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TECHNICAL PAPERS

Effects of Cyclic Pressure on Bone Marrow Cell Cultures

[+] Author and Article Information
Jiro Nagatomi

Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590  

Bernard P. Arulanandam

Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY

Dennis W. Metzger

Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590 Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY  

Alain Meunier

Faculté de Médicine Lariboisiere St-Louis, Universite D.Diderot, Paris, France  

Rena Bizios

Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590e-mail: Bizios@rpi.edu

J Biomech Eng 124(3), 308-314 (May 21, 2002) (7 pages) doi:10.1115/1.1468867 History: Received October 02, 2001; Revised February 05, 2002; Online May 21, 2002
Copyright © 2002 by ASME
Topics: Pressure , Bone
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References

Figures

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Schematic diagram of the computer-operated cyclic pressure system used in this study. A custom-made, cyclic pressure system was designed, fabricated and used to expose cultured cells to controlled cyclic pressure regimes under standard cell culture conditions (that is, a humidified, 37°C, 5% CO2/95% air environment). During experiments, the cells were maintained under Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and 10 nM 1,25(OH)2 vitamin D3 in individual wells of a tissue-culture polystyrene plate, which was placed in the sealed pressure chamber. The pressure of the gas phase above the culture medium was monitored via a pressure transducer, and was controlled by operating inlet and release solenoid valves using computer software specially written for this purpose. In this schematic diagram of the laboratory setup, the electric and pneumatic connections are designated by thin (–) and thick (▪) lines, respectively; electrical signals and gas-flow directions are designated by thin (→) and bold (➛) arrows, respectively.
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Comparison of early versus late cyclic pressure loading on the formation of TRAP-positive osteoclastic cells from bone marrow cells. Rat bone marrow cell populations were cultured on borosilicate glass in Dulbecco’s Modified Eagle Medium (supplemented with 10% fetal bovine serum and 10−8 M1,25(OH)2 vitamin D3) in humidified, 37°C, 5% CO2/95% air environment and were either maintained under control (static) conditions for either 1 or 7 days (post marrow harvesting) and then either maintained under control conditions or exposed to cyclic pressure (10—40 kPa at 1.0 Hz frequency) for 1 hour daily for the following 1, 3, 5, and 7 consecutive days. No TRAP-positive cells were detected within the bone marrow cell populations that were either maintained under control (static) conditions or exposed to cyclic pressure for 1 and 3 days. Compared to controls (□), significantly (**p<0.01,*p<0.05) fewer osteoclastic cells were formed when bone marrow cell populations were exposed to cyclic pressure (■) for 1 h daily for 5 and 7 consecutive days (Frame (a)). In contrast, formation of osteoclastic cells (Frame (b)) was similar when bone marrow cells were either maintained under control (static) conditions (□) for up to 14 days or maintained under control (static) conditions for the first 7 days and then exposed to cyclic pressure (■) for the next 1, 3, 5, and 7 days. Values are mean ± S.E.M.; n=3; **p<0.01,*p<0.05 (t-test).
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Osteoclastic bone resorption under cyclic pressure. These representative scanning electron micrographs illustrate bone resorption observed when rat bone marrow cells were cultured under the conditions of interest to the present study. In all cases the medium was DMEM (supplemented with 10% fetal bovine serum and 10−8 M1,25(OH)2 vitamin D3). Frame (a): surface of devitalized bone substrates following culture of bone marrow cells under control (static) conditions for 14 consecutive days. Frame (b): surface of devitalized bone substrates following culture of bone marrow cells under static conditions for the first 7 days post bone marrow cell harvesting and then under cyclic pressure (10—40 kPa at 1.0 Hz frequency) for 1 h daily for the next 7 consecutive days. Compared to controls (Frame (a)) fewer resorption lacunae (indicated by arrows) were formed by osteoclasts when bone marrow cells were exposed to cyclic pressure (Frame (b)). Bar=100 μm.(Note: The cracks on the surfaces of the bone substrates were formed after the experiments with cells during storage of the specimens.)
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Total planar surface area of the resorption lacunae formed by osteoclasts on devitalized bone. Rat bone marrow cells were cultured on devitalized bone slices under static conditions for the first 7 days post harvesting, and were then either maintained under control (static) conditions or exposed to cyclic pressure (10–40 kPa at 1.0 Hz) for 1 hour daily for the next 7 consecutive days. Intracellular TRAP activity was similar when bone marrow cells were maintained under control (□) conditions or exposed to cyclic pressure (■) (Frame (a)). In contrast and compared to controls (□), total planar area of these resorption lacunae was significantly (p<0.05) reduced when the bone marrow cells were exposed to cyclic pressure (■) for 1 h daily for 7 consecutive days (Frame (b)). Values are mean ±S. E. M; n=3;*p<0.05 (t-test).
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Cytokine mRNA expression of nonosteoclastic cells in bone marrow cells exposed to cyclic pressure. Bone marrow cells cultured on tissue-culture polystyrene in Dulbecco’s Modified Eagle Medium (supplemented with 10% fetal bovine serum and 10−8 M1,25(OH)2 vitamin D3) were maintained under static conditions for the first 7 days post harvesting and were then either maintained under control (static) conditions (C) or exposed to cyclic pressure (P) (10–40 kPa at 1.0 Hz) for 1 h daily for the next 1, 3, 5, and 7 days. This representative autoradiograph illustrates the results of the ribonuclease protection assay (performed in duplicate as described in the “Assessment of Cytokine mRNA Expression” section) which demonstrate that non-osteoclastic bone marrow cells expressed mRNA for interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1) as well as tumor necrosis factor-α (TNF-α). Housekeeping genes: L32=Ribosomal protein L32; GAPDH=Glyceraldehyde-3phosphate Dehydrogenase.
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Quantification of Cytokine mRNA of non–osteoclastic cells in bone marrow cells exposed to cyclic pressure. All bone marrow cell preparations were maintained under static conditions for 7 days post harvesting and were then either maintained under control (static) conditions or exposed to cyclic pressure for 1 h daily for the next 1 day (Frame (a)), 3 (Frame (b)), 5 (Frame (c)), and 7 consecutive days (Frame (d)). Compared to controls (○), mRNA expression for TGF-β was not affected when bone marrow cells were either exposed to cyclic pressure (•) for 1 h daily during the time periods tested in the present study (Frames (a)–(d)). In contrast, IL-6 mRNA expression was lower when these cells were exposed to cyclic pressure (•) for 1 (Frame (a)) and 3 (Frame (b)) consecutive days, but increased when the bone marrow cells were exposed to cyclic pressure (•) for 5 and 7 consecutive days (Frames (c) and (d)). Moreover, compared to controls, mRNA expression for IL-1α, IL-1β, and TNF-α was lower when bone marrow cells were exposed to cyclic pressure for 1 h for 1 day (Frame (a)) and remained lower when these cells were exposed to cyclic pressure for 1 h daily for 3, 5, and 7 consecutive days (Frames (b)–(d)). The bars (–) represent the mean value of two experiments.

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