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

Abstract

Studies performed at tissular (three-dimensional, 3-D) or cellular (two-dimensional, 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) were embedded within collagen type I lattices and studied for 3 weeks. Entrapment and proliferation of cells within the hydrated collagen gel resulted in the generation of contractile forces, which led to contraction of the collagen gel. We used this ability to evaluate the influence of three modes of mechanical stresses on the cell proliferation and differentiation: (1) the freely retracted gels (FRG) were floating in the medium, (2) the tense gels (TG) were stretched statically and isometrically, with contraction prevented in the longitudinal axis, and (3) the dynamic gels (DG) were floating gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed maximum contraction at day 12 in 50 rpm DG, followed by 25 rpm DG, then FRG (88%, 81%, 70%, respectively) and at day 16 in TG (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 extent to cell number. Cells in DG appeared rounder and larger than in other conditions. In TG, cells were elongated and oriented primarily along the tension axis. Scanning electron microscopy (SEM) showed that tension exerted by cells in TG led to reorientation of collagen fibers which, in turn, determined the spatial orientation and morphology of the cells. Transmission electron microscopy (TEM) performed at maximum proliferation showed a vast majority of cells with a distended well-developed RER filled with granular material and numerous mitochondria. Alkaline phosphatase activity peaked close to the proliferation peak in FRG, whereas in TG, a biphasic curve was observed with a small peak at day 4 and the main peak at day 16. In DG, this activity was lower than in the two other conditions. A similar time course was observed for alkaline phosphatase gene expression as assessed by Northern blots. Regardless of the conditions, osteocalcin level showed a triphasic pattern: 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 indicated that mineralization occurred after 14 days and TEM showed crystals within the matrix. We showed that static and dynamic mechanical stresses, in concert with 3-D collagen matrices, played a significant role on the phenotypic modulation of osteoblast-like cells. This experimental model provided a tool to investigate the significance and the mechanisms of mechanical activity of the 3-D cultured osteoblast-like cells.

Nanotopography-guided tissue engineering and regenerative medicine

“…However, the application of mechanical loads has a beneficial effect on the quality and quantity of in vitro generated bone tissues. For example, a study on mechanical loading on osteoblasts in three-dimensional collagen scaffolds under mechanical stress reported that static forces increased cell proliferation and promoted the expression of osteoblastic differentiation markers, such as alkaline phosphatase and osteocalcin [151]. Alternatively, dynamic stresses enhanced the osteocalcin expression but inhibited cell growth and alkaline phosphatase expression [151].…”

“…For example, a study on mechanical loading on osteoblasts in three-dimensional collagen scaffolds under mechanical stress reported that static forces increased cell proliferation and promoted the expression of osteoblastic differentiation markers, such as alkaline phosphatase and osteocalcin [151]. Alternatively, dynamic stresses enhanced the osteocalcin expression but inhibited cell growth and alkaline phosphatase expression [151]. Tanaka et al also reported that mouse osteoblasts seeded in three-dimensional collagen scaffold under a uniaxial mechanical strain reduced the proliferation rate, while the expression of osteocalcin was increased [152].…”

Involvement of 3D osteoblast migration and bone apatite during in vitro early osteocytogenesis

“…Different factors are identified as directing the osteoblast behavior. Among these, a low oxygen tension is understood to promote the osteoblastic differentiation [5] and the presence of mechanical stress is described to modulate the osteoblast proliferation [6] and phenotype [7,8] . Additionally, the fibrillar feature of the collagen network appears crucial since it allows the osteoblasts adhesion via integrin α1β1 and α2β1 [9,10] , favors the physiological-type shape and directs cells toward osteoblast maturation by changing the local topography [11] .…”

From the macroscale to nanostructures: can tissue engineering recreate bone features?

“…The results confirmed that the mechanical stimulus could be as effective as the chemical one to induce bone cell differentiation [134]. Akhouayri and co-workers [135] evaluated the effect of contractive forces onto ROS 17/2.8 osteosarcoma-derived cells. Different contractile conditions were reached in type I collagen gels (freely retracted gels, stretching of the tense gel, periodic stress).…”