O. Akhouayri et al., Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels, J CELL BIOC, 76(2), 2000, pp. 217-230
Studies performed at tissular (three-dimensional, 3-D) or cellular (two-dim
ensional, 2-D) levels showed that the loading pattern plays a crucial role
in the osteoblastic physiology. In this study, we attempted to investigate
the response of a 3-D osteoblastic culture submitted to either no external
stress or static or dynamic stresses. Rat osteosarcoma cells (ROS 17/2.8) w
ere embedded within collagen type I lattices and studied for 3 weeks. Entra
pment and proliferation of cells within the hydrated collagen gel resulted
in the generation of contractile forces, which led to contraction of the co
llagen gel. We used this ability to evaluate the influence of three modes o
f mechanical stresses on the cell proliferation and differentiation: (1) th
e freely retracted gels (FRC) were floating in the medium, (2) the tense ge
ls (TC) were stretched statically and isometrically, with contraction preve
nted in the longitudinal axis, and (3) the dynamic gels (DC) were floating
gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed m
aximum contraction at day 12 in 50 rpm DC, followed by 25 rpm DG, then FRG
(88%, 81%, 70%, respectively) and at day 16 in TC (33%). The proliferation
rate was greater in TG than in FRG (+52%) but remained low in both DGs. Gel
dimensions were related to the collagen concentration and on a minor exten
t to cell number. Cells in DC appeared rounder and larger than in other con
ditions. In TG, cells were elongated and oriented primarily along the tensi
on axis. Scanning electron microscopy (SEM) showed that tension exerted by
cells in TG led to reorientation of collagen fibers which, in turn, determi
ned the spatial orientation and morphology of the cells. Transmission elect
ron microscopy (TEM) performed at maximum proliferation showed a vast major
ity of cells with a distended well-developed RER filled with granular mater
ial and numerous mitochondria. Alkaline phosphatase activity peaked close t
o the proliferation peak in FRG, whereas in TC, a biphasic curve was observ
ed with a small peak at day 4 and the main peak at day 16. In DC, this acti
vity was lower than in the two other conditions. A similar time course was
observed for alkaline phosphatase gene expression as assessed by Northern b
lots. Regardless of the conditions, osteocalcin level showed a triphasic pa
ttern: a first increase at day 2, followed by a decrease from day 4 to 14,
and a second increase above initial values at day 18. Microanalysis-x indic
ated that mineralization occurred after 14 days and TEM showed crystals wit
hin the matrix. We showed that static and dynamic mechanical stresses, in c
oncert with 3-D collagen matrices, played a significant role on the phenoty
pic modulation of osteoblast-like cells. This experimental model provided a
tool to investigate the significance and the mechanisms of mechanical acti
vity of the 3-D cultured osteoblast-like cells. (C) 1999 Wiley-Liss, Inc.