Effects of Acid Treatment on Dental Zirconia: An In Vitro Study

The aim of this study was to evaluate the effects of hydrofluoric (HF) acid, acetic acid, and citric acid treatments on the physical properties and structure of yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) at ambient temperature. In total, 110 bar-shaped zirconia specimens were randomly assigned to 11 groups. The specimens in the control group (C) received no surface treatment, while those in the Cage group were hydrothermally aged at 134°C and 0.2 MPa for 20 h. Ten specimens each were immersed at ambient temperature in 5% and 40% HF acid for 2 h (40HF0), 1 day (5HF1, 40HF1), and 5 days (5HF5, 40HF5), while 10 each were immersed at ambient temperature in 10% acetic acid and 20% citric acid for 7 (AC7, CI7) and 14 days (AC14, CI14). X-ray diffraction (XRD) was used to quantitatively estimate the monoclinic phase. Furthermore, flexural strength, surface roughness, and surface Vickers hardness were measured after treatment. Scanning electron microscopy (SEM) was used to characterize the surface morphology. The Cage group specimens exhibited an increased monoclinic phase and flexural strength. Furthermore, 40% HF acid immersion decreased the flexural strength and surface hardness and deteriorated the surface finish, while 5% HF acid immersion only decreased the surface hardness. All the HF acid-immersed specimens showed an etched surface texture on SEM observations, while the other groups did not. These findings suggest that the treatment of Y-TZP with 40% HF acid at ambient temperature causes potential damage, while treatment with 5% HF acid, acetic acid, and citric acid is safe.     

DNA Transfection of Bone Marrow Stromal Cells Using Microbubble-Mediated Ultrasound and Polyethylenimine: An In Vitro Study

Non-viral vector transfection efficiency is an issue affecting the clinical application of stem cell gene therapy. This study makes use of the synergistic effect of combining ultrasound (US) with microbubbles (MB) and polyethylenimine (PEI) to increase DNA transfection efficiency, which will enhance the efficiency of gene transfer to bone marrow stromal cells (BMSCs). The optimal parameters for primary-cultured rat-BMSC DNA transfection were examined. The study was arranged based on uniform design. Using a construct containing hepatocyte growth factor (HGF) tagged with enhanced green fluorescent protein (pEGFP-HGF) as example, the mixture of BMSCs, MB, and PEI:DNA complex were exposed to US with frequency of 1 MHz and 10 % duty cycle pulses. Other factors such as acoustic intensity (Q), MB dosage, and total treatment time (T) were also tested. The results were analyzed by regression analysis. Using the best match of parameters, Q = 0.6 W/cm², MB = 10⁶/ml, T = 30 s, different groups were compared. The cooperativity of MB-mediated US and PEI enhanced the gene transfection efficiency by nearly 38-times compared to the DNA without US group. Furthermore, the expression of HGF protein was confirmed by Western blot. The eGFP could be not only seen mainly at the cytoplasm, but also seen in the nucleus in a small proportion of the cells (<10 %) for up to 7 observed days. The transfected BMSCs maintained their capability of multi-directional differentiation and reproductive activity. Our results provide useful information in establishing a novel non-viral transfection method, which may be applied to clinical application in stem cell gene therapy.     

Effects of Thimerosal on Lipid Bilayers and Human Erythrocytes: An In Vitro Study

Thimerosal (THI, ethyl-mercury thiosalicylate) is added to vaccines as a preservative; as a consequence, infants may have been exposed to bolus doses of Hg that collectively added up to nominally 200 µg Hg during the first 6 months of life. While several studies report an association between THI-containing vaccines and neurological disorders, other studies do not support the causal relation between THI and autism. With the purpose to understand the molecular mechanisms of the toxic effect of THI it was assayed on human red cells and in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids found in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of THI to interact with DMPC and DMPE was determined by X-ray diffraction and differential scanning calorimetry, whereas intact human erythrocytes were observed by optical, defocusing and scanning electron microscopy. The experimental findings of this study demonstrated that THI interacted in a concentration-dependent manner with DMPC and DMPE bilayers, and in vitro interacted with erythrocytes inducing morphological changes. However, concentrations were considerable higher than those present in vaccines.     

