Fibrin-based scaffolds for dental pulp regeneration: from biology to nanotherapeutics.

Tissue engineering-based endodontic therapies, designed to regenerate the dental pulp (DP) in the devitalised endodontic space, have been proposed to improve tooth longevity compared to conventional root-filling therapies. Their aim is to restore tooth vitality and major DP functions necessary to maintain tooth health such as immunosurveillance, sensitivity and healing/repair/regenerative capacities. Several formulations based on the use of fibrin, the main component of the blood clot matrix, recently gave valuable results in the regeneration of the human DP. This review describes recent fibrin-based scaffolds designed for that purpose. After having presented the various strategies for DP regeneration, the main fibrin-based scaffolds reported so far for clinical use in endodontics were reviewed. Particular emphasis was given to hydrogel devices that may be improved by incorporation of bioactive molecules that stimulate vascularisation and tissue neoformation or provide antibacterial properties. Data indicate that fibrin-based scaffolds constitute a highly favourable environment for mesenchymal stem cells, which is maintained upon functionalisation. Additional knowledge is needed to understand how fibrin and functionalising agents affect adhesion, survival, proliferation, migration and differentiation of cells incorporated in the scaffold or which will colonise it from neighbouring host tissues. This knowledge is needed to adapt the hydrogel formulation for various clinical conditions.

The combination of deep breathing and mental imagery promotes cardiovascular recovery in firefighters.

Firefighters' rescue operations involve intense physical activity associated with a high level of cardiovascular stress. To sustain such intense physical performance whilst maintaining a healthy heart, it is crucial that they benefit from rapid recovery between each intervention. This study aimed at investigating the impacts of a recovery protocol combining deep breathing and mental imagery. Forty firefighters were divided into two experimental groups which undertook two maximum fitness tests separated by either the control recovery protocol (30 min reading time; n = 20) or the experimental recovery protocol (30 min of deep breathing and mental imagery; n = 20). When compared to the pre-tests, the percentage evolution ratios in the post-tests for the Cooper performance, the heart rate recovery and the parasympathetic reactivation were promoted by the experimental protocol, compared to simple reading. In light of these results, we propose the use of practices of deep-breathing combined with mental imagery to improve firefighters' recovery. Practitioner summary: Firefighters' activities involve intense physical activities associated with a high level of psychological stress. Enhancing their recovery after each rescue intervention appears crucial. The results of this study showed that a recovery protocol combining deep breathing and mental imagery promotes heart rate recovery and better maintenance of physical fitness.

Bioassessment of trace element contamination of Mediterranean coastal waters using the seagrass Posidonia oceanica.

A large scale survey of the trace element (TE) contamination of Mediterranean coastal waters was performed from the analysis of Ag, As, Cd, Cu, Hg, Ni and Pb in the bioindicator Posidonia oceanica, sampled at 110 sites differing by their levels of exposure to contaminants. The holistic approach developed in this study, based on the combined utilization of several complementary monitoring tools, i.e. water quality scale, pollution index and spatial analysis, accurately assessed the TE contamination rate of Mediterranean coastal waters. In particular, the mapping of the TE contamination according to a new proposed 5-level water quality scale precisely outlined the contamination severity along Mediterranean coasts and facilitated interregional comparisons. Finally, the reliability of the use of P. oceanica as bioindicator species was again demonstrated through several global, regional and local detailed case studies. NB: The designations employed and the presentation of the information in this document do not imply the expression of any opinion whatsoever on the part of the authors concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.

Assessment of trace element contamination and effects on Paracentrotus lividus using several approaches: Pollution indices, accumulation factors and biochemical tools.

