Addition of nanoscaled bioinspired surface features: A revolution for bone related implants and scaffolds?

Our expanding ability to handle the "literally invisible" building blocks of our world has started to provoke a seismic shift on the technology, environment and health sectors of our society. During the last two decades, it has become increasingly evident that the "nano-sized" subunits composing many materials­living, natural and synthetic­are becoming more and more accessible for predefined manipulations at the nanosize scale. The use of equally nanoscale sized or functionalised tools may, therefore, grant us unprecedented prospects to achieve many therapeutic aims. In the past decade it became clear that nano-scale surface topography significantly influences cell behaviour and may, potentially, be utilised as a powerful tool to enhance the bioactivity and/ or integration of implanted devices. In this review, we briefly outline the state of the art and some of the current approaches and concepts for the future utilisation of nanotechnology to create biomimetic implantable medical devices and scaffolds for in vivo and in vitro tissue engineering,with a focus on bone. Based on current knowledge it must be concluded that not the materials and surfaces themselves but the systematic biological evaluation of these new material concepts represent the bottleneck for new biomedical product development based on nanotechnological principles.

Nitric oxide down-regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells.

Glomerular mesangial cells can produce high amounts of nitric oxide (NO) and reactive oxygen species (ROS). Here we analyzed the impact of NO on the ROS-generating system, particularly on the NADPH oxidase Nox1. Nox1 mRNA and protein levels were markedly decreased by treatment of mesangial cells with the NO-releasing compound DETA-NO in a concentration- and time-dependent fashion. By altering the cGMP signaling system with different inhibitors or activators, we revealed that the effect of NO on Nox1 expression is at least in part mediated by cGMP. Analysis of a reporter construct comprising the 2547 bp of the nox1 promoter region revealed that a stimulatory effect of IL-1beta on nox1 transcription is counteracted by an inhibitory effect of IL-1beta-evoked endogenous NO formation. Moreover, pretreatment of mesangial cells with DETA-NO attenuated platelet-derived growth factor (PDGF)-BB or serum stimulated production of superoxide as assessed by real-time EPR spectroscopy and dichlorofluorescein formation. Transfection of mesangial cells with siRNAs directed against Nox1 and Nox4 revealed that inhibition of Nox1, but not Nox4 expression, is responsible for the reduced ROS formation by NO. Obviously, there exists a fine-tuned crosstalk between NO and ROS generating systems in the course of inflammatory diseases.

Nitric oxide upregulates induction of PDGF receptor-alpha expression in rat renal mesangial cells and in anti-Thy-1 glomerulonephritis.

PDGF and nitric oxide (NO) have been shown to participate in the progression of several forms of glomerulonephritis. A potential influence of NO on PDGF-mediated signaling cascades was therefore examined. Treatment of rat mesangial cells (MC) with the NO donors diethylenetriamine NO (DETA-NO) or spermine-NONOate resulted in a time- and dose-dependent upregulation of PDGF receptor alpha (PDGFRalpha) but not PDGFRbeta mRNA levels. Administration of DETA-NO also induced PDGFRalpha protein expression that was paralleled also by an enhanced receptor phosphorylation. Further experiments using 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), an activator of the soluble guanylyl cyclase (sGC), the membrane-soluble cyclic GMP (cGMP) analog 8-Bromo-PET-cGMP, and the inhibitors of sGC ODQ and NS2028 suggest that elevated cGMP levels are responsible for the effects of NO. Importantly, NO-dependent autophosphorylation of PDGFRalpha drastically augmented PDGF-AA-evoked phosphorylation of PKB/Akt, a classical downstream target of PDGFRalpha signaling. Furthermore, in a rat model of anti-Thy-1 glomerulonephritis, expression and phosphorylation of PDGFRalpha but not PDGFRbeta expression was markedly reduced in nephritic animals that were treated with the inducible NO synthase inhibitor L-N6(1-iminoethyl)lysine(dihydrochloride) (L-NIL) compared with non-L-NIL-treated nephritic rats as demonstrated by Western blotting and immunohistochemistry. Taken together, the data suggest that NO modulates PDGFRalpha-triggered signaling in a cGMP-dependent manner by induction of PDGFRalpha expression in MC and in a rat model of mesangioproliferative glomerulonephritis. The mechanistic details of this regulation have to be elucidated in further experiments.

Identification of manganese superoxide dismutase as a NO-regulated gene in rat glomerular mesangial cells by 2D gel electrophoresis.

The course of inflammatory glomerular diseases is accompanied by changes in the expression of matrix-associated proteins, growth factors, and mediators in renal mesangial cells. Furthermore, the production of nitric oxide (NO) by the inducible isoform of nitric oxide synthase (iNOS) is enhanced after stimulation with pro-inflammatory cytokines. NO has been demonstrated to be a potent modulator of gene expression. To identify NO-regulated genes, we compared the expression patterns of mesangial cells treated for 24h with 500 microM (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) with those of un-stimulated controls by applying a proteomics approach. One protein found to be NO-modulated by 2D gel electrophoresis is the manganese superoxide dismutase (Mn-SOD). Immunoblot and Northern blot analysis demonstrated a dose- and time-dependent induction of Mn-SOD expression by S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and DETA-NO on both the protein and the mRNA levels. An upregulation of Mn-SOD expression by NO was accompanied by an increased Mn-SOD activity. Immunoblots of extracts of IL-1beta-treated cells cultivated with or without the iNOS inhibitor N(G)-monomethyl-L-arginine and the inhibitor of soluble guanylyl cyclase (sGC) 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) demonstrated that the upregulation of the Mn-SOD by NO is due to a NO-dependent activation of sGC. The upregulation of Mn-SOD mRNA expression by NO was confirmed in vivo by Northern blot analysis in kidneys from rats treated with lipopolysaccharide (LPS) either in presence or absence of the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (l-NIL).