Human cartilage was obtained from nine patients (age 35 to 58 years), with ethical approval (East London and The City Research Ethics Committee) and informed patient consent, undergoing total knee arthroplasty at the Royal London Hospital, Barts, and the London NHS Trust, London, UK. Cartilage was removed from the femoral condyles and tibial plateaus. The morphology of the cartilage specimens was graded for gross degenerative changes according to the ICRS classification, and tissues that represent normal (grade 0 or 1) and early (grade 2) OA were used for experiments. Each experimental condition was repeated with chondrocytes from three to four different donors. Cartilage tissue was diced and incubated on rollers for 1 hour at 37°C in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% (vol/vol) Fetal Calf Serum (FCS) + 2 μM L-glutamine, 5 μg/ml penicillin, 5 μg/ml streptomycin, 20 mM Hepes buffer, and 0.05 mg/ml L-ascorbic acid + 700 unit/ml pronase and incubated for a further 16 hours at 37°C in DMEM + 10% FCS, supplemented with 100 units/ml collagenase type XI (Sigma-Aldrich, Poole, UK). The cell suspension was washed and viable chondrocytes counted using a hemocytometer and trypan blue. Cells were finally resuspended in medium at a cell concentration of 8 × 10⁶ cells/ml by using well-established methods 34 35 . In brief, the cell suspension was added to an equal volume of molten 6% (wt/vol) agarose type VII in Earle Balanced Salt Solutions (EBSS) to yield a final cell concentration of 4 × 10⁶ cells/ml in 3% (wt/vol) agarose (Sigma-Aldrich, Poole, UK). The chondrocyte/agarose suspension was transferred into a sterile stainless steel mold, containing holes 5 mm in diameter and 5 mm in height and allowed to gel at 4°C for 20 minutes. Constructs were cultured in a defined culture medium comprising DMEM, 0.1 μM dexamethasone, 0.17 mM ascorbate, 1 mM sodium pyruvate, 0.35 mM proline, 5 μg/ml penicillin, 5 μg/ml streptomycin, 20 mM Hepes buffer, 2 μM L-glutamine, ITS, and supplements (6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 μg/ml seleneous acid, 5.33 μg/ml linoleic acid, and 1.25 μg/ml bovine serum albumin) at 37°C in 5% CO₂ for 24 hours (all from Cambrex Bioscience, Wokingham, UK).

The dose-response effect of CNP was examined in constructs cultured under free-swelling conditions to determine the appropriate concentration for mechanical loading studies. Constructs were cultured in 1 ml of defined media supplemented with either low (0, 0.1, 1, 10, 100 pM) or high (0, 1, 10, 100, 1000 nM) concentrations of CNP in the presence and absence of 10 ng/ml IL-1β and/or 5 μM KT5823 for 48 hours (all from Sigma-Aldrich). KT5823 inhibits PKGII by competing directly with ATP at the catalytic domain. In each case, the medium was additionally supplemented with 1 μCi/ml [³H]-thymidine and 10 μCi/ml ³⁵ SO₄ (both Amersham Biosciences Ltd, Bucks, UK) for the assessment of cell proliferation and proteoglycan synthesis, respectively. At the end of the culture period, the constructs and corresponding media were immediately stored at -20°C before biochemical analysis.

In separate experiments, a fully characterized bioreactor compression system (Zwick Testing Machines, Leominster, UK) was used to determine the effect of CNP and dynamic loading on cell metabolism and gene expression in chondroycte/agarose constructs. The bioreactor has been extensively described previously 34 35 36 . To review briefly, equilibrated constructs were transferred into individual wells of a 24-well culture plate (Costar, High Wycombe, UK) and mounted within the bioreactor. One milliliter of defined media supplemented with 0 or 10 ng/ml IL-1β in the presence and absence of low (100 pM) or high (100 nM) concentrations of CNP and/or 5 μM KT5823 were introduced into each well. Strained constructs were subjected to dynamic compression ranging from 0 to 15% strain in a sinusoidal waveform at a frequency of 1 Hz. The compression regimen was applied in an intermittent manner, with a profile of 1.5 hour compression followed by a 4.5 hour unstrained period for both the 6 and 48 hour culture periods. This resulted in duty cycles equivalent to 5400 and 43200, respectively. Control constructs were maintained in an unstrained state within the bioreactor system and cultured for the same time period. At the end of the culture period, the constructs and corresponding media were immediately stored at -70°C before analysis.