Among the most common contaminants in marine ecosystems, trace elements are recognized as serious pollutants. In Corsica (NW Mediterranean Sea), near the old asbestos mine at Canari, trace elements from the leaching of mine residues have been discharged into the sea for several decades. The aim of this study was to assess the levels of contamination in this area and the potential effects on Paracentrotus lividus (Lamarck, 1816) using pollution indices, accumulation factors and biochemical tools. For this purpose, the concentration of 24 trace elements was measured in sea urchins (gonads and gut content), macroalgae, seawater column and sediment collected at 12 stations nearby the old asbestos mine and at a reference site. The bioaccumulation of trace elements occurs as follows: macroalgae > gut > gonads. TEPI contribute to highlight contamination gradients which are mainly due to the dominant marine currents allowing the migration of mining waste along the coastline. This hypothesis was supported by TESVI, which identified characteristic trace elements in the southern area of the mine. High hydrogen peroxide content, associated with elevated catalase and glutathione-S-transferase enzyme activities, were also identified at these sites and at the reference site. Trace elements contamination as well as several abiotic factors could explain these results (e.g. microbiological contamination, hydrodynamic events, etc.). The results obtained in this study suggest that oxidative stress induced by contamination does not affect the health of Paracentrotus lividus. This work has provided a useful dataset allowing better use of sea urchins and various tools for assessing trace element contamination in coastal ecosystems.

New insights into the functioning and structure of the PE and PP plastispheres from the Mediterranean Sea.

Plastic debris are accumulating in the marine environment and aggregate microorganisms that form a new ecosystem called the plastisphere. Better understanding the plastisphere is crucial as it has self-sufficient organization and carries pathogens or organisms that may be involved in the pollutant adsorption and/or plastic degradation. To date, the plastisphere is mainly described at the taxonomic level and the functioning of its microbial communities still remains poorly documented. In this work, metagenomic and metaproteomic analyzes were performed on the plastisphere of polypropylene and polyethylene plastic debris sampled on a pebble beach from the Mediterranean Sea. Our results confirmed that the plastisphere was organized as self-sufficient ecosystems containing highly active primary producers, heterotrophs and predators such as nematode. Interestingly, the chemical composition of the polymer did not impact the structure of the microbial communities but rather influenced the functions expressed. Despite the fact that the presence of hydrocarbon-degrading bacteria was observed in the metagenomes, polymer degradation metabolisms were not detected at the protein level. Finally, hydrocarbon degrader (i.e., Alcanivorax) and pathogenic bacteria (i.e., Vibrionaceae) were observed in the plastispheres but were not very active as no proteins involved in polymer degradation or pathogeny were detected. This work brings new insights into the functioning of the microbial plastisphere developed on plastic marine debris.

Effects of trace elements contaminations on the larval development of Paracentrotus lividus using an innovative experimental approach.

Several experiments were performed using larvae of Paracentrotus lividus (Lamarck, 1816) in order to determine the consequences of different chronic contamination with mixtures of (i) fifteen trace elements from concentrations measured in the world ocean seawater, and (ii) seven trace elements from contamination resulting from mining. To predict the impact of increased marine pollution, higher concentrations were also used. These bioassays were conducted using spawners collected from Calvi (reference site, Corsica), and Albo (mining area, Corsica). The effects of trace elements have been studied on the entire larval development. The results show wider arms and delayed development as the number and concentration of trace elements increases. Therefore, the synergy between the different trace elements is of paramount importance with regard to the impact on organisms. Probably due to a hormesis phenomenon, larvae contaminated with seven trace elements at average concentrations developed more quickly. This work also highlighted the importance of the origin of spawners in ecotoxicological studies. To our knowledge, this is the first study to investigate the effects of such a broad combination of trace elements for chronic contamination on the entire larval stage of Paracentrotus lividus.

Stress response to trace elements mixture of different embryo-larval stages of Paracentrotus lividus.

This study investigated for the first time the oxidative biomarkers responses in all larval stages of sea urchin. The contamination effects were reproduced by using contaminated seawater to concentrations measured in the area adjacent to an old asbestos mine at factors of 5 and 10. The results suggested that the concentrations were not sufficiently high to induce a major oxidative stress. The biometric differences make this method a more sensitive approach for assessing the effects on sea urchin larvae. Measurements of specific activities of antioxidant enzymes at each stage suggested a high capacity of the larvae to respond to oxidative stress. This normal activity of the organism must be considered in future research. This work also highlighted the importance of spawners provenance in ecotoxicological studies. These data are essential to better understand the stress responses of sea urchin larvae and provide baseline information for later environmental assessment research.

