Eleni Papachristou

Redox activity and in vitro bioactivity of the water-soluble fraction of urban particulate matter in relation to particle size and chemical composition.

Chemical and toxicological characterization of the water-soluble fraction of size-segregated urban particulate matter (PM) (<0.49, 0.49-0.97, 0.97-1.5, 1.5-3.0, 3.0-7.2 and >7.2 μm) was carried out at two urban sites, traffic and urban background, during the cold and the warm period. Chemical analysis of the water-soluble PM fraction included ionic species (NO3(-), SO4(2-), Cl(-), Na(+), NH4(+), K(+), Mg(2+), Ca(2+)), water-soluble organic carbon (WSOC), and trace elements (Al, As, Ba, Cd, Cr, Cu, Fe, Pb, Mn, Ni, Zn, Pt, Pd, Rh, Ru, Ir, Ca, and Mg). The dithiothreitol (DTT) assay was employed for the abiotic assessment of the oxidative PM activity. Cytotoxic responses were investigated in vitro by applying the mitochondrial dehydrogenase (MTT) and the lactate dehydrogenase (LDH) bioassays on human lung cells (MRC-5), while DNA damage was estimated by the single cell gel electrophoresis assay, known as Comet assay. The correlations between the observed bioactivity responses and the concentrations of water-soluble chemical PM constituents in the various size ranges were investigated. The results of the current study corroborate that short-term bioassays using lung human cells and abiotic assays, such as the DTT assay, could be relevant to complete the routine chemical analysis and to obtain a preliminary screening of the potential effects of PM-associated airborne pollutants on human health.

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

The Potential Role of Ribosomal Protein S5 on Cell Cycle Arrest and Initiation of Murine Erythroleukemia Cell Differentiation

Evidence now exists to indicate that some ribosomal proteins besides being structural components of the ribosomal subunits are involved in the regulation of cell differentiation and apoptosis. As we have shown earlier, initiation of erythroid differentiation of murine erythroleukemia (MEL) cells is associated with transcriptional inactivation of genes encoding ribosomal RNAs and ribosomal proteins S5 (RPS5) and L35a. In this study, we extended these observations and investigated whether transfection of MEL cells with RPS5 cDNA affects the onset of initiation of erythroid maturation and their entrance in cell cycle arrest. Stably transfected MEL cloned cells (MEL‐C14 and MEL‐C56) were established and assessed for their capacity to produce RPS5 RNA transcript and its translated product. The impact of RPS5 cDNA transfection on the RPS5 gene expression patterns and the accumulation of RPS5 protein in inducible transfected MEL cells were correlated with their ability to: (a) initiate differentiation, (b) enter cell cycle arrest at G1/G0 phase, and (c) modulate the level of cyclin‐dependent kinases CDK2, CDK4, and CDK6. The data presented indicate that deregulation of RPS5 gene expression (constitutive expression) affects RPS5 protein level and delays both the onset of initiation of erythroid maturation and entrance in cell cycle arrest in inducer‐treated MEL cells. J. Cell. Biochem. 104: 1477–1490, 2008.

Sol-Gel Derived Mg-Based Ceramic Scaffolds Doped with Zinc or Copper Ions: Preliminary Results on Their Synthesis, Characterization, and Biocompatibility

Glass-ceramic scaffolds containing Mg have shown recently the potential to enhance the proliferation, differentiation, and biomineralization of stem cells in vitro, property that makes them promising candidates for dental tissue regeneration. An additional property of a scaffold aimed at dental tissue regeneration is to protect the regeneration process against oral bacteria penetration. In this respect, novel bioactive scaffolds containing Mg2+ and Cu2+ or Zn2+, ions known for their antimicrobial properties, were synthesized by the foam replica technique and tested regarding their bioactive response in SBF, mechanical properties, degradation, and porosity. Finally their ability to support the attachment and long-term proliferation of Dental Pulp Stem Cells (DPSCs) was also evaluated. The results showed that conversely to their bioactive response in SBF solution, Zn-doped scaffolds proved to respond adequately regarding their mechanical strength and to be efficient regarding their biological response, in comparison to Cu-doped scaffolds, which makes them promising candidates for targeted dental stem cell odontogenic differentiation and calcified dental tissue engineering.

Human treated dentin matrices combined with Zn-doped, Mg-based bioceramic scaffolds and human dental pulp stem cells towards targeted dentin regeneration.

