Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography.

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 mum, width: 1 mum), 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. Expression of collagen type I, and alpha1-, beta1-, beta3-integrin were investigated by QPCR. Finally, immunoblotting was applied to visualise MAPK signalling pathways. 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 under simulated microgravity, after 48 h of culturing appeared similar to those cultured at 1g, although cell shape was different. Analysis of variance proved that all main parameters: topography, gravity force, and time were significant. In addition, gene levels were reduced by simulated microgravity particularly those of beta3-integrin and collagen, however alpha-1 and beta-1 integrin levels were up-regulated. ERK1/2 was reduced in RPM, however, JNK/SAPK and p38 remained active. The members of the small GTPases family were stimulated under microgravity, particularly RhoA and Cdc42. The results are in agreement that application of microgravity to fibroblasts promotes a change in their morphological appearance and their expression of cell-substratum proteins through the MAPK intracellular signalling pathways.

The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion.

The differences in morphological behaviour between fibroblasts cultured on smooth and nanogrooved substrata (groove depth: 5-350 nm, width: 20-1000 nm) have been evaluated in vitro. The aim of the study was to clarify to what extent cell guidance occurs on increasingly smaller topographies. Pattern templates were made using electron beam lithography, and were subsequently replicated in polystyrene cell culture material using solvent casting. The replicates were investigated with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. AFM revealed that the nanogroove/ridge widths were replicated perfectly, although at deeper levels the grooves became more concave. The smooth substrata had no distinguishable pattern other than a roughness amplitude of 1 nm. Interestingly, microscopy and image analysis showed that fibroblast after 4 h had adjusted their shape according to nanotopographical features down to cut-off values of 100 nm width and 75 nm depth. After 24 h culturing time, fibroblasts would even align themselves on groove depths as shallow as 35 nm. It appears depth is the most essential parameter in cellular alignment on groove patterns with a pitch ratio of 1:1. On the smooth substrata, cells always spread out in a random fashion. Analysis of variance (ANOVA) demonstrated that both main parameters, topography and culturing time, were significant. We conclude that fibroblast cells cultured on nanotopography experience a threshold feature size of 35 nm, below this value contact guidance does no longer exist.

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

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 combined hypergravity and micro-grooved surface topography on the behaviour of fibroblasts.

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and micro-grooved substrata (groove depth: 1 mum, width: 1, 2, 5, 10 microm), which undergo artificial hypergravity by centrifugation (10, 24 and 50 g; or 1 g control). The aim of the study was to clarify which of these parameters was more important to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell spreading and alignment. Confocal laser scanning microscopy visualised distribution of actin filaments and vinculin anchoring points through immunostaining. Finally, expression of collagen type I, fibronectin, and alpha(1)- and beta(1)-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 (control), cells spread out in a random fashion. The alignment of cells cultured on grooved surfaces increased with higher g-forces until a peak value at 25 g. An ANOVA was performed on the data, for all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, most gene levels were reduced by hypergravity. Still, collagen type 1 and fibronectin are seemingly unaffected by time or force. 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 hypergravity forces.

The effect of combined cyclic mechanical stretching and microgrooved surface topography on the behavior of fibroblasts.

Under the influence of mechanical stress, cultured fibroblasts have a tendency to orient themselves perpendicular to the stress direction. Similar cell alignment can be induced by guiding cells along topographical clues, like microgrooves. The aim of this study was to evaluate cell behavior on microgrooved substrates, exposed to cyclic stretching. We hypothesized that cellular shape is mainly determined by topographical clues. On basis of earlier studies, a 10-microm wide square groove, and a 40-microm wide V-shaped groove pattern were used. Smooth substrates served as controls. Onto all substrates fibroblasts were cultured and 1-Hz cyclic stretching was applied (0, 4, or 8%) for 3-24 h. Cells were prepared for scanning electron microscopy, immunostaining of filamentous actin, alignment measurements, and PCR (collagen-I, fibronectin, alpha1- and beta1-integrins). Results showed that cells aligned on all grooved surfaces, and fluorescence microscopy showed similar orientation of intracellular actin filaments. After 3 h of stretch, cellular orientation started to commence, and after 24 h the cells had aligned themselves almost entirely. Image analysis showed better orientation with increasing groove depth. Statistical testing proved that the parameters groove type, groove orientation, and time all were significant, but the variation of stretch force was not. Substrates with microgrooves perpendicular to the stretch direction elicit a better cell alignment. The expression of beta1-integrin and collagen-I was higher in the stretched samples. In conclusion, we can maintain our hypothesis, as microgrooved topography was most effective in applying strains relative to the long axis of the cell, and only secondary effects of stretch force were present.

Effectiveness of the soluble form of the interleukin-1 receptor accessory protein as an inhibitor of interleukin-1 in collagen-induced arthritis.

OBJECTIVE: To investigate whether the soluble form of interleukin-1 (IL-1) receptor accessory protein (sIL-1RAcP), whose physiologic function remains to be established, can serve as a specific inhibitor of IL-1 signaling in vitro, and to evaluate its applicability in collagen-induced arthritis (CIA). METHODS: Soluble IL-1RAcP was cloned from murine liver complementary DNA and expressed by the use of either an adenoviral vector (AdRGD) for sIL-1RAcP or a stable-transfected NIH3T3 fibroblast cell line. The ability of affinity-purified sIL-1RAcP to inhibit IL-1 signaling was tested on NF-kappaB luciferase reporter fibroblasts and quantified by luminometer. To investigate therapeutic efficacy, sIL-1RAcP was both locally (knee joint) and systemically overexpressed in collagen-immunized male DBA/1 mice. Severity of arthritis was monitored visually, and the pathologic process in the joint was examined histologically. Serum was obtained from mice to quantify IL-6 and anti-bovine type II collagen (BCII) antibody levels. RESULTS: Incubation of the NF-kappaB reporter fibroblast with purified sIL-1RAcP protein showed a marked reduction of IL-1-induced, but not tumor necrosis factor-induced, NF-kappaB activation. This showed a novel role for sIL-1RAcP as a specific inhibitor of IL-1 signaling. Local transplantation of sIL-1RAcP-producing NIH3T3 fibroblasts into the knee before onset of CIA had little or no effect on general disease severity in these mice. Histologic evaluation of the knee joints receiving sIL-1RAcP cell transplantation showed a marked reduction in both joint inflammation and bone and cartilage erosion. Local treatment with sIL-1RAcP had no profound effect on serum levels of IL-6 and anti-BCII antibodies, which is indicative of the ongoing presence of arthritis in distal joints. In contrast to local treatment, systemic treatment with the AdRGD for sIL-1RAcP markedly ameliorated CIA in all joints. CONCLUSION: In this study we demonstrated that sIL-1RAcP is a biologically active and innovative inhibitor of IL-1, and treatment of mice with sIL-1RAcP had a profound prophylactic effect on collagen-induced arthritis.