Development of an antibacterial nanocomposite hydrogel for human dental pulp engineering.

Hydrogel-based regenerative endodontic procedures (REPs) are considered to be very promising therapeutic strategies to reconstruct the dental pulp (DP) tissue in devitalized human teeth. However, the success of the regeneration process is limited by residual bacteria that may persist in the endodontic space after the disinfection step and contaminate the biomaterial. The aim of this work was to develop an innovative fibrin hydrogel incorporating clindamycin (CLIN)-loaded Poly (d,l) Lactic Acid (PLA) nanoparticles (NPs) to provide the hydrogel with antibacterial properties. CLIN-PLA-NPs were synthesized by a surfactant-free nanoprecipitation method and their microphysical properties were assessed by dynamic light scattering, electrophoretic mobility and scanning electron microscopy. Their antimicrobial efficacy was evaluated on Enteroccocus fæcalis by the determination of the minimal inhibitory concentration (MIC) and the minimal biofilm inhibition and eradication concentrations (MBIC and MBEC). Antibacterial properties of the nanocomposite hydrogel were verified by agar diffusion assays. NP distribution into the hydrogel and release from it were evaluated using fluorescent PLA-NPs. NP cytotoxicity was assessed on DP mesenchymal stem cells (DP-MSCs) incorporated into the hydrogel. Type I collagen synthesis was investigated after 7 days of culture by immunohistochemistry. We found that CLIN-PLA-NPs displayed a drug loading of 10 ± 2 µg per mg of PLA polymer and an entrapment efficiency of 43 ± 7%. Antibiotic loading did not affect NP size, polydispersity index and zeta potential. The MIC for Enterococcus fæcalis was 32 µg mL-1. MBIC50 and MBEC50 were 4 and 16 µg mL-1, respectively. CLIN-PLA-NPs appeared homogenously distributed throughout the hydrogel. CLIN-PLA-NP-loaded hydrogels clearly inhibited E. faecalis growth. DP-MSC viability and type I collagen synthesis within the fibrin hydrogel were not affected by CLIN-PLA-NPs. In conclusion, CLIN-PLA-NP incorporation into the fibrin hydrogel gave the latter antibacterial and antibiofilm properties without affecting cell viability and function. This formulation could help establish an aseptic environment supporting DP reconstruction and, accordingly, might be a valuable tool for REPs.

A new agarose-based microsystem to investigate cell response to prolonged confinement.

Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and its porosity enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all cell lines tested. The soft cell confiner is thus a valuable tool to decipher the effects of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer.

Spatial variations in trace element concentrations of the sea urchin, Paracentrotus lividus, a first reference study in the Mediterranean Sea.

A study on Trace Elements (TE) from sea urchin gonads has been conducted in the western Mediterranean Sea. Contamination data were used to determine a Trace Method Pollution Index (TEPI). TE concentrations varied considerably depending on the location of the sampling stations. The results showed that five trace elements (Zn, Fe, As, Al, Cu) are ubiquitous. The geographical area considered (Corsica) represents an important range of environmental conditions and types of pressure that can be found in the western Mediterranean Sea. TEPI was used to classify the studied sites according to their degree of contamination and allowed reliable comparison of TE contamination between local and international sites. TE contamination of the western Mediterranean Sea displayed a north-to-south gradient, from the Italian coasts down through the insular Corsican coasts to the north African littoral. Due to the increasing environmental pressure on the Mediterranean Sea, a regular monitoring of TE levels in marine organisms is necessary to prevent any further environmental deterioration.

Trace element concentrations in the apex predator swordfish (Xiphias gladius) from a Mediterranean fishery and risk assessment for consumers.

Swordfish (Xiphias gladius L., 1758) is an apex predator, highly migratory meso-pelagic fish widely distributed in the Atlantic Ocean and Mediterranean. As top predators, this fish may be the end reservoir of the bioaccumulation of trace elements in a food chain because they occupy higher trophic levels and are an important food source, causing them to be potentially hazardous to consume. This study aims to investigate the concentration of 18 trace elements of Swordfish, caught in the Mediterranean Sea and to discuss human exposure risks. The mean element levels in the fish muscles were clearly below the maximum allowable concentrations established by International food safety regulations. The data suggested that the risk is minor and acceptable for human health. The findings of this study amplify the scarce database on contaminants available, especially new data on "emerging elements", for this species from the Mediterranean Sea.

