Fluid shear stress magnitude, duration, and total applied load regulate gene expression and nitric oxide production in primary calvarial osteoblast cultures.
BACKGROUND
Successful bone engineering requires an understanding of the effects of mechanical stress on osteoblast differentiation. Therefore, we examined the effects of varying magnitude and duration of fluid shear stress on factors associated with osteoblastic differentiation.


METHODS
Using a cone viscometer, primary neonatal rat calvarial osteoblasts were exposed to continuous fluid shear stress at varying doses: 0.21, 0.43, and 0.85 Pa for varying time periods. Gene expression was analyzed using Northern blots and nitric oxide production was quantified with the colorimetric Griess reaction.


RESULTS
Fluid shear stress stimulated comparable transient increases in TGF-beta1 and TGF-beta3 expression by 3 hours. TGF-beta1 expression returned to baseline by 12 hours at all shear doses. In contrast, TGF-beta3 expression decreased by 22 percent and 47 percent at 12 hours in response to 0.43 Pa and 0.85 Pa, respectively. Osteopontin and Msx-2 expression patterns were consistent with a more differentiated phenotype at all shear levels. The maximum level of shear stress increased nitric oxide production 2.5-fold at 12 hours and 6.0-fold at 24 hours.


CONCLUSIONS
These data demonstrate differential regulation of TGF-beta1 and TGF-beta3 isoforms with fluid shear stress. Furthermore, because osteopontin and Msx-2 changes were consistent with progressive differentiation at all levels of shear stress, dosage appears to be less important than the presence of an effective physical stimulus. Lastly, nitric oxide does not appear to be the primary regulator of early transcriptional changes found in this study.
González
Octavio
O
Stanford, Calif.; and New York, N.Y. From the Department of Surgery and the Orthopaedic Research Laboratory, Stanford University School of Medicine; and the Institute of Reconstructive Plastic Surgery, New York University Medical Center.
Fong
Kenton D
KD
Trindade
Michael C D
MCD
Warren
Stephen M
SM
Longaker
Michael T
MT
Smith
R Lane
RL
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D005786:Gene Expression Regulation / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics; D006874:Hydrostatic Pressure; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis / Q000502:physiology*; D053495:Osteopontin / Q000235:genetics; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D012886:Skull / Q000166:cytology*; D013314:Stress, Mechanical; D023822:Tissue Engineering / Q000379:methods*; D053773:Transforming Growth Factor beta1 / Q000235:genetics; D053782:Transforming Growth Factor beta3 / Q000235:genetics; D014783:Viscosity
D018398:Homeodomain Proteins; C084119:MSX2 protein; C506897:Spp1 protein, rat; D053773:Transforming Growth Factor beta1; D053782:Transforming Growth Factor beta3; D053495:Osteopontin; D009569:Nitric Oxide