Reclamation efficiency of composts produced from the mixture of municipal waste (Radiowo and ZUSOK), urban green waste composts (Complex), housing estate lawn mowing waste (plants) and sewage sludge, ...as well as mineral fertilizer (NPK – without the use of any organic fertilizer), was assessed in a model experiment on an ash soil. The experiment was launched in 2005 and continued until the end of 2013. Crops were collected and subjected to the analysis in 2005, 2006 and between 2011 and 2012. In the period between 2007 and 2010 no agrotechnical treatments were carried out, with the experiment being limited to mere observation of the natural (spontaneous) succession of plants. Reclamation doses of composts and sewage sludge were measured quantitatively, therefore they differed in the content of dry matter as well as in the contents of organic substances and minerals in the dry matter. The largest aggregated dry matter yield of plants (2005–2013) was reported in the Complex variant, and similarly, in the Radiowo and plant variants, whereas the ZUSOK variant reported the lowest aggregated dry matter field of plants. The yield-forming efficiency of NPK fertilizer was lower than in the case of compost and sludge variants. However, the field-forming efficiency of sewage sludge was lower than its fertilization potential as it was unstable and difficult to mix with the experimental soil. The results showed the yield-forming potential of plants on the experimental coal combustion waste deposits fertilized with composts and sewage sludge as well as the examples of the sites where sewage sludge could be used for the biological reclamation of landfills and spoil tips.
► OxLDL promotes induction of pATM and p21 in fibroblasts/endothelial cells. ► OxLDL activates ATM-kinase by a doubled-stranded DNA break-independent mechanism. ► ATM-deficient fibroblasts exhibit ...low cell viability but increased oxidative stress towards oxLDL. ► ATM expression exerts protective effects against ox-LDL-induced cellular toxicity.
Chronic oxidative stress is involved in the pathogenesis of multiple inflammatory diseases, including cardiovascular disease and atherosclerosis. The rare autosomal recessive disorder Ataxia-telangiectasia (A-T) is characterized by progressive cerebellar ataxia secondary to Purkinje cell death, immunodeficiency, and increased cancer incidence. ATM, the protein mutated in A-T, plays a key role in cellular DNA-damage responses. A-T cells show poor cellular anti-oxidant defences and increased oxidant sensitivity compared to normal cells, and ATM functions, in part, as an oxidative stress sensor. The oxidation of low-density lipoprotein (oxLDL) and its uptake by macrophages is an initiating step in the development of atherosclerosis. We demonstrate that oxLDL activates ATM and downstream p21 expression in normal fibroblasts and endothelial cells. In ATM-deficient fibroblasts oxLDL induces DNA double-strand breaks, micronuclei formation and causes chromosome breaks. Furthermore, oxLDL decreases cell viability and inhibits colony formation in A-T fibroblasts more effectively as compared to normal controls. Formation of oxLDL-induced reactive oxygen species is significantly higher in A-T, than normal fibroblasts. Last, pre-treatment of cells with ammonium pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of transcription factor nuclear factor κB, reduces oxLDL-induced reactive oxygen species formation. Our data indicates that ATM functions in the defence against oxLDL-mediated cytotoxicity.
The corneal epithelium is renewed by stem cells located at the limbus, the so-called limbal stem cells (LSCs). Absence, damage or loss of the LSC population leads to the painful and blinding ...condition of LSC deficiency (LSCD). Ex vivo expansion of LSCs is an increasingly well recognized treatment modality for LSCD. One method of ex vivo expansion of LSCs involves the culture of limbal explants on amniotic membrane (AM). The purpose of this study was to analyze the outgrowths from human cadaveric limbal explants cultured on AM for properties associated with LSCs. In particular, the expression of putative stem cell markers and the colony-forming efficiency of the different zones of the outgrowth were studied.