Entropic Imaging of Cataract Lens: An In Vitro Study

Phacoemulsification is a common surgical method for treating advanced cataracts. Determining the optimal phacoemulsification energy depends on the hardness of the lens involved. Previous studies have shown that it is possible to evaluate lens hardness via ultrasound parametric imaging based on statistical models that require data to follow a specific distribution. To make the method more system-adaptive, nonmodel-based imaging approach may be necessary in the visualization of lens hardness. This study investigated the feasibility of applying an information theory derived parameter – Shannon entropy from ultrasound backscatter to quantify lens hardness. To determine the physical significance of entropy, we performed computer simulations to investigate the relationship between the signal-to-noise ratio (SNR) based on the Rayleigh distribution and Shannon entropy. Young''s modulus was measured in porcine lenses, in which cataracts had been artificially induced by the immersion in formalin solution in vitro. A 35-MHz ultrasound transducer was used to scan the cataract lenses for entropy imaging. The results showed that the entropy is 4.8 when the backscatter data form a Rayleigh distribution corresponding to an SNR of 1.91. The Young''s modulus of the lens increased from approximately 8 to 100 kPa when we increased the immersion time from 40 to 160 min (correlation coefficient r = 0.99). Furthermore, the results indicated that entropy imaging seemed to facilitate visualizing different degrees of lens hardening. The mean entropy value increased from 2.7 to 4.0 as the Young''s modulus increased from 8 to 100 kPa (r = 0.85), suggesting that entropy imaging may have greater potential than that of conventional statistical parametric imaging in determining the optimal energy to apply during phacoemulsification.     

The Effects of Mechanical Loading on Tendons - An In Vivo and In Vitro Model Study

Mechanical loading constantly acts on tendons, and a better understanding of its effects on the tendons is essential to gain more insights into tendon patho-physiology. This study aims to investigate tendon mechanobiological responses through the use of mouse treadmill running as an in vivo model and mechanical stretching of tendon cells as an in vitro model. In the in vivo study, mice underwent moderate treadmill running (MTR) and intensive treadmill running (ITR) regimens. Treadmill running elevated the expression of mechanical growth factors (MGF) and enhanced the proliferative potential of tendon stem cells (TSCs) in both patellar and Achilles tendons. In both tendons, MTR upregulated tenocyte-related genes: collagen type I (Coll. I ∼10 fold) and tenomodulin (∼3–4 fold), but did not affect non-tenocyte-related genes: LPL (adipocyte), Sox9 (chondrocyte), Runx2 and Osterix (both osteocyte). However, ITR upregulated both tenocyte (Coll. I ∼7–11 fold; tenomodulin ∼4–5 fold) and non-tenocyte-related genes (∼3–8 fold). In the in vitro study, TSCs and tenocytes were stretched to 4% and 8% using a custom made mechanical loading system. Low mechanical stretching (4%) of TSCs from both patellar and Achilles tendons increased the expression of only the tenocyte-related genes (Coll. I ∼5–6 fold; tenomodulin ∼6–13 fold), but high mechanical stretching (8%) increased the expression of both tenocyte (Coll. I ∼28–50 fold; tenomodulin ∼14–48 fold) and non-tenocyte-related genes (2–5-fold). However, in tenocytes, non-tenocyte related gene expression was not altered by the application of either low or high mechanical stretching. These findings indicate that appropriate mechanical loading could be beneficial to tendons because of their potential to induce anabolic changes in tendon cells. However, while excessive mechanical loading caused anabolic changes in tendons, it also induced differentiation of TSCs into non-tenocytes, which may lead to the development of degenerative tendinopathy frequently seen in clinical settings.     

High-Frequency Ultrasound Assessment of Antimicrobial Photodynamic Therapy In Vitro

Ultrasound imaging is proving to be an important tool for medical diagnosis of dermatological disease. Backscatter spectral profiles using high-frequency ultrasound (HFUS, 10–100 MHz) are sensitive to subtle changes in eukaryotic cellular morphology and mechanical properties that are indicative of early apoptosis, the main type of cell death induced following photodynamic therapy (PDT). We performed experiments to study whether HFUS could also be used to discern changes in bacteria following PDT treatment. Pellets of planktonic Staphylococcus aureus were treated with different PDT protocols and subsequently interrogated with HFUS. Changes in ultrasound backscatter response were found to correlate with antimicrobial effect. Despite their small size, distinct changes in bacterial morphology that are indicative of cell damage or death are detectable by altered backscatter spectra from bacterial ensembles using HFUS. This highlights the potential for HFUS in rapidly and non-invasively assessing the structural changes related to antimicrobial response.     

