in 6-well plates within 6–8 h. Subsequently, an SA-β-Gal staining assay was performed according to the manufacturer's instructions (Beyotime Institute of Biotechnology, Haimen, China). Finally, SA-β-Gal stain-positive cells were observed under an Olympus BX51 light microscope (Olympus Corp., Tokyo, Japan) and SA-β-Gal activity expressed as the percentage of SA-β-Gal stain-positive cells to the total cells was analyzed using the Image-Pro Plus software (Version 5.1.0.20; Media Cybernetics, Inc., Rockville, MD, USA).

Following compression, NP cells seeded in the scaffold were harvested as described above. NP cells (3×10³ cells per group) were seeded in a 96-well plate and NP cell proliferation was detected at 6, 24 and 48 h using a Cell Counting Κit-8 (CCK-8; C0037; Beyotime Institute of Biotechnology).

Subsequent to compression, NP cells seeded in the scaffold were harvested as described above. The NP cell pellets were incubated with RIPA lysis buffer (Beyotime Institute of Biotechnology) and centrifuged (12,000 × g at 4°C for 5 min) to collect the supernatant. Then, a telomerase ELISA kit (ml-003023; Mlbio, Shanghai, China) was used to measure telomerase activity (IU/l) according to the manufacturer's instructions.

Following compression, NP cells seeded in the scaffold were harvested as described above. The NP cell pellets were fixed with 75% ethanol overnight at 4°C and stained with propidium iodide solution (50 µg/ml; Beyotime Institute of Biotechnology) and RNase A (100 µg/ml; Beyotime Institute of Biotechnology) for 30 min at 4°C. Subsequently, NP cells were subjected to flow cytometry assay and the fraction of each cell cycle phase (G₀/G₁, G₂/M and S) was calculated using the multicycle software (FlowJo 7.6.1, Engine 2.79000; Phenix Co., Ltd., Tokyo, Japan).

Gene expression of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II) was analyzed by qPCR assay. Briefly, total RNA was extracted using TriPure Isolation Reagent (11667157001, Roche Applied Science, Penzberg, Germany) and synthesized into cDNA using a First Strand cDNA Synthesis kit (04379012001, Roche Applied Science). Then, qPCR was performed using a reaction system containing cDNA, SYBR Green Mix (Toyobo Life Science, Osaka, Japan) and primers (Table I). The thermal cycling conditions for all reactions were as follows: 5 min at 95°C, followed by 35 amplification cycles of 30 sec at 95°C, 20 sec at 56°C and 15 sec at 72°C. β-actin served as an internal reference and the relative gene expression was expressed as 2^−ΔΔCq (19).

Protein expression levels of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II) were analyzed by western blotting assay. Briefly, after the total protein was extracted using RIPA lysis solution (Beyotime Institute of Biotechnology) and the protein concentration was measured using a BCA kit (P0009, Beyotime Institute of Biotechnology), protein samples were subjected to an 12% SDS-PAGE system and transferred to a polyvinylidene difluoride (PVDF) membrane (100 V for 60 min). Then, the PVDF membrane was incubated with primary antibodies [β-actin: ProteinTech Group, Inc., Chicago, IL, USA (cat. no. 60008–1-Ig); p16: Novus Biologicals, LLC, Littleton, CO, USA (cat. no. NBP2-37740); p53: ProteinTech Group, Inc. (cat. no. 10442-1-AP); aggrecan: Santa Cruz Biotechnology Inc. (cat. no. sc-16492); collagen II: Abcam, Cambridge, MA, USA. (cat. no. ab34712); all diluted 1:1,000] at 4°C overnight and the corresponding secondary antibodies (OriGene Technologies, Inc., Beijing, China; 1:2,000) at 37°C for 2 h. Protein bands were developed using the SuperSignal West Pico Trial kit (34080, Pierce; Thermo Fisher Scientific, Inc.) and analyzed using Image J software (v Java 1.6.0_20 32-bit, National Institutes of Health, Bethesda, MA, USA).

All data are expressed as means ± standard deviation and each experiment was performed in triplicate. After the homogeneity test for variance, comparisons between groups were performed by one-way analysis of variance using SPSS 13.0 software, and the post hoc test was determined by the least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of N-CDH in regulating NP cell senescence under high-magnitude compression, N-CDH expression in NP cells was enhanced by recombinant lentiviral vectors. Predictably, N-CDH expression in NP cells under high-magnitude compression also increased following N-CDH overexpression (Fig. 2).

Senescent cells often exhibit increased SA-β-Gal activity (20), decreased cell proliferation potency (21), aggravated G₁ cell cycle arrest (22), decreased telomerase activity (23) and upregulated expression levels of senescence markers (p16 and p53) (24). Compared with NP cells without N-CDH overexpression under high-magnitude compression, N-CDH overexpressed NP cells exhibited significantly decreased SA-β-Gal activity (Fig. 3A), increased cell proliferation potency (Fig. 3B), decreased percentage of cells arrested in the G₁ phase of the cell cycle (Fig. 3C), increased telomerase activity (Fig. 3D), and downregulated gene (Fig. 3E) and protein (Fig. 3F) expression levels of senescence markers (p16 and p53).

Senescent cells demonstrate altered matrix metabolism and matrix catabolism is often promoted in senescent cells (25,26). qPCR indicated that the gene expression of matrix macromolecules (aggrecan and collagen II) in N-CDH overexpressed NP cells was higher than that in NP cells without N-CDH overexpression under high-magnitude compression (Fig. 4A). Additionally, protein expression levels of these matrix macromolecules presented a similar trend (Fig. 4B).