Role of metallothioneins in metal regulation by the guillemot Uria aalge.

Guillemots, like other seabird species living in the North Sea, appear to be heavily contaminated by copper. Metallothioneins are present in both liver and kidney but, at least in the specimens stranded along the Belgian coast, fail to maintain constant the copper, zinc and cadmium load of the high molecular weight soluble proteins of both organs, stressing the potential toxic role of these metals, mainly copper.

A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips.

The present study gives a summary using state-of-the-art technology to monitor Posidonia oceanica and Mytilus galloprovincialis as bioindicators of the pollution of the Mediterranean littoral with trace elements (TEs), and discusses their complementarity and specificities in terms of TE bioaccumulation. Furthermore, this study presents two complementary indices, the Trace Element Spatial Variation Index (TESVI) and the Trace Element Pollution Index (TEPI): these indices were shown to be relevant monitoring tools since they led to the ordering of TEs according to the overall spatial variability of their environmental levels (TESVI) and to the relevant comparison of the global TE pollution between monitored sites (TEPI). In addition, this study also discusses some underestimated aspects of P. oceanica and M. galloprovincialis bioaccumulation behaviour, with regard to their life style and ecophysiology. It finally points out the necessity of developing consensual protocols between monitoring surveys in order to publish reliable and comparable results.

Experimental in situ exposure of the seagrass Posidonia oceanica (L.) Delile to 15 trace elements.

The Mediterranean seagrass Posidonia oceanica (L.) Delile has been used for trace element (TE) biomonitoring since decades ago. However, present informations for this bioindicator are limited mainly to plant TE levels, while virtually nothing is known about their fluxes through P. oceanica meadows. We therefore contaminated seagrass bed portions in situ at two experimental TE levels with a mix of 15 TEs (Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Ag, Cd, Pb and Bi) to study their uptake and loss kinetics in P. oceanica. Shoots immediately accumulated pollutants from the beginning of exposures. Once contaminations ended, TE concentrations came back to their original levels within two weeks, or at least showed a clear decrease. P. oceanica leaves exhibited different uptake kinetics depending on elements and leaf age: the younger growing leaves forming new tissues incorporated TEs more rapidly than the older senescent leaves. Leaf epiphytes also exhibited a net uptake of most TEs, partly similar to that of P. oceanica shoots. The principal route of TE uptake was through the water column, as no contamination of superficial sediments was observed. However, rhizomes indirectly accumulated many TEs during the overall experiments through leaf to rhizome translocation processes. This study thus experimentally confirmed that P. oceanica shoots are undoubtedly an excellent short-term bioindicator and that long-term accumulations could be recorded in P. oceanica rhizomes.

Establishing research strategies, methodologies and technologies to link genomics and proteomics to seagrass productivity, community metabolism, and ecosystem carbon fluxes.

A complete understanding of the mechanistic basis of marine ecosystem functioning is only possible through integrative and interdisciplinary research. This enables the prediction of change and possibly the mitigation of the consequences of anthropogenic impacts. One major aim of the European Cooperation in Science and Technology (COST) Action ES0609 "Seagrasses productivity. From genes to ecosystem management," is the calibration and synthesis of various methods and the development of innovative techniques and protocols for studying seagrass ecosystems. During 10 days, 20 researchers representing a range of disciplines (molecular biology, physiology, botany, ecology, oceanography, and underwater acoustics) gathered at The Station de Recherches Sous-marines et Océanographiques (STARESO, Corsica) to study together the nearby Posidonia oceanica meadow. STARESO is located in an oligotrophic area classified as "pristine site" where environmental disturbances caused by anthropogenic pressure are exceptionally low. The healthy P. oceanica meadow, which grows in front of the research station, colonizes the sea bottom from the surface to 37 m depth. During the study, genomic and proteomic approaches were integrated with ecophysiological and physical approaches with the aim of understanding changes in seagrass productivity and metabolism at different depths and along daily cycles. In this paper we report details on the approaches utilized and we forecast the potential of the data that will come from this synergistic approach not only for P. oceanica but for seagrasses in general.