Production and Characterization of Single Chain Nimotuzumab: An In Vitro Study

Recombinant expression and EGFR-binding activity assay of single chain nimotuzumab (nimotuzumab scFv) is reported in this study. The scFv was produced in VH-linker-VL format in Origami^? 2(DE3)pLysS bacterial cells and purified using Ni-TNA resin column. 3-D structure prediction using I-TASSER (Iterative Threading Assembly Refinement) server and analyzing the predicted models using YASARA (Yet Another Scientific Artificial Reality Application) viewer revealed that VH and VL domains assemble into a correctly-folded single chain antibody that is able to bind EGFR. The scFv was evaluated in ELISA and western blot tests and proven to be able to bind EGFR-overexpressing cancer cells (A-431 cells in an efficient manner but unable to recognize cancer cells expressing low levels of EGFR (MCF-7 breast cancer cells).     

Release of Metal Ions from Orthodontic Appliances: An In Vitro Study

In this paper, we report the results of an in vitro experiment on the release of metal ions from orthodontic appliances composed of alloys containing iron, chromium, nickel, silicon, and molybdenum into artificial saliva. The concentrations of magnesium, aluminum, silicon, phosphorus, sulfur, potassium, calcium, titanium, vanadium, manganese, iron, cobalt, copper, zinc, nickel, and chromium were significantly higher in artificial saliva in which metal brackets, bands, and wires used in orthodontics were incubated. In relation to the maximum acceptable concentrations of metal ions in drinking water and to recommended daily doses, two elements of concern were nickel (573 vs. 15 μg/l in the controls) and chromium (101 vs. 8 μg/l in the controls). Three ion release coefficients were defined: α, a dimensionless multiplication factor; β, the difference in concentrations (in micrograms per liter); and γ, the ion release coefficient (in percent). The elevated levels of metals in saliva are thought to occur by corrosion of the chemical elements in the alloys or welding materials. The concentrations of some groups of dissolved elements appear to be interrelated.     

Naringin Mitigates Erythrocytes Aging Induced by Paclitaxel: An In Vitro Study

In this study, the protective role of naringin (NAR) against paclitaxel (PTX)‐induced erythrocytes aging has been investigated using human erythrocyte as an in vitro model. Erythrocytes were incubated with PTX in the presence and absence of NAR. Incubation of erythrocytes with PTX resulted in increased protein carbonyl content and malondialdehyde and hemolysis percentage compared with control. Furthermore, a significant increase in the ratios of glutathione peroxidase/glutathione reductase, superoxide dismutase/glutathione peroxidase, and superoxide dismutase/catalase in PTX‐treated cells was observed, compared with control cells. In contrast, reduced glutathione/oxidized glutathione ratio and glucose‐6‐phosphate dehydrogenase activity were decreased upon PTX treatment. The simultaneous incubation of erythrocytes with PTX and NAR restored these variables to values similar to those of control erythrocytes. These results suggest that NAR inhibited PTX‐induced aging by lessening the PTX‐induced oxidative stress.     

Migration of Salmonella typhi through Intestinal Epithelial Monolayers: An In Vitro Study