The limbal explants were expanded in the standard way used for clinical transplantation and the outgrowths were divided into three zones depending on proximity to the explant (inner, middle and outer zones). The colony-forming efficiencies (CFEs) of cells from each zone were calculated. In addition, the expression of DeltaNp63, ABCG2 (both putative positive LSC markers) and cytokeratin K3 (marker of corneal differentiation) were assessed using quantitative reverse transcription PCR (RT-PCR). Immunohistochemistry on paraffin-embedded sections was also performed to demonstrate protein localization and allow further confirmation of the quantitative RT-PCR results.
Successful cultures for both the explant outgrowths and the CFE calculations were obtained in every case. CFE showed a successive decline in zones further away from the explant (p < 0.00005). Quantitative RT-PCR revealed that the expression of the positive putative LSC markers DeltaNp63 and ABCG2 also showed a steady decrease in the zones furthest from the explant (p < 0.05 and p < 0.005, respectively). The expression of cytokeratin K3 was increased in zones furthest from the explant (p < 0.005). Immunohistochemistry on paraffin-embedded sections of intact ex vivo-expanded limbal epithelium for the putative positive marker p63 and cytokeratin K3 confirmed the findings of the quantitative RT-PCR and CFE results.
We demonstrate for the first time that outgrowths from human limbal explants, a widely used technique in ex vivo expansion of LSCs for clinical transplantation, show a steady decline in a wide range of stem cell properties with distance from the central explant. These findings support the importance of proximity of stem cells to their niche environment in maintaining their undifferentiated state. These findings suggest the need for modifications of existing techniques to ensure maximum numbers of LSCs following ex vivo expansion protocols, which will then ensure the success of subsequent engraftment.
A brief comparative analysis of different approaches to cell viability testing in cytogerontological experiments is performed with a focus on problems in constructing survival curves for cultured ...cells in the “stationary phase aging” model. It is emphasized that the choice of methods to this end depends mainly on the researchers’ ideas about molecular and cellular mechanisms of aging. A note is made that the evaluation of colonyforming efficiency, though optimal for cell viability assessment, is unfortunately not applicable to postmitotic or very slowly propagating cells. Consideration is also given to some problems encountered when using the most popular molecular probes designed for live/dead cell viability assays.
This study was designed to demonstrate the effects of pluripotin on the proliferation, senescence and colony formation efficiency of rabbit limbal epithelial cells (RLECs) in vitro. Rabbit primary ...limbal epithelial cells were harvested and cultured in the presence of pluripotin. The cell proliferation was measured using the 3-(4,5)-dimethylthiahiazo(-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and was also observed by confocal microscopy with Ki67 staining, whereas cell senescence was detected by senescence-associated β-galactosidase (SA-β-gal) staining. The colony morphology, colony-forming efficiency and colony size were observed and compared. The characteristics of the proliferating cells were examined by immunofluorescent staining using antibodies against deltaNP63, ABCG2 and Keratin 3/12. The results showed that pluripotin significantly promoted the proliferation of RLECs and increased the dividing cells with positive Ki67 staining at the concentrations lower than 400 nM. The colony-forming efficiency increased from 13.5% in the control cells to 26.4% in the 200 or 400 nM pluripotin-treated cells. The number of colonies of moderate size (600–900 μm) increased significantly in the presence of pluripotin (above 60.0% at 200 nM or 400 nM) compared with the untreated normal cells (18.6%), whereas the number of small-sized colonies (<600 μm) decreased from 79.5% for the control cells to lower than 35.0% at 200 nM or 400 nM pluripotin. Moreover, the cells treated with pluripotin stained negative with SA-β-gal, while the untreated cells showed visible positive staining. Immunofluorescent staining suggested that the pluripotin treatment resulted in higher positive staining for the limbal stem cell markers (deltaNP63 and ABCG2) and down-regulated of differentiated corneal epithelial cell marker (Keratin 3/12). This study confirmed that the small molecular compound pluripotin promoted the proliferation of rabbit limbal epithelial cells by improving the expansion of limbal stem/progenitor cells in vitro.
