Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels

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.