Volume 127, Issue 3, Pages 792-801 (September 2004) Interleukin 10-deficient mice develop osteopenia, decreased bone formation, and mechanical fragility of long bones  Rivka Dresner-Pollak, Nir Gelb, Daniel Rachmilewitz, Fanny Karmeli, Miron Weinreb  Gastroenterology  Volume 127, Issue 3, Pages 792-801 (September 2004) DOI: 10.1053/j.gastro.2004.06.013 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 IL-10−/− mice develop osteopenia (decreased bone mass). Femora were cleaned of soft tissue, dried at 60°C overnight, and ashed at 700°C for 24 hours to determine ash weight, an index of total bone mass. Ash weight, corrected for femoral length, is reduced in 8- and 12-week-old IL-10−/− mice compared with WT mice. Data represent mean ± SEM. There were 10 mice per group. Gastroenterology 2004 127, 792-801DOI: (10.1053/j.gastro.2004.06.013) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 IL-10−/− mice develop osteopenia of both cancellous and cortical bone. Cancellous bone area and surface were determined in sections of the tibial proximal metaphysis, using the Bioquant Nova Image Analysis System (R&M Biometrics). Cortical bone area and width were determined in sections of the tibial diaphysis. (A) Cancellous bone area, an index of cancellous bone mass, is decreased in 8- and 12-week-old IL-10−/− mice. (B) Trabecular surface and number, indices of cancellous bone mass, are decreased in 12-week-old IL-10−/− mice. (C) Trabecular width, an index of cancellous bone mass, is decreased in 8- and 12-week-old IL-10−/− mice. (D) Cortical bone area and width, indices of cortical bone mass, are decreased in 12-week-old IL-10−/− mice. Data represent mean ± SEM. There were 10 mice per group. Gastroenterology 2004 127, 792-801DOI: (10.1053/j.gastro.2004.06.013) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Osteopenia in IL-10−/− mice results from decreased bone formation. To label bone-forming surfaces, mice were administered with subcutaneous calcein (15 mg/kg) 8 and 2 days before death. The extent of cancellous bone surface with single- or double-calcein labels was determined as the percentage of the total bone surface. The mineralizing surface and mineral apposition rate (mar) were measured as indicated in the Materials and Methods section. (A) Extent of cancellous double labels and mineralizing surface is decreased significantly in 8- and 12-week-old IL-10−/− mice. (B) Cancellous MAR and bone formation rate are decreased significantly in 8-week-old IL-10−/− mice. Femora were used to generate bone marrow stromal-cell cultures. After a culture period of 21 days, mineralized nodules, an index of osteoblastogenesis and bone formation, were stained using the Von-Kossa method, and counted. Alkaline phosphatase activity, a marker of osteoblast differentiation, was determined in culture with a colorimetric assay. (C) The number of mineralized nodules per well is decreased significantly in 8-week-old IL-10−/− mice. (D) Alkaline phosphatase activity is decreased significantly in 8-week-old IL-10−/− mice. Data represent mean ± SEM. There were 10 mice per group in A and B, and 6 mice per group in C and D. Gastroenterology 2004 127, 792-801DOI: (10.1053/j.gastro.2004.06.013) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Osteopenia in IL-10−/− mice is accompanied by mechanical fragility. Femora were cleaned of soft tissue, and the middiaphysis was subjected to a 3-point biomechanical bending test. The maximal load at fracture and stiffness were determined from the load/deflection plots. Femora of 8- and 12-week-old IL-10−/− mice were more fragile than those of age-matched WT mice, exhibiting lower maximal load at fracture and lower stiffness. Data represent mean ± SEM. There were 10 mice per group. Gastroenterology 2004 127, 792-801DOI: (10.1053/j.gastro.2004.06.013) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 The correlation between body weight and ash weight corrected for femoral length (an index of total bone mass) in 12-week-old IL-10−/− and WT mice is shown. ■, WT mice; •, IL-10−/− mice. The Pearson correlation coefficients were r = 0.9, P < 0.005, and r = 0.86, P < 0.001 in WT and IL-10−/− mice, respectively. Gastroenterology 2004 127, 792-801DOI: (10.1053/j.gastro.2004.06.013) Copyright © 2004 American Gastroenterological Association Terms and Conditions