RNA was isolated from chondrocytes cultured in agarose by using protocols described in the QIAquick Spin gel extraction and RNeasy kits, as previously described (Qiagen, West Sussex, UK) 28 37 . By following manufacturer's instructions, Ambion's DNA-free DNase treatment and removal reagents were used to eliminate any contaminating DNA from the RNA sample (Ambion Applied Biosystems, Warrington, UK). RNA was quantified on the Nanodrop ND-1000 spectrophotometer (LabTech, East Sussex, UK), and reverse transcription was performed by using manufacturer's protocols from the Enhanced Avian RT First Strand cDNA synthesis kit, oligo(dT)₂₃ primer, and a total of 200 ng of RNA (Sigma Genosys, Cambridge, UK). Real-time quantitative PCR assays coupled with LNA probes were performed in 25-μl reaction mixtures containing 1 μl cDNA, 12.5 μl JumpStart Taq PCR Master Mix, primer pairs, and probes detailed in Table 1 and nuclease-free PCR-grade water to 25 μl (Sigma Genosys, Cambridge, UK). Each sample was run in duplicate on the 96-well thermal system of the Mx3000P quantitative PCR instrument (Stratagene, Amsterdam, The Netherlands). Thermocycling conditions comprised an initial polymerase activation step at 95°C for 3 minutes, followed by denaturation of 35 cycles at 95°C for 30 seconds, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute. PCR efficiencies for optimal primer pair and probe concentrations were derived from standard curves (n = 3) by preparing a 10-fold serial dilution of cDNA from a sample that represented the untreated control at time-zero conditions. The real-time PCR efficiencies (E) of amplification for each target was defined according to the relation, E = 10^[-1/slope]. The R² value of the standard curve exceeded 0.9998 and revealed efficiency values presented in Table 1.

Fluorescence data were collected during the annealing stage of amplification, and data were analyzed on the MxPro qPCR software (version 3, Stratagene). Baselines and thresholds were automatically set by the RG-3000 qPCR software and used after manual inspection. The cycle threshold (C_t) value for each duplicate reaction was expressed as the mean value, and the results were exported into Microsoft Excel for further analysis. The data obtained by PCR assay for GAPDH were validated as a reference gene by displaying the C_t values as box-and-whisker plots, and the distribution examined under mechanical loading conditions (data not shown). The C_t values for GAPDH remained stable, with no changes detected under all culture conditions, suggesting its suitability as a reference gene. Relative quantification of iNOS, COX-2, aggrecan, and collagen type II signals were accomplished by normalizing each target to the reference gene, GAPDH, and to the calibrator sample by a comparative C_t approach. For each sample, the ratio of target ΔCt and reference ΔCt was calculated, as previously described 28 37 .

The production of NO was determined in media by converting nitrate to nitrite by using 1 unit/ml nitrate reductase in 40 μM NAPDH, 500 μM glucose 6-phosphate, 160 unit/ml glucose 6-phosphate dehydrogenase and 20 mM Tris-HCL for 15 minutes at 37°C. Total nitrite was assayed spectrophotometrically at 540 nm by using the Griess reaction. PGE₂ production was measured in the culture media by using a high-sensitivity enzyme immunoassay according to manufacturer's instructions (Amersham Biosciences Ltd, Bucks, UK). [³H]-thymidine incorporation was measured in constructs digested overnight at 37°C with 10 U/ml agarase followed by 1 hour at 60°C with 2.8 U/ml papain (both Sigma Chemical Co., Poole, UK) and analyzed with 10% trichloroacetic acid precipitation onto filters by using a Millipore Multiscreen system (Millipore, Watford, UK), as previously described 29 34 35 . Incorporation of ³⁵SO₄ was determined in both agarase/papain digests and the culture media by using the Alcian blue precipitation method, as previously described 29 34 35 . Total DNA content remained stable throughout the culture conditions was assayed by using the Hoescht dye 33258 in agarose/papain digests.

For dose-response studies, data represent the mean and SEM values of six replicates from three separate experiments. For the mechanical loading experiments, biochemical and gene-expression data represent the mean and SEM values of eight replicates from three separate experiments. Statistical analysis was performed with a two-way analysis of variance (ANOVA) and the multiple post hoc Bonferroni-corrected t tests to compare differences between the various treatment groups, as indicated in the figure legend. For gene-expression data, ratio values were log transformed before analysis by a two-way ANOVA and the post hoc Bonferroni-corrected t test. In all cases, a level of 5% was considered statistically significant (P < 0.05).

Chondrocytes cultured in agarose constructs produce significant amounts of NO and PGE₂ release in response to IL-1β (both P values < 0.001; Figure 1a and 1b, respectively). To examine whether CNP and the selective PKGII inhibitor could influence the IL-1β-induced NO and PGE₂ release, constructs were cultured with IL-1β and CNP at concentrations ranging from 0.1 to 100 pM in the presence and absence of KT5823. It was evident that CNP reduced IL-1β induced NO and PGE₂ release in a dose-dependent manner, with maximal inhibition at 10 and 100 pM when compared with IL-1β-treated constructs (all P < 0.001). Treatment with IL-1β and the PKGII inhibitor did not influence NO and PGE₂ levels in constructs cultured with CNP (Figures 1a and 1b). In the absence of IL-1β, CNP increased [³H]-thymidine incorporation in a dose-dependent manner (P < 0.01; Figure 1c), whereas this effect of CNP was reduced by IL-1β and/or KT5823 (P < 0.01). At 100 pM, CNP increased ³⁵SO₄ incorporation (P < 0.05; Figure 1d), and this effect was reduced by IL-1β (P < 0.001) and not further influenced with KT5823.