► Small molecule pluripotin promoted the proliferation of limbal epithelial cells. ► Pluripotin prevented the early senescence of rabbit limbal epithelial cells. ► Pluripotin promoted the colony-forming efficiency of limbal epithelial cells. ► Pluripotin enhanced the clonal expansion of limbal stem/progenitor cells. ► Pluripotin up-regulated the expression of limbal stem/progenitor cell markers.
Because inhibition of integrin signaling induces apoptosis, we investigated whether keratinocytes expressing β1 and α6β4 integrins (enriched for stem cells) are protected from cell death. ...Keratinocytes rapidly adhering to type IV collagen expressed highest levels of β1 and α6β4 and of the anti-apoptotic stem cell marker p63. Apoptotic cells were significantly higher in slowly adhering than in rapidly adhering keratinocytes. Anti-β1 integrin caused a significant increase in apoptotic cells, while it decreased Bcl-2 levels in stem keratinocytes. Bax and Bad proteins were higher in slowly adhering than in rapidly adhering cells. By contrast, Bcl-2, Bcl-x and Mcl-1 proteins were highest in rapidly adhering keratinocytes and nearly absent in slowly adhering cells. After addition of anti-β1 integrin, the apoptotic rate was significantly higher in HaCaT cells not expressing Bcl-2 than in controls. These results indicate that keratinocytes enriched for stem cells are protected from apoptosis via β1 integrin, in a Bcl-2 dependent manner.
: The aim of the study was to investigate the effect of low‐level laser therapy (LLLT) on attachment and proliferation of human gingival fibroblasts (HGF) cultured on titanium implant material. HGF ...were exposed to gallium–aluminum–arsenide diode laser at dosages of 1.5 or 3 J/cm2 and then cultured on commercially pure titanium discs. Cell profile areas were measured after 1, 3 and 24 h, using scanning electron microscopy and an automatic image analyzer. The results were expressed as percentage of attachment. In order to investigate the effect of LLLT on cellular growth after 8 and 10 days, HGF were cultured on titanium discs for 24 h and then exposed to laser irradiation on 3 consecutive days. Colony‐forming efficiency (CFE) and clonal growth rates (CGR) were measured. Cell viability was determined by Hoechst and prodidium iodide staining. Non‐lased cultures served as controls. Morphologically, the cells spread well on all titanium surfaces, indicating good attachment by both irradiated and non‐irradiated cells. Fibroblasts exposed to laser irradiation had significantly higher percentages of cell attachment than the non‐exposed cells (P<0.05). CFE and CGR were also enhanced for the irradiated cells (P<0.05). Cell viability was high (>90%) in the irradiated and control groups, without significant differences. It is concluded that in vitro LLLT enhances the attachment and proliferation of HGF on titanium implant material.
Résumé
Le but de l'étude présente a été d'analyser l'effet du traitement par laser à bas niveau (LLLT) sur l'attache et la prolifération des fibroblastes gingivaux humains (HGF) mis en culture sur du matériel implantaire en titane. HGF ont été exposés à des lasers d'iode GaA1As à des doses de 1,5 ou 3 J/cm2 et ensuite mis en culture sur des disques de titane commercialement purs. Des aires de profils cellulaires ont été mesurés après une, trois et 24 h en utilisant le MEB et l'analyse d'image automatique. Les résultats ont été exprimés en pourcentage d'attache. Afin d'analyser l'effet du LLLT sur la poussée cellulaire après huit et dix jours, HGF ont été mis en culture sur des disques en titane pour 24 h et ensuite exposés à l'irradiation par laser trois jours consécutifs. L'efficacitéà former des colonies (CFE) et les taux de croissance de clônes (CGR) ont été mesurés. La viabilité cellulaire a été déterminée par la coloration iodure de prodidium et Hoechst. Des cultures sans utilisation du laser ont servi de contrôles. Morphologiquement les cellules s'étalaient bien sur toutes les surfaces en titane indiquant une bonne attache des cellules tant irradiées que non‐irradiées. Des fibroblastes exposés à l'irradiation laser avaient significativement des pourcentages plus élevés d'attache cellulaire que les cellules non‐exposées (P<0.05). Les taux de CFE et de CGR augmentaient également pour les cellules irradiées (P<0.05). La vitalité cellulaire était importante (>90%) dans les groupes irradiés et contrôles sans aucune différence significative. Le LLLT in vitro augmente l'attache et la prolifération des HGF sur le matériel implantaire en titane.
