Search

Objectives Periodontal ligament stem cells (PDLSCs) have an underlined significance as their high proliferative capacity and multipotent differentiation provide an important therapeutic potential. The integrity of these cells is frequently disturbed by the routinely used irrigative compounds applied as periodontal or endodontic disinfectants (e.g., hydrogen peroxide (H2O2) and chlorhexidine (CHX)). Our objectives were (i) to monitor the cytotoxic effect of a novel dental irrigative compound, chlorine dioxide (ClO2), compared to two traditional agents (H2O2, CHX) on PDLSCs and (ii) to test whether the aging factor of PDLSC cultures determines cellular responsiveness to the chemicals tested. Methods Impedimetry (concentration-response study), WST-1 assays (WST = water soluble tetrazolium salt), and morphology analysis were performed to measure changes in cell viability induced by the 3 disinfectants; immunocytochemistry of stem cell markers (STRO-1, CD90, and CD105) measured the induced mesenchymal characteristics. Results Cell viability experiments demonstrated that the application of ClO2 does not lead to a significant decrease in viability of PLDSCs in concentrations used to kill microbes. On the contrary, traditional irrigants, H2O2, and CHX are highly toxic on PDLSCs. Aging of PLDSC cultures (passages 3 vs. 7) has characteristic effects on their responsiveness to these agents as the increased expression of mesenchymal stem cell markers turns to decreased. Conclusions and clinical relevance While the active ingredients of mouthwash (H2O2, CHX) applied in endodontic or periodontitis management have a serious toxic effect on PDLSCs, the novel hyperpure ClO2 is less toxic providing an environment favoring dental structure regenerations during disinfectant interventions.

Bortezomib (BOZ) is a proteasome inhibitor chemotherapeutic agent utilized to treat multiple myeloma and recently offered to cure melanoma. Bortezomib-induced neuropathy is one of the dose-limiting side-effects, which can be treated with antioxidants (e.g. alpha-lipoic acid—ALA and Vitamin B1—vit B1). We hypothesized that these antioxidants may counteract the antitumor activity by disrupting the BOZ-induced pathways (e.g. proteasome inhibition or reactive oxygen species generation). The objectives were: (i) to verify the anti-proliferative effect of BOZ; (ii) to compare the influence of the antioxidants on the antitumor effect of BOZ in melanoma (A2058) and myeloma (U266) cells. At first, the reduction in the anti-proliferative effect of BOZ by ALA was proved in melanoma cells. Analysis of p53 phosphorylation and the cell cycle progression revealed that ALA failed to counteract these effects of BOZ. Nevertheless, a good correlation was found between the inhibition of the anti-proliferative effect, the anti-proteasome activity and the oxidative stress level after the co-treatment with 20 ng/mL BOZ + 100 μg/mL ALA. Downregulation of apoptotic proteins such as HO-1 and Claspin along with the inhibition of the cleavage of Caspase-3 indicated the proteomic background of the altered responsiveness of the melanoma cells exposed to BOZ + ALA. This phenomenon draws attention to the proper application of cancer supportive care to avoid possible interactions.

Periodontal ligament stem cells (PDLSCs) possess extensive regeneration potential. However, their therapeutic application demands a scaffold with appropriate properties. HydroMatrix (HydM) is a novel injectable peptide nanofiber hydrogel developed recently for cell culture. Our aim was to test whether HydM would be a suitable scaffold for proliferation and osteogenic differentiation of PDLSCs. PDLSCs were seeded on non-coated or HydM-coated surfaces. Both real-time impedance analysis and cell viability assay documented cell growth on HydM. PDLSCs showed healthy, fibroblast-like morphology on the hydrogel. After a 3-week-long culture in osteogenic medium, mineralization was much more intense in HydM cultures compared to control. Alkaline phosphatase activity of the cells grown on the gels reached the non-coated control levels. Our data provided evidence that PDLSCs can adhere, survive, migrate, and proliferate on HydM and this gel also supports their osteogenic differentiation. We first applied impedimetry for dental stem cells cultured on a scaffold. HydM is ideal for in vitro studies of PDLSCs. It may also serve not only as a reference material but also in the future as a promising biocompatible scaffold for preclinical studies.