Status and origin of Haemonchinae (Nematoda: Trichostrongylidae) in deer: a survey conducted in France from 1985 to 1998.

During our investigations on helminthofauna in roe deer (Capreolus capreolus) and red deer (Cervus elaphus) in France (1985-1998) we isolated nematodes not only of the genus Haemonchus Cobb, 1898 but also of the genus Ashworthius Le Roux 1930, both of which belong to the same subfamily of Haemonchinae. The prevalence of Ashworthius was 22% (65/294) in roe deer and 40% (10/25) in red deer. H. contortus was not found in red deer, whereas its prevalence was only 3% (9/294) in roe deer. These data contrast with those observed in other European countries. The presence of A. sidemi in red and roe deers in France suggests a case of parasitism imported via sika deer. A hypothesis of their spread is proposed.

The signal transduction pathway of erythropoietin involves three forms of mitogen-activated protein (MAP) kinase in UT7 erythroleukemia cells.

The survival and proliferation of the UT-7 human leukemic cell line is strictly dependent on the presence of either interleukin 3, granulocyte-macrophage colony-stimulating factor or erythropoietin. In these cells, erythropoietin stimulation led to the rapid phosphorylation of several proteins including the erythropoietin receptor and proteins with molecular masses around 45 kDa which could be mitogen-activated protein (MAP) kinases. Separation of cytosol from resting or erythropoietin-stimulated UT-7 cells by anion-exchange chromatography revealed two peaks of myelin basic protein kinase activity. The kinase activity of the first peak was independent of erythropoietin treatment of the cells and corresponded to an unidentified 50-kDa kinase, whereas the second peak was only present in erythropoietin-stimulated cells and corresponded to three forms of MAP kinases with molecular masses of 45, 44 and 42 kDa. The three forms were separated by hydrophobic chromatography and were shown to be activated in erythropoietin-stimulated cells. The 44-kDa and 42-kDa forms corresponded to extracellular signal-regulated kinase (ERK)-1 and ERK-2, respectively. Evidence was obtained showing that the 45-kDa form is not a shifted form of ERK-1 but corresponded to a less well defined form of MAP kinase which may be the previously described ERK-4. MAP kinase activation was detected after 1 min erythropoietin stimulation and remained detectable after more than 1 hour. A role for MAP kinase activation in erythropoietin-stimulated cell proliferation was suggested by the simultaneous inhibition of erythropoietin-induced MAP kinase stimulation and cell proliferation. The potential activator of MAP kinase, RAF-1, was hyperphosphorylated in erythropoietin-stimulated cells and its autophosphorylation activity was strongly increased. The protein adaptor Shc was heavily phosphorylated in UT-7 erythropoietin-stimulated cells and associated strongly with a unidentified 145-kDa protein. However, Shc bound poorly to the activated erythropoietin receptor and most Shc proteins were cytosolic in both unstimulated and erythropoietin-stimulated cells. In contrast, Grb2 associated efficiently with the activated erythropoietin receptor and a significant part of Grb2 was associated to a particulate subcellular fraction upon erythropoietin stimulation.

Isolation of human simple repeat loci by hybridization selection.

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Structure of the erythropoietin receptor.

Despite extensive studies, the structure of the erythropoietin receptor remains little understood. cDNAs encoding the human and murine erythropoietin receptors have been cloned and the structure of these proteins is discussed. Although the proteins encoded by these cDNAs play key roles in erythropoietin binding and in erythropoietin signal transduction, increasing evidence strongly suggests that the erythropoietin receptor is a multimeric complex. The murine erythropoietin receptor has been solubilized under mild conditions and the molecular mass of the native receptor has been shown to be significantly higher than the molecular mass of the cloned chain. Cross-linking experiments have revealed the presence of three proteins covalently bound to erythropoietin by the cross-linking reagents; however, only one of them seems to derive from the cloned chain. Moreover, functional evidence also suggests the presence of other erythropoietin receptor subunits.