Figure 5
The importance of the RANKL-RANK-OPG pathway in bone resorption induced by synovial fluids (SF) from patients with osteoarthritis (OA) or with a loose prosthesis. (a) Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis on samples obtained by incubating five bones in control medium and five in medium containing SF (10%) from a patient with a loose prosthesis. RNA from the five different bones in each group was pooled and used for RT-PCR analysis. The expressions of the genes of interest were compared to that of GAPDH, and the values below each gel show the number of cycles in the PCRs. (b) Quantitative real-time PCR analysis of rankl mRNA in mouse calvarial bones stimulated by SFs from either patients with OA OK or patients with a loose prosthesis. (c) Quantitative real-time PCR analysis of rank mRNA in mouse calvarial bones stimulated by SFs from either patients with OA or patients with a loose prosthesis. (d) Quantitative real-time PCR analysis of opg mRNA in mouse calvarial bones stimulated by SFs from either patients with OA OK or patients with a loose prosthesis. In (b-d), one calvarial bone was incubated for 48 hours with SF (10%) from an OA patient or with SF (10%) from a patient with a loose prosthesis and the effects were compared to those obtained in unstimulated bones (controls = 100%) or in bones stimulated by D3 (10-8 M). Data shown represent the effects by SFs from seven patients with OA and seven with a loose prosthesis. Effects were compared to the means of two unstimulated bones and two bones treated with D3. The mRNA expression of the gene of interest was expressed in relation to that of β-actin, used as a housekeeping gene. Data shown in (b-d) for the different SFs represent the values obtained in individual bones, and the asterisk denotes a statistically significant (p < 0.05) effect between averages of SFs from patients with OA and those from patients with a loose prosthesis. (e) Addition of OPG to culture medium inhibits calcium 45 (45Ca) release induced by SFs from patients with OA. (f) Addition of OPG to culture medium inhibits 45Ca release induced by SFs from patients with a loose prosthesis. In (e,f), five or six calvarial bones were incubated with SF (10%) from one patient and five or six bones with the same SF and OPG (300 ng/ml). In total, seven patients in each category were tested with and without OPG. At the end of the experiment (96 hours), 45Ca release was analysed and compared to that in unstimulated control bones (100%). OPG significantly (p < 0.05) inhibited the effect of seven of seven SFs from patients with OA and five of seven OKSFs from patients with a loose prosthesis. (g) Quantitative real-time PCR analysis of nfat2 mRNA in mouse calvarial bones stimulated by SFs from either patients with OA or patients with a loose prosthesis. (h) Quantitative real-time PCR analysis of oscar mRNA in mouse calvarial bones stimulated by SFs from either patients with OA or patients with a loose prosthesis. In (g,h), experiments and analysis were performed as described for (b-d) above. Co, control; D3, 1,25(OH)2-vitamin D3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NFAT2, nuclear factor of activated T cells 2; OPG, osteoprotegerin; OSCAR, osteoclast-associated receptor; PCR, polymerase chain reaction; RANK, receptor activator of nuclear factor-kappa-B; RANKL, receptor activator of nuclear factor-kappa-B ligand.