Background: Cardiomyopathy induced by the chemotherapeutic agents doxorubicin and daunorubicin is a major limiting factor for their application in cancer therapy. Chemotactic drug targeting potentially increases the tumor selectivity of drugs and decreases their cardiotoxicity. Increased expression of gonadotropin-releasing hormone (GnRH) receptors on the surface of tumor cells has been reported. Thus, the attachment of the aforementioned chemotherapeutic drugs to GnRH-based peptides may result in compounds with increased therapeutic efficacy. The objective of the present study was to examine the cytotoxic effect of anticancer drug–GnRH-conjugates against two essential cardiovascular cell types, such as cardiomyocytes and endothelial cells. Sixteen different previously developed GnRH-conjugates containing doxorubicin, daunorubicin and methotrexate were investigated in this study. Their cytotoxicity was determined on primary human cardiac myocytes (HCM) and human umbilical vein endothelial cells (HUVEC) using the xCELLigence SP system, which measures impedance changes caused by adhering cells on golden electrode arrays placed at the bottom of the wells. Slopes of impedance–time curves were calculated and for the quantitative determination of cytotoxicity, the difference to the control was analysed.Results: Doxorubicin and daunorubicin exhibited a cytotoxic effect on both cell types, at the highest concentrations tested. Doxorubicin-based conjugates (AN-152, GnRH-III(Dox-O-glut), GnRH-III(Dox-glut-GFLG) and GnRH-III(Dox=Aoa-GFLG) showed the same cytotoxic effect on cardiomyocytes. Among the daunorubicin-based conjugates, [4Lys(Ac)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-YRRL), {GnRH-III(Dau=Aoa-YRRL-C)}2 and {[4N-MeSer]-GnRH-III(Dau-C)}2 had a significant but decreased cytotoxic effect, while the other conjugates – GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-K(Dau=Aoa)), [4Lys(Dau=Aoa)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-GFLG), {GnRH-III(Dau-C)}2 and [4N-MeSer]-GnRH-III(Dau=Aoa) – exerted no cytotoxic effect on cardiomyocytes. Mixed conjugates containing methotrexate and daunorubicin – GnRH-III(Mtx-K(Dau=Aoa)) and [4Lys(Mtx)]-GnRH-III(Dau=Aoa) – showed no cytotoxic effect on cardiomyocytes, as well.Conclusion: Based on these results, anticancer drug–GnRH-based conjugates with no cytotoxic effect on cardiomyocytes were identified. In the future, these compounds could provide a more targeted antitumor therapy with no cardiotoxic adverse effects. Moreover, impedimetric cytotoxicity analysis could be a valuable technique to determine the effect of drugs on cardiomyocytes.

Intercellular communication via extracellular vesicles (EVs) and their target cells, especially immune cells, results in functional and phenotype changes that consequently may play a significant role in various physiological states and the pathogenesis of immune-mediated disorders. Monocytes are the most prominent environment-sensing immune cells in circulation, skilled to shape their microenvironments via cytokine secretion and further differentiation. Both the circulating monocyte subset distribution and the blood plasma EV pattern are characteristic for preeclampsia, a pregnancy induced immune-mediated hypertensive disorder. We hypothesized that preeclampsia-associated EVs (PE-EVs) induced functional and phenotypic alterations of monocytes. First, we proved EV binding and uptake by THP-1 cells. Cellular origin and protein cargo of circulating PE-EVs were characterized by flow cytometry and mass spectrometry. An altered phagocytosis-associated molecular pattern was found on 12.5 K fraction of PE-EVs: an elevated CD47 “don’t eat me” signal (p