Zusammenfassung
Das Ziel dieser Studie war, den Einfluss der Low Level Laser Therapie (LLLT) auf die Anhaftung und Proliferation von humanen gingivalen Fibroblasten (HGF), welche auf Titan Implantatmaterial gezüchtet wurden, zu untersuchen. HGF wurden einem GaAlAs Diodenlaser mit einer Dosis von 1.5 oder 3 J/cm2 ausgesetzt und dann auf im Handel erhältlichen reinen Titanscheiben gezüchtet. Die Zellprofilareale wurden nach 1, 3 und 24 Stunden mit einem Rasterelektronenmikroskop und einer automatischen Bildanalyse ausgemessen. Die Resultate wurden als Prozent der Anhaftung ausgedrückt. Um den Effekt der LLLT auf das Zellwachstum nach 8 und 10 Tagen zu untersuchen, wurden HGF auf Titanscheiben während 24 Stunden gezüchtet und dann an den drei darauf folgenden Tagen der Laserbestrahlung ausgesetzt. Es wurden die Koloniebildungseffizienz (CFE) und die klonalen Wachstumsraten (CGR) gemessen. Die Zelllebensfähigkeit wurde durch die Hoechst und Prodidiumjodid Färbung bestimmt. Als Kontrolle dienten nicht mit Laser bestrahlte Kulturen. Morphologisch breiteten sich die Zellen gut auf den Titanoberflächen aus, was eine gute Anhaftung sowohl für die bestrahlten als auch für die unbestrahlten Zellen bedeutete. Fibroblasten, welche der Laser Bestrahlung ausgesetzt waren, zeigten höhere Prozentwerte bezüglich Zellanhaftung als die nicht bestrahlten Zellen (P<0.05). Die Koloniebildungseffizienz und die klonalen Wachstumsraten waren bei den bestrahlten Zellen gesteigert (P>0.05). Die Zelllebensfähigkeit war bei der bestrahlen Gruppe und bei der Kontrollgruppe hoch (>90%). Es bestanden keine signifikanten Unterschiede. Es wird die Schlussfolgerung gezogen, dass LLLT in vitro die Anhaftung und die Proliferation von HGF auf Titan Implantatmaterial fördert.
Resumen
La intención del estudio fue investigar el efecto del tratamiento con láser de bajo nivel (LLLT) en la inserción y la proliferación de fibroblastos humanos (HGF) cultivados en material de implantes de titanio Se expusieron HGF a láser de diodo GaA1As en dosis de 1.5 o 3 J/cm2 y posteriormente cultivados en ciscos de titanio comercialmente puro. Se midieron los perfiles de las áreas de células tras 1, 3 y 24 h, usando microscopía electrónica de barrido y un analizador de imágenes automático. Los resultados se expresaron en porcentajes de inserción. En orden a investigar el efecto de LLLT en el crecimiento celular tras ocho y diez días, se cultivaron HGF en discos de titanio durante 24 h y Lugo se expusieron a irradiación láser durante tres días consecutivos. Se midieron la eficiencia en formar colonias (CFE) y los índices de crecimiento clonal (CGR). Se determinó la viabilidad celular por medio de tinción de Hoechst y Prodidium iodide. Cultivos no expuestos al láser sirvieron de control. Morfológicamente, las células se expendieron bien en todas las superficies de titanio, indicando una buena inserción tanto las células irradiadas como las no irradiadas. Los fibroblastos expuestos a la irradiación láser tuvieron unos mayores porcentajes de inserción celular que las células no expuestas (P<0.05). La eficiencia en la formación de colonias y los índices de crecimiento clonal fueron también realzados en las células irradiadas (P<0.05). La viabilidad celular fue alta (>90%) en los grupos irradiados y de control, sin diferencias significativas. Se concluye que in vitro el LLLT realza la inserción y la proliferación de HGF en material de implante de titanio.