OBJECTIVE This study aimed to investigate the potential of Mg-based bioceramic scaffolds combined with human treated-dentin matrices (hTDMs) and dentinogenesis-related morphogens to promote odontogenic differentiation and dentin-like tissue formation by Dental Pulp Stem Cells-DPSCs. METHODS DPSC cultures were established and characterized by flow cytometry. Experimental cavities were prepared inside crowns of extracted teeth and demineralized by EDTA (hTDMs). Zn-doped, Mg-based bioceramic scaffolds, synthesized by the sol-gel technique, were hosted inside the hTDMs. DPSCs were spotted inside the hTDMs/scaffold constructs with/without additional exposure to DMP-1 or BMP-2 (100ng/ml, 24h). Scanning Electron Microscopy-SEM, live/dead fluorescence staining and MTT assay were used to evaluate cell attachment and viability; Real time PCR for expression of osteo/odontogenic markers; Inductively Coupled Plasma-Atomic Emission Spectrometry-ICP/AES for scaffold elemental release analysis; ELISA for hTDM growth factor release analysis; SEM and X-ray Diffraction-XRD for structural/chemical characterization of the regenerated tissues. RESULTS Scaffolds constantly released low concentrations of Mg(2+), Ca(2+), Zn(2+) and Si(4+), while hTDMs growth factors, like DMP-1, BMP-2 and TGFβ-1. hTDMs/scaffold constructs supported DPSC viability, inducing their rapid odontogenic shift, indicated by upregulation of DSPP, BMP-2, osteocalcin and osterix expression. Newly-formed Ca-P tissue overspread the scaffolds partially transforming into bioapatite. Exposure to DMP-1 or BMP-2 pronouncedly enhanced odontogenic differentiation phenomena. SIGNIFICANCE This is the first study to validate that combining the bioactivity and ion releasing properties of bioceramic materials with growth factor release by treated natural dentin further supported by exogenous addition of key dentinogenesis-related morphogens (DMP-1, BMP-2) can be a promising strategy for targeted dentin regeneration.

Genetic manipulation of RPS5 gene expression modulates the initiation of commitment of MEL cells to erythroid maturation: Implications in understanding ribosomopathies

Impairment of ribosome biogenesis contributes to the molecular pathophysiology of ribosomopathies by deregulating cell-lineage specific proliferation, differentiation and apoptosis decisions of haematopoietic progenitor cells. Here, using pro-erythroblast-like murine erythroleukemia (MEL) cells, a model system of erythroid maturation, we aimed to investigate whether genetic manipulation of RPS5 expression affects the capacity of cells to grow and differentiate in culture. Parental MEL cells stably transfected with full length RPS5 cDNA in sense (MEL-C14 culture) or antisense (MEL-antisenseRPS5 culture) orientation, as well as MEL cells transiently transfected with siRNAs specific for RPS5 gene silencing (MEL-RPS5siRNA culture) were assessed for their ability to fully execute their erythroid maturation program in culture. The data obtained thus far indicate that: a) MEL-antisenseRPS5 exhibit a pronounced delay in the initiation of differentiation, as well as an impairment of commitment, since the continuous presence of the inducer in culture is required for the cells to fully execute their erythroid maturation program. b) RNAi-mediating silencing of RPS5 gene expression resulted in the inability of MEL cells to differentiate; however, when these cells were allowed to recapitulate normal RPS5 gene expression levels they regained their differentiation capacity by accumulating high proportion of erythroid mature cells. c) Interestingly the latter, is accompanied by morphological changes of cells and an impairment of their proliferation and apoptosis potential. Such data for the first time correlate the RPS5 gene expression levels with the differentiation capacity of MEL cells in vitro, a fact that might also have implications in understanding ribosomopathies.

Effective Cell Growth Potential of Mg-Based Bioceramic Scaffolds towards Targeted Dentin Regeneration

SUMMARY New emerging approaches in tissue engineering include incorporation of metal ions involved in various metabolic processes, such as Cu, Zn, Si into bioceramic scaffolds for enhanced cell growth and differentiation of specific cell types. The aim of the present work was to investigate the attachment, morphology, growth and mineralized tissue formation potential of Dental Pulp Stem Cells (DPSCs) seeded into Mg-based glassceramic scaffolds with incorporated Zn and Cu ions. Bioceramic scaffolds containing Si 60%, Ca 30%, Mg 7.5% and either Zn or Cu 2.5%, sintered at different temperatures were synthesized by the foam replica technique and seeded with DPSCs for up to 21 days. Scanning Electron Microscopy with associated Energy Dispersive Spectroscopy (SEM-EDS) was used to evaluate their ability to support the DPSCs’s attachment and proliferation, while the structure of the seeded scaffolds was investigated by X-Ray Diffraction Analysis (XRD). Zn-doped bioceramic scaffolds promoted the attachment and growth of human DPSCs, while identically fabricated scaffolds doped with Cu showed a cytotoxic behaviour, irrespective of the sintering temperature. A mineralized tissue with apatite-like structure was formed on both Cu-doped scaffolds and only on those Zn-doped scaffolds heat-treated at lower temperatures. Sol-gel derived Zn-doped scaffolds sintered at 890oC support DPSC growth and apatite-like tissue formation, which renders them as promising candidates towards dental tissue regeneration.