Is there a Trojan-horse effect during magnetic nanoparticles and metalloid cocontamination of human dermal fibroblasts?

This study investigates the issue of nanoparticles/pollutants cocontamination. By combining viability assays, physicochemical and structural analysis (to probe the As speciation and valence), we assessed how γFe(2)O(3) nanoparticles can affect the cytotoxicity, the intra- and extracellular speciation of As(III). Human dermal fibroblasts were contaminated with γFe(2)O(3) nanoparticles and As(III) considering two scenarios: (i) a simultaneous coinjection of the nanoparticles and As, and (ii) an injection of the nanoparticles after 24 h of As adsorption in water. In both scenarios, we did not notice significant changes on the nanoparticles surface charge (zeta potential ∼ -10 mV) nor hydrodynamic diameters (∼950 nm) after 24 h. We demonstrated that the coinjection of γFe(2)O(3) nanoparticles and As in the cellular media strongly affects the complexation of the intracellular As with thiol groups. This significantly increases at low doses the cytotoxicity of the As nonadsorbed at the surface of the nanoparticles. However, once As is adsorbed at the surface the desorption is very weak in the culture medium. This fraction of As strongly adsorbed at the surface is significantly less cytotoxic than As itself. On the basis of our data and the thermodynamics, we demonstrated that any disturbance of the biotransformation mechanisms by the nanoparticles (i.e., surface complexation of thiol groups with the iron atoms) is likely to be responsible for the increase of the As adverse effects at low doses.

Preclinical safety study of a combined therapeutic bone wound dressing for osteoarticular regeneration.

The extended life expectancy and the raise of accidental trauma call for an increase of osteoarticular surgical procedures. Arthroplasty, the main clinical option to treat osteoarticular lesions, has limitations and drawbacks. In this manuscript, we test the preclinical safety of the innovative implant ARTiCAR for the treatment of osteoarticular lesions. Thanks to the combination of two advanced therapy medicinal products, a polymeric nanofibrous bone wound dressing and bone marrow-derived mesenchymal stem cells, the ARTiCAR promotes both subchondral bone and cartilage regeneration. In this work, the ARTiCAR shows 1) the feasibility in treating osteochondral defects in a large animal model, 2) the possibility to monitor non-invasively the healing process and 3) the overall safety in two animal models under GLP preclinical standards. Our data indicate the preclinical safety of ARTiCAR according to the international regulatory guidelines; the ARTiCAR could therefore undergo phase I clinical trial.

The bioconjugation mechanism of purine cross-linkers affects microstructure and cell response to ultra rapidly gelling purine-chitosan sponges.

As a cell carrier, cross-linking is one of the most common approaches used to provide chitosan with greater structural integrity. We introduced a cross-linking strategy by using two purines, guanosine 5'-diphosphate (GDP) or adenosine 5'-diphosphate (ADP), as cross-linkers. The rationale for this approach is that both GDP and ADP have an important physiological role and act as intercellular signaling molecules in numerous biological processes. The slight difference between the chemical structure of guanine and adenosine in GDP and ADP, respectively, affect the cross-linking mechanism. This resulted in a different scaffold microstructure and thus, altered the response of encapsulated cells to the scaffold. FTIR and solid-state 13C-NMR revealed the formation of a quadruplex structure among the four GDP molecules confined between the chitosan backbone. This resulted from the ability of guanine to form intermolecular hydrogen bonds, while adenosine in ADP lacks this capacity. The formation of a more organized structure in GDP-chitosan sponges also increased the crystallinity of the sponge as shown by X-ray diffraction data. Further, physicochemical analyses with SEM and µCT indicated a more open-pore architecture and increased porosity. Although an active population of encapsulated cells was maintained in all chitosan sponges overtime, the GDP-based sponges provided a 6-fold increase in the activity of MC-3T3 cells and significantly enhanced their proliferation due to a more appropriate microstructure. Overall, these findings suggest that slight changes in the chemical structure of the cross-linker in the preparation of chitosan-based biomaterials will have a significant impact on the structural properties of the chitosan. This important parameter can be utilized to modulate cell response and to understand the cell signaling pathway of chitosan-based biomaterials in the context of their applications in tissue engineering.

