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Tissue engineering requires the response of the cells to different stimuli inducing the synthesis of the extracellular matrix (ECM). It was been shown that mechanical and biochemical stimuli acted on the synthesis of ECM, particularly type I and III collagens. Growth factors implied in transduction pathways are multiple, but the main is TGF-β. Member of the transforming growth factor-β (TGF-β) family bind to type II and type I serine/threonine kinase receptors, which initiate intracellular signals through activation of SMADs proteins. Nevertheless, the effects of mechanical stress of this pathway remain unknown. The aim of this work was to study the pathway of TGF-β via the SMADs proteins under mechanical (stretching) and biochemical (TGF-β) stimulations. Endogenous SMADs expression and its modulation by biochemical and mechanical stimulations were evaluated by both flow cytometry and confocal microscopy. Our results demonstrate that 10 ng of TGF-β and stretching (5%, 1 Hz) applied during 15 min induced a negative feed back loop which blocks the signalling pathway to control TGF-β activity. This inhibition effect was raised after 1 h of stimulation. Nevertheless, these preliminary studies should be continued by study of expression and localization of inhibitory SMADs (SMAD7). [ABSTRACT FROM AUTHOR]

Nowadays, synthetic biodegradable polymers, such as aliphatic polyesters, are largely used in tissue engineering. They provide several advantages compared to natural materials which use is limited by immunocompatibility, graft availability, etc. In this work, poly(L-lactic) acid (PLLA), poly(DL-lactic) acid (PDLA), poly-ℇ-caprolactone (PCL), poly(L-lactic)-co-caprolactone (molar ratio 70/30) (PLCL) were selected because of their common use in tissue engineering. The membranes were elaborated by solvent casting. Membrane morphology was investigated by atomic force microscopy. The membranes were seeded with human fibroblasts from cell line CRL 2703 in order to evaluate the biocompatibility by the Alamar blue test. The roughness of the membranes ranged from 4 nm for PDLA to 120 nm and they presented very smooth surface except for PCL which beside a macroscopic structure due to its hydrophobicity. Human fibroblasts proliferated over 28 days on the membranes proving the non-in vitro toxicity of the materials and of the processing method. A further step will be the fabrication of three-dimensional scaffold for tissue engineering and the treatment of the scaffolds to augment cell adhesion. [ABSTRACT FROM AUTHOR]

The aim of this work was to study the influences of Ca2+ (medium free calcium, with BAPTA, with 100 mM Ca2+, 100 mM Ca2+ + 10 μM ionomycin) on the expression of caveolin-1 (structural protein of caveolae) of endothelial cells (ECs) submitted to mechanical forces (shear stress) or biochemical stimulation (TNF-α). We found that shear stress enhanced the caveolin-1 expression. Simultaneously, the caveolin-1 expression is dependant on [Ca2+]i: [Ca2+]free medium+100 μM BAPTA < [Ca2+]free medium < [Ca2+]100 mM < [Ca2+]100 mM+10 μM ionomycin. In contrast, TNF-α induced a decrease of caveolin-1 in the cells, whatever the [Ca2+]i. These results suggest that there could be a synergistic effect between shear stress and Ca2+ on caveolin-1 expression but an inflammatory stimulation (TNF-α) induces a down regulation of caveolin-1 expression. [ABSTRACT FROM AUTHOR]

Tissue engineering requires the response of the cells to different stimuli inducing the synthesis of the extracellular matrix (ECM). It was been shown that mechanical and biochemical stimuli acted on the synthesis of ECM, particularly type I and III collagens. Growth factors implied in transduction pathways are multiple, but the main is TGF-beta. Member of the transforming growth factor-beta (TGF-beta) family bind to type II and type I serine/threonine kinase receptors, which initiate intracellular signals through activation of SMADs proteins. Nevertheless, the effects of mechanical stress of this pathway remain unknown. The aim of this work was to study the pathway of TGF-beta via the SMADs proteins under mechanical (stretching) and biochemical (TGF-beta) stimulations. Endogenous SMADs expression and its modulation by biochemical and mechanical stimulations were evaluated by both flow cytometry and confocal microscopy. Our results demonstrate that 10 ng of TGF-beta and stretching (5%, 1 Hz) applied during 15 min induced a negative feed back loop which blocks the signalling pathway to control TGF-beta activity. This inhibition effect was raised after 1 h of stimulation. Nevertheless, these preliminary studies should be continued by study of expression and localization of inhibitory SMADs (SMAD7).

In this work, we analysed the effect of growth factors on in vitro cell proliferation and collagens synthesis by fibroblasts cultured for 72 h on different substrates (silicon sheet with or without 1% gelatin, and glass as control surface) for ligament tissue engineering. A human fibroblast cell line (CRL-2703) was used. The synthesis of type I and type III collagens were evaluated qualitatively and quantitatively by RT-PCR and confocal microscopy, respectively. Cell proliferation was evaluated by two methods: (1) MTT assay (2) cell cycle analysis. It was found that PDGF-AB stimulate the proliferation of fibroblast cultured on gelatin coated silicon sheet in dose dependant manner with a maximum effect at 10 ng ml(-1). The exogenous TGF-beta1 induced the expression of type I and type III collagens in a dose and substrate-dependant manner. We deduce from this work that biochemical conditions and substrates have an important impact for optimisation of the tissue neo synthesis.

The aim of this work was to study the influences of Ca2+ (medium free calcium, with BAPTA, with 100 mM Ca2+, 100 mM Ca2++10 microM ionomycin) on the expression of caveolin-1 (structural protein of caveolae) of endothelial cells (ECs) submitted to mechanical forces (shear stress) or biochemical stimulation (TNF-alpha). We found that shear stress enhanced the caveolin-1 expression. Simultaneously, the caveolin-1 expression is dependant on [Ca2+]i: [Ca2+]free medium+100 microM BAPTA<[Ca2+]free medium<[Ca2+]100 mM<[Ca2+]100 mM+10 microM ionomycin. In contrast, TNF-alpha induced a decrease of caveolin-1 in the cells, whatever the [Ca2+]i. These results suggest that there could be a synergistic effect between shear stress and Ca2+ on caveolin-1 expression but an inflammatory stimulation (TNF-alpha) induces a down regulation of caveolin-1 expression.

We studied the effect of mechanical forces (shear stress) on the kinetics of internalization of native LDL and ox-LDL in endothelial cell line ECV304. This study was performed by using Confocal microscopy and FRET with two carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiO) as the donor and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiI) as the receptor. The cells were incubated with a culture medium containing either 10 microg/ml DiI-LDL or DiO-LDL in static conditions or subjected to a laminar flow under a Confocal Laser Scanning Microscope (SP2 Leica, Germany). The results showed: (1) the possibility to evaluate the kinetics of LDL endocytosis in living cells, (2) shear stress in comparison with control group more effectively enhanced LDL uptake, (3) ox-LDL (>50 microg/ml) >4 hours incubation was found to affect the cells as reflected by their detachment at low shear stress.