The effect of combined simulated microgravity and microgrooved surface topography on fibroblasts

Abstract

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 microm, width: 1, 2, 5, and 10 microm), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment and area. Confocal laser scanning microscopy visualised distribution of actin filaments and focal adhesion points. Finally, expression of collagen type I, fibronectin, and alpha1- and beta1-integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces decreased under simulated microgravity, especially after 24 h of culturing. Cell surface area on grooved substrata were significantly smaller than on smooth substrata, but simulated microgravity on the grooved groups resulted in an enlargement of cell area. ANOVA was performed on all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, gene levels were reduced by microgravity particularly those of beta1-integrin and fibronectin. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by microgravity conditions.

The effect of different surface roughnesses on the differentiation of MC3T3-E1 mouse osteoblasts in vitro

“…However, cells cultured on wider microgrooves of either 5 μm or 10 μm in width attached to the substrate just the same as on smooth surfaces, and there was no acceleration of calcification. Loesberg et al (9,10) used the same size microgrooves as Matsuzaka et al (11) and suggested that cell differentiation was accelerated on microgrooves of 1 μm to 2 μm in width, because cells were suspended in the groove as a cellular bridge and exposed receptors existing on the cell membrane, making it easier to get molecular signals such as dexamethasone into the culture medium and cause (or induce) the cells to differentiate into mature osteoblasts.…”

Growing Tissues in Real and Simulated Microgravity: New Methods for Tissue Engineering

“…Due to the construction of an RPM a broad variety of sample enclosures are applied in RPM studies. This ranges from regular T25 flasks 31,32 to multiwell plates, 33 flask on slides, 34 or more dedicated devices. 35 Performing RPM studies, where the best simulation of microgravity is in the center of rotation of the two axes, has limits for the size of the preferred volume for the samples.…”

Mechanomics and Physicomics in Gravisensing

“…The rods would deflect in response to internal outwarddirected forces. Loesberg et al (2006Loesberg et al ( , 2007 used an isotropic microstructured surface in combination with hypergravity stress or simulated microgravity in a random positioning machine, RPM, to reveal the forces involved in cell orientation and morphology. Properties of standard plastic culture flasks are often taken for granted.…”