Abstract
It is our hypothesis that interstitial fluid flow plays a role in the bone remodeling response to mechanical loading. The fluid flow-induced expression of three proteins (collagen, osteopontin, and alkaline phosphatase) involved in bone remodeling was investigated. Rat calvarial osteo-blasts subjected to pulsatile fluid flow at an average shear stress of 5 dyne/cm2 showed decreased alkaline phosphatase (AP) mRNA expression after only 1 hour of flow. After 3 hours of flow, AP mRNA levels had decreased to 30% of stationary control levels and remained at this level for an additional 5 hours of flow. Steady flow (4 dyne/cm2 fluid shear stress), in contrast, resulted in a delayed and less dramatic decrease in AP mRNA expression to 63% of control levels after 8 hours of flow. The reduced AP mRNA expression under pulsatile flow conditions was followed by reduced AP enzyme activity after 24 hours. No changes in collagen or osteopontin mRNA expression were detected over 8 hours of pulsatile flow. This is the first time fluid flow has been shown to affect gene expression in osteoblasts.
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References
Nillson BE, Westlin NE (1971) Bone density in athletes. Clin Orthop 77: 179–182
Arnaud SB, Sherrard DJ, Maloney N, Whalen RT, Fung P (1992) Effects of 1-week head-down tilt bed rest on bone formation and the calcium endocrine system. Aviation Space Env Med Jan: 14–20
Donaldson CL, Hulley SB, Vogel JM, Hattner RS, Bayers JH, McMillan DE (1970) Effect of prolonged bed rest on bone mineral. Metabolism 19(12): 1071–1084
Roer RD, Dillaman RM (1990) Bone growth and calcium balance during simulated weightlessness in the rat. J Appl Physiol 68(1): 13–20
Schneider VS, McDonald J (1984) Skeletal calcium homeostasis and countermeasures to prevent disuse osteoporosis. Calcif Tissue Int 36: S151-S153
Whedon GD (1984) Disuse osteoporosis: physiological aspects. Calcif Tissue Int 36: S146-S150
Reich KM, Gay CV, Frangos JA (1990) Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. J Cell Physiol 143: 100–104
Hillsley MV, Frangos JA (1994) Bone tissue engineering: the role of interstitial fluid flow. Biotech Bioeng 43: 573–581
Kelly PJ, Montgomery RJ, Bronk JT (1990) Reaction of the circulatory system to injury and regeneration. Clin Orthop Rel Res 254: 275–288
Wray JB, Lynch CJ (1959) The vascular response to fracture of the tibia in the rat. J Bone Joint Surg 41A(6): 1143–1148
Simmons KJ, Kunin AS (1979) Skeletal research-an experimental approach. Academic Press, New York
Welch RD, Johnston CE, Waldron MJ, Poteet B (1993) Bone changes associated with intraosseous hypertension in the caprine tibia. J Bone Joint Surg 75A(1): 53–60
Kelly PJ, Bronk JT (1990) Venous pressure and bone formation. Microvasc Res 39: 364–375
Dillaman RM (1984) Movement of ferritin in the 2-day-old chick femur. Anat Rec 209: 445–453
Doty SB, Schofield BH (1972) Metabolic and structural changes within osteocytes of rat bones. In; Talmage RV, Munson PL (eds) Calcium, parathyroid hormone and the calcitonins. Excerpta Medica, Amsterdam, pp 353–364
Montgomery RJ, Sutker BD, Bronk JT, Smith SR, Kelly PJ (1988) Interstitial fluid flow in cortical bone. Microvasc Res 35: 295–307
Seliger WG (1967) Tissue fluid movement in compact bone. Anat Rec 166: 247–256
Kufahl RH, Saha S (1990) A theoretical model for stressgenerated fluid flow in the canaliculi-lacunae network in bone tissue. J Biomech 23(2): 171–180
Salzstein RA, Pollack SR, Mak AFT, Petrov N (1987) Electromechanical potentials in cortical bone I: a continuum approach. J Biomech 20(3): 261–270
Weinbaum S, Cowin SC, Zeng Y (1994) A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stress. J Biomech 27(3): 339–360