A combinatorial approach towards achieving an injectable, self-contained, phosphate-releasing scaffold for promoting biomineralization in critical size bone defects.

An injectable, guanosine 5'-diphosphate (GDP)-crosslinked chitosan sponge was investigated as a drug delivery system (DDS) for accelerating biomineralization in critical size bone defects (CSBDs). Two approaches were examined both individually, and in combination, in order to achieve this goal. The first approach involved the encapsulation and release of Bone Morphogenetic Protein 7 (BMP-7), a powerful mineralization stimulant. Results confirmed that the rapid gelation of the chitosan sponge prompted high encapsulation of BMP-7 and provided a controlled release over a period of 30 days with no burst release. The second approach was aimed at encapsulating pyrophosphatase (PPtase) in the chitosan sponge to cleave pyrophosphate (PPi) - a mineralization inhibitor and a degradation by-product of the chitosan sponge - into phosphate ions (Pi). PPtase was successfully encapsulated in the chitosan sponge and was able to completely eliminate PPi from the media by cleaving them to Pi. Chitosan sponges releasing Pi into the media were shown to increase overall biomineralization fourfold as compared to controls, an amount equivalent to biomineralization caused by direct injection of 1µg of free BMP-7 to the cells. Even though the combined encapsulation of 1µg BMP-7 and PPtase in the sponges did not demonstrate an additional increase in biomineralization, encapsulation of low concentrations of BMP-7 can promote mesenchymal stem cell migration into the sponge after application in vivo. The findings suggest that the sponge-PPtase system likely allows excellent bone regeneration with lower concentrations of BMP-7, reducing risks and expense of the treatment. STATEMENT OF SIGNIFICANCE: There are bone defects, known as critical size defects, which do not heal on their own and require a therapeutic intervention. The current commercially-available therapies use large quantities of growth factors, such as Bone Morphogenetic Proteins (BMPs), which makes them expensive and a source for a myriad of unwanted side effects. In this manuscript we demonstrate, for the first time, the use of an injectable chitosan-based sponge that contains no inorganic components, but can nonetheless act as a source of phosphate ions to improve bone mineralization. We also demonstrate that this sponge can entrap small concentrations of BMP-7 and provide controlled release over time. The ability to release phosphate ions and low concentrations of BMP-7 makes this therapeutic intervention clinically-relevant, affordable, and safe.

Toward an understanding of mechanism of aging-induced oxidative stress in human mesenchymal stem cells.

Under physiological conditions, there is a production of limited range of free radicals. However, when the cellular antioxidant defence systems, overwhelm and fail to reverse back the free radicals to their normal basal levels, there is a creation of a condition of redox disequilibrium termed "oxidative stress", which is implicated in a very wide spectrum of genetic, metabolic, and cellular responses. The excess of free radicals can, cause unfavourable molecular alterations to biomolecules through oxidation of lipids, proteins, RNA and DNA, that can in turn lead to mutagenesis, carcinogenesis, and aging. Mesenchymal stem cells (MSCs) have been proven to be a promising source of cells for regenerative medicine, and to be useful in the treatment of pathologies in which tissue damage is linked to oxidative stress. Moreover, MSCs appeared to efficiently manage oxidative stress and to be more resistant to oxidative insult than normal somatic cells, making them an interesting and testable model for the role of oxidative stress in the aging process. In addition, aging is accompanied by a progressive decline in stem cell function, resulting in less effective tissue homeostasis and repair. Also, there is an obvious link between intracellular reactive oxygen species levels and cellular senescence. To date, few studies have investigated the promotion of aging by oxidative stress on human MSCs, and the mechanism by which oxidative stress induce stem cell aging is poorly understood. In this context, the aim of this review is to gain insight the current knowledge about the molecular mechanisms of aging-induced oxidative stress in human MSCs.