Figure 2 presents the dose-response effects of CNP at concentrations ranging from 0.1 to 1000 nM in constructs cultured with IL-1β and/or KT5823 (Figure 2). IL-1β-induced NO and PGE₂ release was reduced by CNP in a dose-dependent manner (Figure 2a). However, the effect was most pronounced in the presence of 1 nM CNP, which completely abolished the IL-1β-induced PGE₂ release (P < 0.001; Figure 2b). Treatment with CNP and the PKGII inhibitor had no further effect on the reduction of IL-1β-induced release of both NO and PGE₂. At 1 nM, CNP increased [³H]-thymidine incorporation when compared with untreated controls (P < 0.05; Figure 2c). The presence of IL-1β inhibited [³H]-thymidine incorporation and the response was not significantly influenced by CNP and/or the PKGII inhibitor (Figure 2c). In contrast, CNP increased ³⁵SO₄ incorporation in a dose-dependent manner with maximal stimulation at 100 and 1000 nM (P < 0.001; Figure 2d). The presence of IL-1β inhibited ³⁵SO₄ incorporation, and the levels were enhanced with CNP at 100 and 1000 nM, only. However, stimulation of ³⁵SO₄ incorporation by CNP in IL-1β-treated constructs was abolished in the presence of the PKGII inhibitor (P < 0.001; Figure 2d).

In separate experiments, the effects of CNP and dynamic compression were examined in constructs cultured with IL-1β and/or the PKGII inhibitor by using either low (100 pM) or high (100 nM) concentrations of the peptide (Figure 3). In the absence and presence of CNP, dynamic compression reduced NO release (P < 0.001; Figure 3a) but had no significant effect on PGE₂ levels (Figure 3b). In unstrained constructs, the presence of IL-1β increased NO and PGE₂ release, and the response was reduced by dynamic compression, CNP, and/or both stimuli (all P < 0.001). Stimulation with CNP and/or dynamic compression in the presence of the PKGII inhibitor further reduced NO and PGE₂ release in IL-1β-treated constructs (both P < 0.01; Figure 3a and 3b, respectively). In contrast, dynamic compression increased [³H]-thymidine incorporation in the presence and absence of CNP (P < 0.001; Figure 3c). This effect was inhibited with IL-1β and could be reversed by stimulation with 100 pM CNP, dynamic compression, or both. The presence of the PKGII inhibitor blocked CNP-induced stimulation of [³H]-thymidine incorporation in IL-1β-treated constructs. The opposite effect was found for ³⁵SO₄ incorporation, whereby stimulation with 100 nM CNP and dynamic compression induced the greatest response when compared with untreated controls or constructs cultured with 100 nM CNP (P < 0.001; Figure 3d). The IL-1β-induced inhibition of ³⁵SO₄ incorporation was reversed by both 100 nM CNP and dynamic compression (P < 0.001) and inhibited with KT5823 (Figure 3d).

To investigate the effect of CNP on the expression of catabolic and anabolic genes, constructs were subjected to dynamic compression over 6 and 48 hour period in the presence and absence of low (100 pM) and high (100 nM) concentrations of the peptide (Figure 4). In unstrained constructs, IL-1β induced iNOS and COX-2 expression at 6 and 48 hours (all P < 0.001; Figure 4a and 4b, respectively). At 6 and 48 hours, the IL-1β-induced iNOS and COX-2 expression was inhibited by dynamic compression (both P < 0.001) or by the presence of low (both P < 0.01) or high concentrations of CNP (both P < 0.01). A combination of dynamic compression and CNP reduced iNOS and COX expression at 6 hours, with levels returning to basal values with IL-1β at 48 hours. This effect was not significantly influenced further with KT5823 in constructs cultured with IL-1β and CNP. In contrast, dynamic compression increased aggrecan and collagen type II expression at 6 hours but not at 48 hours (both P < 0.05; Figure 4c and 4d, respectively). In unstrained constructs, stimulation with CNP increased aggrecan and collagen type II expression in a concentration-dependent manner and the effect was further enhanced with dynamic compression at either 6 or 48 hours. In unstrained constructs, IL-1β inhibited aggrecan and collagen type II expression and the effect was reversed with dynamic compression (P < 0.001), 100 nM CNP (P < 0.05), or both at 6 (P < 0.01) and 48 hours (P < 0.05) for aggrecan expression. In contrast, stimulation by 100 pM or 100 nM CNP and dynamic compression reversed the IL-1β-induced inhibition of collagen type II expression at 6 (both P < 0.01) and 48 hours (both P < 0.05; Figure 4d). The compression-induced stimulation of aggrecan and collagen type II expression was inhibited with KT5823 in constructs cultured with IL-1β and CNP.