Mechanical loading highly increases IL-6 production and decreases OPG expression by osteoblasts  C. Sanchez, Ph.D., O. Gabay, Ph.D., C. Salvat, B.Sc., Y.E. Henrotin, Ph.D., F. Berenbaum, M.D., Ph.D.  Osteoarthritis and Cartilage  Volume 17, Issue 4, Pages 473-481 (April 2009) DOI: 10.1016/j.joca.2008.09.007 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Three-dimensional-osteoblasts membrane and Flexercell apparatus used for compression application. (A–C) Calvarias were harvested from 1l of 6-day-old Swiss mice. After, enzymatic digestion, osteoblasts were plated in 12-wells plates and cultured for 28-days. (C) After 28 days of culture, osteoblasts were embedded in an extensive extracellular matrix and expressed membrane AP. (D) Collagen membrane containing osteoblasts were collected. Two membranes were pooled and put into a Biopress 6-wells culture plate. (E) 1.5ml of media was added and each well was hermetically sealed with a specific cap. (F) The compression was applied by the Flexercell Compression Plus system. The cyclic compression was applied at the magnitude of 1.67MPa (corresponding at 10%) and at the frequency of 1Hz with a sinusoidal waveform. Microscopic view of light green/hematoxylin stained 3D-osteoblasts membrane in the control conditions (G) or after 4h of 10% loading (H) (magnification 40×). Osteoarthritis and Cartilage 2009 17, 473-481DOI: (10.1016/j.joca.2008.09.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effect of compression duration on PGE2 metabolism. (A) COX-2 and (B) 15-PGDH gene expressions as well as (C) PGE2 production were determined after 1, 2, 4, 6 and 8h of 10% compression applied at the frequency of 1Hz. The mRNA copy numbers were normalized against the corresponding copy number of HPRT mRNA and expressed as the fold of increase compared to unloaded 3D-osteoblasts membrane. The PGE2 production is the accumulation of PGE2 in the supernatant and is expressed in pg per μg of DNA. The results are the mean±s.e.m. of ten experiments performed in duplicate. Comparison of mean values was performed by multivariate ANOVA test. Loaded and unloaded culture were significantly different with a *P<0.05, **P<0.01 and ***P<0.001. ND, not detectable. Osteoarthritis and Cartilage 2009 17, 473-481DOI: (10.1016/j.joca.2008.09.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of compression duration on IL-6 and OPG expression and IL-6 production. (A) IL-6 gene expression, (B) IL-6 protein production and (C) OPG gene expression were determined after 1, 2, 4, 6 or 8h of 10% compression applied at the frequency of 1Hz. The mRNA copy numbers were normalized against the corresponding copy number of HPRT mRNA and expressed as the fold of increase compared to unloaded 3D-osteoblasts membrane. The protein production is the accumulation of the protein in the supernatant and is expressed in pg of IL-6 per μg of DNA. The results are the mean±s.e.m. of 10 experiments performed in duplicate. Comparison of mean values was performed by multivariate ANOVA test. Loaded and unloaded cultures were significantly different with a *P<0.05, **P<0.01 and ***P<0.001. ND, not detectable. Osteoarthritis and Cartilage 2009 17, 473-481DOI: (10.1016/j.joca.2008.09.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effects of compression duration on MMPs synthesis. (A) MMP-2, -3 and -13 and (B) MMP-3 production was determined after 1, 2, 4, 6 or 8h of 10% compression applied at the frequency of 1Hz. The mRNA copy numbers were normalized against the corresponding copy number of HPRT mRNA and expressed as the fold of increase compared to unloaded 3D-osteoblasts membrane. The protein production is the accumulation of the protein in the supernatant and is expressed in pg of MMP-3 per μg of DNA. The results are the mean±s.e.m. of 10 experiments performed in duplicate. Comparison of mean values was performed by multivariate ANOVA test. Loaded and unloaded cultures were significantly different with a *P<0.05, **P<0.01 and ***P<0.001. ND, not detectable. Osteoarthritis and Cartilage 2009 17, 473-481DOI: (10.1016/j.joca.2008.09.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of compression amplitude on COX-2, 15-PGDH, IL-6 and MMP-3 gene expressions by Ob. The osteoblasts were compressed during 4h at the amplitude of 6 or 10%. The mRNA copy numbers were normalized against the corresponding copy number of HPRT mRNA and expressed as the fold of increase compared to unloaded 3D-osteoblasts membrane. The results are the mean±s.e.m. of three experiments performed in duplicate. Comparison of mean values was performed by multivariate ANOVA test. Loaded and unloaded cultures were significantly different with a *P<0.05 and ***P<0.001. Osteoarthritis and Cartilage 2009 17, 473-481DOI: (10.1016/j.joca.2008.09.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Effect of inhibitors on (A) IL-6, (B) COX-2 and (C) OPG/RANKL gene expression induced by 4h of 10% compression applied at the frequency of 1Hz. The mRNA copy numbers were normalized against the corresponding copy number of HPRT mRNA and expressed as the fold of increase compared to unloaded 3D-osteoblasts membrane with the same treatment (with or without inhibitors). The results are the mean±s.e.m. of three experiments performed in duplicate. Comparison of mean values was performed by multivariate ANOVA test. The inhibitors significantly modified gene expression compared to controls with a *P<0.05, **P<0.01 or ***P<0.001. Osteoarthritis and Cartilage 2009 17, 473-481DOI: (10.1016/j.joca.2008.09.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions