1 9 10 11 12 14 15 16 17 18 19 18 20 21 18 18 22 23 18 22 23 24 in vivo 1 2 7 25 in situ Materials and Methods Animals n In vivo Mechanical Loading 1 7 25 in vivo 25 in vivo 26 27 2 2 15 Tissue After fixation, the tibiae were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) with 0.5% paraformaldehyde in PBS at 4°C for 4.5 weeks. Finally, the tibiae were washed in PBS and dehydrated through a series of ethanol and xylene at room temperature and embedded in paraffin. 28 Reagents All restriction enzymes and modifying enzymes were purchased from Roche Molecular Biochemicals (Mannheim, Germany), as well as digoxigenin-uridine triphosphate (UTP), antidigoxigenin Fab fragments, nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and blocking reagent. Nylon membranes were purchased from Qiagen (Hilden, Germany). Polyvinyl alcohol was obtained from Aldrich (Milwaukee, WI). Euparal mounting medium was purchased from Chroma Gesellschaft (Waldeck, Division Chroma, Münster, Germany). Silane-coated glass slides were obtained from Sigma-Aldrich (St. Louis, MO). Mouse IGFBP-2 cDNA was kindly provided by Dr. S. L. S. Drop and Dr. J. W. van Neck (Department of Pediatrics, Sophia Children’s Hospital, Erasmus University, Rotterdam, The Netherlands) via Dr. S. C. van Buul-Offers (Department of Metabolic and Endocrine Diseases, University Medical Center, Utrecht, The Netherlands). Synthesis of Digoxigenin-Labeled Complementary RNA (cRNA) Probes in vitro 29 28 30 31 Nonradioactive In Situ Hybridization In situ 32 Hybridization was performed in a solution containing 50% formamide, 2× SSC, 1× Denhardt’s solution, 250 μg/mL transfer RNA, 480 μg/mL herring sperm RNA, 10% dextran-sulfate, and the mouse IGFBP-2 digoxigenin-labeled cRNA probe at concentrations of 500 pg/μL (brain) and 1,000 pg/μL (tibia). Sections were hybridized overnight at 53°C. After hybridization, sections were washed with 50% formamide in 2× SSC at the hybridization temperature for 30 minutes and treated with RNase A (1 unit/mL) for 30 minutes at 37°C. Subsequently, sections were rinsed in 2× SSC, treated with 1% blocking reagent for 30 minutes, and incubated with sheep antidigoxigenin Fab fragments conjugated with alkaline phosphatase (1:1,500) overnight at 4°C. 33 Quantification and Statistics in situ Effects of mechanical loading were expected in the shaft of the tibiae. Therefore, a second quantitative evaluation of the endocortical osteocytes expressing IGFBP-2 mRNA was performed with Lucia G Version 4.82 (Uvikon) using coded slides of sections II and III. The defined area of interest, which comprised the loading zone, started below the primary spongiosa at the endosteal side of the shaft following the total length of the shaft to the distal side (total length maximal 3,852 μm). At the endosteal side of the shaft, all osteocytes, which were positioned within 100 μm of the endosteal surface, were included in the area of interest. Within the area of interest, total osteocyte number and total IGFBP-2 mRNA-positive osteocyte number were measured. t t P Photography Brightfield photographs with different magnifications (objectives ×10, ×20, and ×40) were made using a Leica microscope (DM4000B) with a digital camera (Leica DC500) and Leica software IM50 (Leica Microsystems, Rijswijk, The Netherlands). Results Endogenous IGFBP-2 mRNA Expression in Tissue 1A 1C 1C, E 1G 1G 1G 1D 1G 1D 1E Fig. 1 A, B C-G H J A, B D, E, G J C, F) A B C D E F G arrowheads H arrowheads J arrowheads  1B 1F Effect of Mechanical Loading on IGFBP-2 mRNA Expression in Osteocytes of the Tibial Shaft n 1 n 1H n 1G n n 1G 2A n n 1H 2B P P 2A, B P P Fig. 2 A ** P n B * P n P P  Effects of Mechanical Loading on IGFBP-2 mRNA Expression in Osteoblasts, Chondrocytes, and Bone Marrow Cells No differences in IGFBP-2 mRNA expression between loaded, sham-loaded, and control tibiae were observed in the osteoblasts, chondrocytes, and bone marrow cells by semiquantitative screening. No differences in morphology of the cells were observed between groups. Discussion in vivo 25 27 in vivo 1 In addition, this study showed that the sham-loaded tibiae have increased IGFBP-2 mRNA production in endocortical osteocytes 6 hours after sham loading. Apparently, besides bending, squeezing alone is also responsible for the upregulation of IGFBP-2 mRNA in the endocortical osteocytes. However, squeezing did not induce lamellar bone formation. Remarkably, there is a large range in the data for the percentage of IGFBP-2 mRNA-positive osteocytes in the contralateral control tibiae of the load group in comparison to the data of the contralateral tibiae of the sham group. This could be caused by the biological variation between the individuals because the animals were randomly divided between the groups and there is no significant statistical difference between those tibiae. 1 7 in situ in situ 28 34 Nevertheless, this study has some limitations. The applied load of 60 N is supraphysiological, and 6 hours after a single loading session is too early to demonstrate bone formation. in vivo