Analysis of metallic traces from the biodegradation of endomedullary AZ31 alloy temporary implants in rat organs after long implantation times.

AZ31 alloy has been tested as a biodegradable material in the form of endomedullary implants in female Wistar rat femurs. In order to evaluate the accumulation of potentially toxic elements from the biodegradation of the implant, magnesium (Mg), aluminium (Al), zinc (Zn), manganese (Mn) and fluorine (F) levels have been measured in different organs such as kidneys, liver, lungs, spleen and brain. Several factors that may influence accumulation have been taken into account: how long the implant has been in place, whether or not the bone is fractured, and the presence of an MgF2 protective coating on the implant. The main conclusions and the clinical relevance of the study have been that AZ31 endomedullary implants have a degradation rate of about 60% after 13 months, which is fully compatible with fracture consolidation. Neither bone fracture nor an MgF2 coating seems to influence the accumulation of trace elements in the studied organs. Aluminium is the only alloying element in this study that requires special attention. The increase in Al recovered from the sampled organs represents 3.95% of the amount contained in the AZ31 implant. Al accumulates in a statistically significant way in all the organs except the brain. All of this suggests that in long-term tests AZ31 may be a suitable material for osteosynthesis.

Fracture bone healing and biodegradation of AZ31 implant in rats.

The ideal temporary implant should offer enough mechanical support to allow healing of the fracture and then biodegrade and be resorbed by metabolic mechanisms without causing any toxic effect. The aim of this research has been to simultaneously study in situ bone healing and the biodegradation of AZ31 Mg alloy as an osteosynthesis material. The in vivo study was carried out in AZ31 implants with and without Mg-fluoride coating inserted in un-fractured and fractured femurs of Wistar rats for long experimentation time, from 1 to 13 months, by means of computed tomography, histological and histomorphometric analysis. Tomography analysis showed the bone healing and biodegradation of AZ31 implants. The fracture is healed in 100% of the animals, and AZ31 maintains its mechanical integrity throughout the healing process. Biodegradation was monitored, quantifying the evolution of gas over time by 3D composition of tomography images. In all the studied groups, gas pockets disappear with time as a result of the diffusion process through soft tissues. Histomorphometric studies reveal that after 13 months the 46.32% of AZ31 alloy has been resorbed. The resorption of the coated and uncoated AZ31 implants inserted in fractured femurs after 1, 9 and 13 months does not have statistically significant differences. There is a balance between the biodegradation of AZ31 and bone healing which allows the use of AZ31 to be proposed as an osteosynthesis material.

Anti-inflammatory action of type I interferons deduced from mice expressing interferon beta.

Type I interferons (IFN) are widely used for the therapeutic treatment of viral infections, tumor growth and various chronic diseases such as multiple sclerosis. Antagonism between type I IFNs and IFN-gamma has been described in cells of the immune system, in particular in the activation of macrophages. To study the systemic effects of type I IFNs we used transgenic mice carrying a human IFN-beta (hIFN-beta) gene under the control of the rat insulin I promoter. These animals expressed high levels of hIFN-beta in beta-pancreatic cells, and the ability of the macrophages to respond to pro-inflammatory stimuli was analyzed. Transgenic mice exhibited an increased extravasation of cells to the peritoneal cavity after eliciting with thioglycollate broth. The expression of the inducible form of nitric oxide synthase and cyclooxygenase-2, two enzymes involved in inflammation, was impaired in transgenic animals challenged with lipopolysaccharide and IFN-gamma. Analysis of the mechanisms leading to this attenuated inflammatory response showed a decrease in the serum levels of TNF-alpha and an inhibition of the activation of the transcription factor NF-KB in various tissues. These results indicate that systemic administration of IFN-beta might influence the response to pro-inflammatory stimuli, in particular through the antagonism of IFN-gamma signaling.

Inhibitory effect of IGF-I on type 2 nitric oxide synthase expression in Ins-1 cells and protection against activation-dependent apoptosis: involvement of phosphatidylinositol 3-kinase.