Human mesenchymal stem cells (MSCs) are capable of repairing pulmonary disorders, but their efficacy is limited by poor engraftment. A strategy is proposed to augment MSC migration to lung tissue by ...antagonizing macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine. Recombinant MIF (85 ng/ml) inhibited in vitro chemokinesis of multipotent MSCs by nearly 50 and 20% for donor preparations with colony-forming efficiencies of 22 ± 4% and 66 ± 3%, respectively (P < 0.05). The small-molecule MIF antagonist, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1, 85 μg/ml), restored MSC migration for all donors to levels found in the absence of MIF. At this concentration, ISO-1 increased migration to conditioned medium from bronchial epithelial cell cultures by ⩾3-fold for all donor MSC preparations (P < 0.05). Transcript levels for the MIF receptor, CD74, in MSCs were independent of colony-forming efficiency. These data suggest that MIF and its antagonists may be relevant to the control of MSC homing and efficacy of stem cell therapies in a variety of clinical scenarios.
The effect of an aqueous solution of hydrated C₆₀-fullerene (HyFn) on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during the slowing down of their ...proliferation within a single passage and the subsequent “aging” in the stationary phase of growth) of transformed B11-dii FAF28 Chinese hamster cells was studied. The final calculated concentration of HyFn in the growth medium was 10⁻¹⁹ M. A paradoxical result contrasting the available data on the absence of HyFn cytotoxicity at higher concentrations was obtained in our experiments: namely, HyFn decelerated cell proliferation (estimated by the growth of mass culture, as well as by the efficiency of colony formation) and accelerated the “stationary phase aging” of the cell culture. Moreover, repeated addition of an aqueous solution of HyFn (to the final calculated concentration of 10⁻¹⁹ M) to the cells that had already reached the stationary phase of growth caused a rapid (within no more than 24 h) death of a significant part of the cell population. The observed effect of HyFn at ultralow concentration is supposed to arise from the alterations in the properties of the water surrounding the fullerene molecule: namely, water becomes a donor and acceptor of electrons and regulates redox processes (especially those involving oxygen) in aqueous systems. This effect of HyFn at an ultralow concentration may be specific for transformed cells, and, therefore, experiments on normal fibroblasts with limited mitotic potential are planned as a continuation of the present study. It is also possible that the reported antiaging effect of HyFn in experimental animals is due to its anticancer, immunostimulatory, antiviral, and antibacterial properties manifested only at the whole-organism level.
AIM: To determine the proliferative potential and the maintenance of stem cell activity in stored human limbal tissues, and correlate this with the preservation time, cell viability and the ...expression of stem cell markers.METHODS: Thirty limbal rims were split into 4 parts and stored in corneal preservation medium at 4℃ for 0, 1, 4, or 7 days. The limbal stem cell and mitotic markers P63, CK19, proliferating cell nuclear antigen (PCNA), and Ki67 were determined by immunohistochemical staining. The proliferative potential of limbal epithelial cells was assessed by cell viability, the ability of generating stratified epithelium, and colony forming assay. RESULTS: The stored tissues maintained limbal stratified structure to 7 days and exhibited comparable expression level of stem cell and mitotic markers. The proportion of viable cells decreased with the prolonged preservation time, while colony forming efficiency decreased from the 1st day and disappeared at the 4th day. When inoculated on amniotic membrane, the cells preserved for 1 day formed a stratified epithelium, while the cells from 4 days’ preservation formed a discontinuous layer. CONCLUSION: The colony forming efficiency of limbal epithelial stem/progenitor cells decreased rapidly with the increasing preservation time, while the expression level of markers and capacity of forming epithelial monolayer on amniotic membrane decreased gradually. The limbal epithelial stem cells lost their function earlier than the lost expression level of stem cell markers. This may help us to better choose the appropriate preservation grafts for future limbal stem cell transplantation.
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