IVDs were obtained from lumbar spines of mature New Zealand white rabbits immediately postmortem. The nucleus pulposus was harvested from these specimens, washed with Hank’s balanced salt solution (HBSS), and transported to the laboratory within 30 min of harvesting. The nucleus pulposus tissue was rinsed 3 times in HBSS and then dissected into small fragments ~1 mm³ in size. Cells were isolated from the nucleus pulposus using sequential enzyme digestion with 0.25% trypsin for 30 min, washed with HBSS and incubated in 0.1% collagenase type II at 37°C for 2–3 h. The cells were collected by filtering through a 200-mesh nylon cell strainer and subjected to centrifugation of 1,000 × g for 5 min. The cells were then washed twice with phosphate-buffered saline, resuspended and grown in medium containing Dulbecco’s modified Eagle’s medium with Ham’s F-12 nutrient mixture (DMEM-F12) supplemented with 10% (vol/vol) fetal bovine serum (FBS) plus 1% penicillin and streptomycin. The culture medium was changed every 2–3 days. The phenotype of the nucleus pulposus cell was confirmed using positive immunostaining for type II collagen and toluidine blue staining of glycosaminoglycans. First-passage chondrocytes were used in our experiments.

Cells were grown in 80% confluency, first-passage chondrocytes were digested using trypsin and then transferred to 6-well plates to grow to a density of 1×10⁵/ml in DMEM-F12 supplemented with 10% (vol/vol) FBS. After 24 h of adherence, the medium was changed to DMEM-F12 without FBS, and the cells were cultured for another 24 h. At that time, the medium was removed, and the cells were grown in DMEM-F12 containing IL-1β (100 μg/l) with or without IGF-1 (500 μg/l) for 24 h. A subset of cells grown in DMEM-F12 without FBS for 24 h served as controls.

Animal care was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80–23; revised 1996) and was approved by the Bengbu Medical College Animal Care Committee of the Use of Laboratory Animals.

The cells were digested using trypsin and cultured in 6-well plates, each of which contained a 1×1-cm cover glass. The slides were removed 12 h later. The cells growing on each slide were fixed in methanol for 2 min. Giemsa staining solution was then applied for 10 min, and the cells were made transparent using xylene. The cells were then covered and observed using light microscopy.

The cells were again subjected to trypsinization and cultured in 6-well plates, each containing a 1×1-cm cover glass. The glass slides were removed 12 h later, and the cells growing on them were fixed in 4% paraformaldehyde for 60 min. Methanol containing 3% H₂O₂ was then applied for 5 min to inactivate endogenous peroxidase. The cells were then exposed to 0.1% Triton X-100 at 4°C for 2 min, incubated with 50 μl of the TUNEL reaction mixture (5 μl TdT-enzyme solution + 45 μl of nucleotide mixture solution) in the dark at 37°C for 60 min, exposed to 3,3′-diaminobenzidine (DAB), and then counterstained with hematoxylin. The cells were then dehydrated using graded ethanol and covered with a xylene-based mounting medium. The percentage of TUNEL-positive cells from the control, IL-1β and L-1β⁺ IGF-1 groups were determined by counting the TUNEL-positive cells under 10 non-continuous low-power fields (magnification, ×100).

Cells were cultured in DMEM-F12 containing IL-1β 100 μg/l with or without IGF-1 500 μg/l for 24 h. Cells cultured in DMEM-F12 without FBS for 24 h served as controls. The cells were then digested using trypsin, collected, washed in FCM buffer, and resuspended in FCM wash buffer. To detect cell apoptosis, the cells were incubated with 5 μl of Annexin V-FITC incubation reagent in the dark for 15 min at 4°C, followed by incubation with propidium iodine (PI)-PE of 10 μl for 5 min at 4°C. Samples were analyzed within 30 min using FCM.

Data were presented as the means ± SD (n=3). Groups of data were compared statistically using the Mann-Whitney U test. Values were considered statistically significant when P<0.05.

Primary nucleus pulposus cells were obtained by sequential enzyme digestion and cultured in DMEM-F12 10% FBS (Fig. 1A). Following culture for 7–10 days, the cells formed a complete monolayer (Fig. 1B). To identify the nucleus pulposus phenotype cell, toluidine blue staining was used to identify glycosaminoglycans and immunostaining was used to detect type II collagen (Fig. 1C and D). The results showed that these cells expressed both type II collagen and glycosaminoglycans, thus demonstrating the phenotype of nucleus pulposus cells.

The result obtained from Giemsa staining indicates that nuclear fragmentation occurred following culture of nucleus pulposus cells in the presence of 100 μg/l IL-1β (Fig. 2), suggesting that IL-1β induced apoptosis of nucleus pulposus cells. TUNEL staining also showed that the percentage of TUNEL-positive cells was markedly greater in the IL-1β-treated group than that in the controls (Fig. 3A-C).

To determine whether IGF-1 affected IL-1β-induced apoptosis of nucleus pulposus cells, both 500 μg/l IGF-1 and 100 μg/l IL-1β were added into culture medium simultaneously. The result of TUNEL indicated that IL-1β-induced apoptosis was significantly suppressed (P<0.01) in the presence of IGF-1 (Fig. 3A and D), suggesting that IGF-1 inhibits apoptosis of nucleus pulposus cells induced by IL-1β.

The results of TUNEL were confirmed by FCM. The percentage of nucleus pulposus cells with signs of early and late stages of apoptosis was significantly higher in the IL-1β group compared with the controls. However, the treatment of IGF-1 reduced IL-1β-induced apoptosis of nucleus pulposus cells (Fig. 4A-C, P<0.01).