Challenge of Ins-1 cells, a rat beta-pancreatic cell line, with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) promoted the expression of type 2 nitric oxide synthase (NOS-2) in a cooperative way. Treatment of Ins-1 cells with IGF-I significantly inhibited the expression of NOS-2, especially at subsaturating concentrations of LPS and IFN-gamma. The inhibitory effect of IGF-I on NOS-2 expression was abrogated when cells were incubated with wortmannin or LY294002, two inhibitors of phosphatidylinositol 3-kinase. Transient expression of the p110 subunit of phosphatidylinositol 3-kinase impaired the LPS and IFN-gamma-dependent NOS-2 promoter activity in cells transfected with a 1-kb fragment corresponding to the 5'-flanking region of the NOS-2 gene. However, expression of a dominant negative form of p85 abolished the inhibitory action of IGF-I on the NOS-2 promoter activity. Analysis of the decreased NOS-2 promoter activity in cells incubated with IGF-I showed a lower nuclear factor KB binding as determined by electrophoretic mobility shift assays. The synthesis of NO, produced after LPS and IFN-gamma challenge, triggered an apoptotic response in these cells. IGF-I reduced apoptosis mainly through the decreased synthesis of NO. However, in activated cells treated with N-[3-(aminomethyl)benzyl]acetamidine, a specific NOS-2 inhibitor, IGF-I completely abolished the NO-independent apoptosis. This protection from apoptosis was dependent on phosphatidylinositol 3-kinase activity. These results suggest an important anti-inflammatory and anti-apoptotic role for IGF-I in beta-pancreatic cells, with both actions depending on the activation of phosphatidylinositol 3-kinase.

Implication of glutamate in the expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices.

Nitric oxide synthesis by inducible nitric oxide synthase (iNOS) has been postulated to contribute to ischemia-reperfusion neurotoxicity. The expression of this enzyme has been demonstrated in cells present in the postischemic brain. The mechanisms of iNOS expression after cerebral ischemia are a subject of current research. We therefore decided to investigate whether glutamate, which is released in ischemia and is implicated in neurotoxicity, might be involved in the mechanisms by which oxygen and glucose deprivation (OGD) leads to the expression of iNOS in rat forebrain slices. In this model, we have shown previously that 20 min of OGD causes the expression of iNOS. We have now found that the NMDA receptor antagonist MK-801 blocks the expression of iNOS, suggesting that the activation of the NMDA subtype of glutamate receptor is implicated in the mechanisms that lead to the expression of this isoform. Moreover, we have found that glutamate alone could trigger the induction process, as shown by the appearance of a Ca(2+)-independent NOS activity and by the detection of iNOS mRNA and protein in slices exposed to glutamate. Glutamate-dependent iNOS expression was concentration-dependent and was blocked by EGTA and by the inhibitors of nuclear factor kappaB (NF-kappaB) activation pyrrolidine dithiocarbamate and MG132. In addition, glutamate induced NF-kappaB translocation to the nucleus, an effect that was inhibited by MG132. Taken together, our data suggest that activation of NMDA receptors by glutamate released in ischemia is involved in the expression of iNOS in rat forebrain slices via a Ca(2+)-dependent activation of the transcription factor NF-kappaB. To our knowledge, this is the first report showing an implication of excitatory amino acids in the expression of iNOS caused by ischemia.

Up-regulation of protein kinase C-epsilon promotes the expression of cytokine-inducible nitric oxide synthase in RAW 264.7 cells.

Stimulation of the murine macrophage RAW 264.7 cell line with phorbol esters fails to promote nitric oxide synthesis as occurs in rat hepatocytes or peritoneal macrophages. Transfection of RAW 264.7 cells with plasmids harboring protein kinase C (PKC) -epsilon isotype but not with PKC-alpha, -beta1, -delta, or constitutively active -alpha and -beta1 isotypes resulted in the expression of nitric oxide synthase type II (iNOS), as reflected by the synthesis of nitric oxide measured in the culture medium of transfected cells. cotransfection of RAW 264.7 cells with the -1592 to +121-base pair promoter region of the murine iNOS gene and PKC isotypes specifically induced the transactivation of this promoter in the case of the plasmids containing the PKC-epsilon isotype. The mechanism by which PKC-epsilon induced iNOS expression involved the activation of nuclear factor binding to kappaB sites (NF-kappaB) as deduced by the suppressive effect of pyrrolidine dithiocarbamate on nitric oxide synthesis, an inhibitor of NF-kappaB activation, and by the activation of kappaB sites in cells transfected with a vector containing a kappaB motif linked to a chloramphenicol acetyltransferase reporter gene. These results suggest that PKC-epsilon can regulate a pathway that promotes iNOS expression in macrophages in response to phorbol ester activation.