Reich KM, Frangos JA (1991) Effect of flow on prostaglandin E2 and inositol trisphospate levels in osteoblasts. Am J Physiol 261(Cell Physiol 30): C429-C432
Reich KM, Frangos JA (1993) Protein kinase C mediates flow-induced prostaglandin E2 production in osteoblasts. Calcif Tissue Int 52: 62–66
Hung CT, Reilly TM, Pollack SR, Brighton CT (1994) Heterogenous [Ca2+]i response in bone cells exposed to fluid flow. Am J Physiol 262: c1411-c1417
Williams JL, Iannotti JP, Ham A, Bleuit J, Chen JH (1994) Effects of fluid shear stress on bone cells. Biorheology 31(2): 163–170
Klein-Nulend J, van der Plas A, Semeins CM, Ajubi NE, Frangos JA, Nijweide PJ, Burger EH (1994) Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9: 441–445
Ecarot-Charrier B, Glorieux H, van der Rest M, Pereira G (1983) Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture. J Cell Biol 96: 639–643
Hillsley MV (1990) The effects of fluid shear stress and 1,25-dihydroxy-vitamin D3 on collagen and osteoclacin production by osteoblasts. M.S. Thesis, The Pennsylvania State University
Frangos JA, Eskin SG, McIntire LV, Ives CL (1985) Flow effects on prostacyclin production by cultured human endothelial cells. Science 227: 1477–1479
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analy Biochem 162: 156–159
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current protocols in molecular biology. John Wiley & Sons, London, New York pp 4.2.1–4.2.5, pp 4.9–4.9.1
Tso JY, Sun XH, Kao TH, Reece KS, Wu R (1985) Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs. Nucl Acids Res 13: 2485–2502
Noda M, Yoon K, Thiede M, Buenaga R, Weiss M, Henthorn P, Harris H, Rodan GA (1987) cDNA cloning of alkaline phosphatase from rat osteosarcoma (ROS 17/2.8) cells. J Bone Miner Res 2(2): 161–164
Genovese C, Rowe D, Kream B (1984) Construction of DNA sequences complementary of rat α1 and α2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 23: 6210–6216
Harrington CH, Chikaraishi DM (1983) Identification and sequence of the initiation site for rat 45S ribosomal RNA synthesis. Nucleic Acid Res 11(10): 3317–3332
Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L, Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS (1990) Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 143: 420–430
Fedarko NS, Bianco P, Vetter U, Gehron Robey P (1990) Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle. J Cell Physiol 144: 115–121
Levesque MJ, Nerem RM, Sprague EA (1990) Vascular endothelial cell proliferation in culture and the influence of flow. Biomaterials 11(9): 702–707
Ibaraki K, Termine JD, Whitson SW, Young MF (1992) Bone matrix mRNA expression in differentiating fetal bovine osteoblasts. J Bone Miner Res 7(7): 743–754
Akamine T, Jee WSS, Ke HZ, Li XJ, Lin BY (1992) Prostaglandin E2 prevents bone loss and adds extra bone to immobilized distal femoral metaphysis in female rats. Bone 13: 11–22
Jee WSS, Ke HZ, Li XJ (1991) Long-term anabolic effects of prostaglandin E2 on tibial diaphyseal bone in male rats. Bone Miner 15: 33–55
Binderman I, Shimshoni Z, Somjen D (1984) Biochemical pathways involved in the translation of physical stimulus into biological message. Calcif Tissue Int 36: S82-S85
Ozawa H, Imamura K, Abe E, Takahashi N, Hiraide T, Shibasaki Y, Fukuhara T, Suda T (1990) Effect of a continuously applied compressive pressure on mouse osteoblast-like cells (MC3T3-E1) in vitro. J Cell Physiol 142: 177–185
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Hillsley, M.V., Frangos, J.A. Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow. Calcif Tissue Int 60, 48–53 (1997). https://doi.org/10.1007/s002239900185
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DOI: https://doi.org/10.1007/s002239900185