DNA damage and oxidative stress induced by CeO2 nanoparticles in human dermal fibroblasts: Evidence of a clastogenic effect as a mechanism of genotoxicity.

The broad range of applications of cerium oxide (CeO2) nanoparticles (nano-CeO2) has attracted industrial interest, resulting in greater exposures to humans and environmental systems in the coming years. Their health effects and potential biological impacts need to be determined for risk assessment. The aims of this study were to gain insights into the molecular mechanisms underlying the genotoxic effects of nano-CeO2 in relation with their physicochemical properties. Primary human dermal fibroblasts were exposed to environmentally relevant doses of nano-CeO2 (mean diameter, 7 nm; dose range, 6 × 10(-5)-6 × 10(-3) g/l corresponding to a concentration range of 0.22-22 µM) and DNA damages at the chromosome level were evaluated by genetic toxicology tests and compared to that induced in cells exposed to micro-CeO2 particles (mean diameter, 320 nm) under the same conditions. For this purpose, cytokinesis-blocked micronucleus assay in association with immunofluorescence staining of centromere protein A in micronuclei were used to distinguish between induction of structural or numerical chromosome changes (i.e. clastogenicity or aneuploidy). The results provide the first evidence of a genotoxic effect of nano-CeO2, (while not significant with micro-CeO2) by a clastogenic mechanism. The implication of oxidative mechanisms in this genotoxic effect was investigated by (i) assessing the impact of catalase, a hydrogen peroxide inhibitor, and (ii) by measuring lipid peroxidation and glutathione status and their reversal by application of N-acetylcysteine, a precusor of glutathione synthesis in cells. The data are consistent with the implication of free radical-related mechanisms in the nano-CeO2-induced clastogenic effect, that can be modulated by inhibition of cellular hydrogen peroxide release.

EPR spin trapping evaluation of ROS production in human fibroblasts exposed to cerium oxide nanoparticles: evidence for NADPH oxidase and mitochondrial stimulation.

To better understand the antioxidant (enzyme mimetic, free radical scavenger) versus oxidant and cytotoxic properties of the industrially used cerium oxide nanoparticles (nano-CeO(2)), we investigated their effects on reactive oxygen species formation and changes in the antioxidant pool of human dermal and murine 3T3 fibroblasts at doses relevant to chronic inhalation or contact with skin. Electron paramagnetic resonance (EPR) spin trapping with the nitrone DEPMPO showed that pretreatment of the cells with the nanoparticles dose-dependently triggered the release in the culture medium of superoxide dismutase- and catalase-inhibitable DEPMPO/hydroxyl radical adducts (DEPMPO-OH) and ascorbyl radical, a marker of ascorbate depletion. This DEPMPO-OH formation occurred 2 to 24 h following removal of the particles from the medium and paralleled with an increase of cell lipid peroxidation. These effects of internalized nano-CeO(2) on spin adduct formation were then investigated at the cellular level by using specific NADPH oxidase inhibitors, transfection techniques and a mitochondria-targeted antioxidant. When micromolar doses of nano-CeO(2) were used, weak DEPMPO-OH levels but no loss of cell viability were observed, suggesting that cell signaling mechanisms through protein synthesis and membrane NADPH oxidase activation occurred. Incubation of the cells with higher millimolar doses provoked a 25-60-fold higher DEPMPO-OH formation together with a decrease in cell viability, early apoptosis induction and antioxidant depletion. These cytotoxic effects could be due to activation of both the mitochondrial source and Nox2 and Nox4 dependent NADPH oxidase complex. Regarding possible mechanisms of nano-CeO(2)-induced free radical formation in cells, in vitro EPR and spectrophotometric studies suggest that, contrary to Fe(2+) ions, the Ce(3+) redox state at the surface of the particles is probably not an efficient catalyst of hydroxyl radical formation by a Fenton-like reaction in vivo.

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei.

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C-MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C-MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.