Introduction Thrombogenesis plays an important role in today's morbidity and mortality. Antithrombotics are among the most frequently prescribed drugs. Thorough knowledge of platelet function is needed for optimal clinical care. Platelet adhesion is a separate subprocess of platelet thrombus formation; still, no well-standardized technique for the isolated measurement of platelet adhesion exists. Impedimetry is one of the most reliable, state-of-art techniques to analyze cell adhesion, proliferation, viability, and cytotoxicity. We propose impedimetry as a feasible novel method for the isolated measurement of 2 significant platelet functions: adhesion and spreading. Methods Laboratory reference platelet agonists (epinephrine, ADP, and collagen) were applied to characterize platelet functions by impedimetry using the xCELLigence SP system. Platelet samples were obtained from 20 healthy patients under no drug therapy. Standard laboratory parameters and clinical patient history were also analyzed. Results Epinephrine and ADP increased platelet adhesion in a concentration-dependent manner, while collagen tended to have a negative effect. Serum sodium and calcium levels and age had a negative correlation with platelet adhesion induced by epinephrine and ADP, while increased immunoreactivity connected with allergic diseases was associated with increased platelet adhesion induced by epinephrine and ADP. ADP increased platelet spreading in a concentration-dependent manner. Conclusion Impedimetry proved to be a useful and sensitive method for the qualitative and quantitated measurement of platelet adhesion, even differentiating between subgroups of a healthy population. This novel technique is offered as an important method in the further investigation of platelet function.

Transglutaminase-2 (TG2) is best known as a Ca2+-dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent of its catalytic activity and include matrix remodelling, adhesion and migration. S100A4 belongs to the Ca2+-binding EF-hand S100 protein family and acts both intra- and extra-cellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. It has recently been suggested that TG2 mediates S100A4-dependent tumour cell migration. In the present study we provide evidence that S100A4 is an interacting partner and also a specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys100 in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell-surface receptor in this state and increases cell adhesion.

Inspired by the well-established clinical evidence about the interplay between apoptotic TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) mechanism and reactive oxygen species (ROS)-mediated oxidative stress, a set of novel ONC201 hybrids containing the impiridone core and one or two differently positioned ferrocenylalkyl groups were synthesised in our present work. These two types of residues have been implicated in the aforementioned mechanisms associated with cytotoxic activity. A straightforward, primary amine-based synthetic approach was used allowing the introduction of a variety of N-substituents into the two opposite regions of the heterocyclic skeleton. Reference model compounds with benzyl and halogenated benzyl groups were also synthesised and tested. The in vitro assays of the novel impiridones on five malignant cell lines disclosed characteristic structure-activity relationship (SAR) featuring significant substituent-dependent activity and cell-selectivity. A possible contribution of ROS-mechanism to the cytotoxicity of the novel metallocenes was suggested by density functional theory (DFT)studies on simplified models. Accordingly, unlike the mono-ferrocenylalkyl-substituted products, the compounds containing two ferrocenylalkyl substituents in the opposite regions of the impiridone core display a much more pronounced long-term cytotoxic effect against A-2058 cell line than do the organic impiridones including ONC201 and ONC212. Furthermore, the prepared bis-metallocene derivatives also present substantial activity against COLO-205- and EBC-1 cell lines.

BACKGROUND Experiments on porcine heart scaffold represent significant assays in development of immunoneutral materials for cardiac surgery. Characterization of cell-cell and cell-scaffold interactions is essential to understand the homing process of cardiac cells into the scaffolds. MATERIAL AND METHODS In the present study, the highly sensitive and real-time impedimetric technique of xCELLigence SP was used to monitor cell adhesion, which is the key process of recellularization in heart scaffolds. Our objectives were: (i) to characterize the effect of decellularized porcine heart scaffold on cell adhesion of human cardiovascular cells potentially used in the recellularization process; and (ii) to investigate cell-extracellular matrix element interactions for building artificial multi-layer systems, applied as cellular models of recellularization experiments. Human fibrosarcoma, endothelial, and cardiomyocyte cells were investigated and the effect of decellularized porcine heart scaffold (HS) and fibronectin on cell adhesion was examined. Adhesion was quantified as slope of curves. RESULTS Heart scaffold had neutral effect on cardiomyocytes as well as on endothelial cells. Adhesion of cardiomyocytes was increased by fibronectin (1.480±0.021) compared to control (0.745±0.029). The combination of fibronectin and HS induced stronger adhesion of cardiomyocytes (2.407±0.634) than fibronectin alone. Endothelial and fibrosarcoma cells showed similarly strong adhesion profiles with marked enhancer effect by fibronectin. CONCLUSIONS Decellularized porcine HS does not inhibit adhesion of human cardiovascular cells at the cell biological level, while fibronectin has strong cell adhesion-inducer effect, as well as an enhancer effect on activity of HS. Consequently, decellularized porcine hearts could be used as scaffolds for recellularization with cardiomyocytes and endothelial cells with fibronectin acting as a regulator, leading to construction of working bioartificial hearts.

Dynamic properties of cancer cells, most notably their ability to migrate, have been correlated successfully with their invasive nature in vivo. To establish a stronger experimental basis for such a correlation we subjected five different cancer cell lines of well-defined metastatic potential to a sequence of three independent assays reporting on three different aspects of cell dynamics, namely (1) the kinetics of cell spreading, (2) cell shape fluctuations, and (3) cell migration. The sequentially applied assays correspond to different measuring modes of the well-established ECIS technique that is based on non-invasive and label-free impedance readings of planar gold-film electrodes that serve as the growth substrate for the cells under study. Every individual assay returned a characteristic parameter describing the behavior of the cell lines in that particular assay quantitatively. The parameters of all three assays were ranked to establish individual profiles of cell dynamics for every cell line that correlate favorably with the cells' invasive properties. The sequence of impedance-based assays described here requires only small cell populations (< 10.000 cells), it is highly automated and easily adapted to 96-well formats. It provides an in-depth dynamic profile of adherent cells that might be useful in other areas besides cancer research as well.

The cytotoxicity of glass ionomer cements (GICs) was investigated using a novel, cost-effective, easy-to-perform and standardized test. GIC rings were made using in-house designed, custom-made moulds under sterile conditions; 10 with Fuji Equia and 10 with Fuji Triage capsules, placed in direct contact with primary human gingival fibroblasts (HGF) and immortalized human fibroblasts (HFF1). On day 1, 4, 14 and 21, an AlamarBlue® (resazurin) assay was completed towards determining the effects of the GICs on metabolic activities of the cells, whilst cell morphology was examined by light microscopy. The influence of the compounds released from the GIC rings on cell physiological effects (viability, proliferation and adhesion) during 24 h incubation was further investigated by impedimetry. Result trends obtained from this battery of techniques were complementary. At 100 v/v% concentration, the released compounds from Equia were strongly cytotoxic, while at lower concentration (0, 4, 20 v/v%) they were not cytotoxic. In contrast, Triage elicited only slightly transient cytotoxicity. The method proposed has been proved as being efficient, reliable and reproducible and may be useful in quick testing of the cytotoxicity of similar biomaterials by using an immortalized cell line.

Adrenomedullin (AMD), proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) were studied for chemotaxis, chemotactic selection and G-actin/F-actin transition in Tetrahymena. The aim of the experiments was to study the effects of two different peptides encoded by the same gene compared to a peptide related to one of the two, but encoded by a different gene, at a low level of phylogeny. The positive, chemotactic effect of ADM and the strong negative, chemorepellent effect of PAMP suggest that in Tetrahymena, the two peptides elicit their chemotactic effects via different signalling mechanisms. The complexity of swimming behaviour modulated by the three peptides underlines that chemotaxis, chemokinesis and some characteristics of migratory behaviour (velocity, tortuosity) are working as a sub-population level complex functional unit. Chemotactic responsiveness to ADM and CGRP is short-term, in contrast to PAMP, which as a chemorepellent ligand, has the ability to select sub-populations with negative chemotactic responsiveness. The different effects of ADM and PAMP on the polymerization of actin networks show that the microtubular structure of cilia is more essential to chemotactic response than are transitions of the actin network. The results draw attention to the characteristic effects of vasoactive peptides at this low level of phylogeny.