This study characterizes the transmigration of enteroinvasive Salmonella typhi in vitro, using a human intestinal epithelial cell line as a model of small intestinal epithelium. C2BBe cells, a subclone of CACO-2 with a highly differentiated enterocytic phenotype, were grown to maturity on Transwell filters. S. typhi Ty2 and the vaccine strain, Ty21a, the S. typhi mutant X7344 and parent strain SB130, and S. typhimurium 5771 in logarithmic phase were introduced to the upper chamber of the filter units. Numbers of bacteria in the lower chamber, TER and permeability of the monolayer to mannitol were measured over time. Monolayers were examined by light, electron and confocal microscopy to determine the pathway of bacterial transmigration, and intracellular bacteria were estimated by gentamicin assay. Epithelial cell injury was quantified by light microscopy. S. typhi transmigrated earlier and in larger numbers than S. typhimurium, inducing marked changes in electrical resistance and permeability. Unlike S. typhimurium, S. typhi selected epithelial cells in small number and caused their death and extrusion from the monolayers leaving holes through which S. typhi transmigrated. Ty2 consistently transmigrated in larger numbers and with more injury to monolayers than Ty21a. S. typhi crosses the monolayers of C2BBe cells by a paracellular route in contrast to the transcellular pathway described for other Salmonellae. This may be related to the unique pathophysiology of S. typhi infection and the restricted host specificity of this pathogen. In these assays the vaccine strain, Ty21a, is slightly less invasive than its parent, though more invasive than S. typhimurium.     

Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current definition of a probiotic.     

Interactions of Anaerobic Bacteria with Dental Stem Cells: An In Vitro Study

Background

In patients with periodontitis, it is highly likely that local (progenitor) cells encounter pathogenic bacteria. The purpose of this in vitro study was to elucidate how human dental follicle stem cells (hDFSC) react towards a direct challenge with anaerobic periodontal pathogens under their natural oxygen-free atmosphere. HDFSC were compared to human bone marrow mesenchymal stem cells (hBMSC) and differentiated primary human gingival fibroblasts (hGiF), as well as permanent gingival carcinoma cells (Ca9-22).

Methodology/Principal Findings

The different cell types were investigated in a co-culture system with Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). The viability of the cells and pathogens under anaerobic conditions, as well as interactions in terms of adherence and internalization, were examined. Additionally, the release of pro-inflammatory interleukin-8 (IL-8) and anti-inflammatory interleukin-10 (IL-10) was quantified via enzyme-linked immunosorbent assay. The bacteria adhered less efficiently to hDFSC compared to Ca9-22 (P. gingivalis: 0.18% adherence to hDFSC; 3.1% adherence to Ca9-22). Similar results were observed for host cell internalization (F. nucleatum: 0.002% internalization into hDFSC; 0.09% internalization into Ca9-22). Statistically significantly less IL-8 was secreted from hDFSC after stimulation with F. nucleatum and P. gingivalis in comparison with hGiF (F. nucleatum: 2080.0 pg/ml – hGiF; 19.7 pg/ml – hDFSC). The IL-10 response of the differentiated cells was found to be low in relation to their pro-inflammatory IL-8 response.

Conclusions/Significance

The results indicate that dental stem cells are less prone to interactions with pathogenic bacteria than differentiated cells in an anaerobic environment. Moreover, during bacterial challenge, the stem cell immune response seems to be more towards an anti-inflammatory reaction. For a potential future therapeutic use of hDFSC, these findings support the idea of a save application.     

Anti-hemolytic and Peroxyl Radical Scavenging Activity of Organoselenium Compounds: An In Vitro Study

Selenium-containing amino acids, selenocystine (CysSeSeCys), methylselenocysteine (MeSeCys), and selenomethionine (SeMet) have been examined for anti-hemolytic and peroxyl radical scavenging ability. Effect of these compounds on membrane lipid peroxidation, release of hemoglobin, and loss of intracellular K⁺ ion as a consequence of peroxyl radicals-induced oxidation of human red blood cells were used to evaluate their anti-hemolytic ability. The peroxyl radicals were generated from thermal degradation of 2,2′-azobis(2-methylpropionamidine) dihydrochloride. Significant delay (t _eff) was observed in oxidative damage in the presence of the selenium compounds. From the IC₅₀ values for the inhibition of hemolysis, lipid peroxidation, and K⁺ ion leakage, the relative anti-hemolytic ability of the compounds were found to be in the order of CysSeSeCys > MeSeCys > SeMet. The anti-hemolytic abilities of the compounds, when compared with sodium selenite (Na₂SeO₃) under identical experimental conditions, were found to be better than Na₂SeO₃. Relative rate constants estimated for the reaction of MeSeCys and SeMet with peroxyl radicals by competition kinetics using ABTS²⁻ as a reference confirmed that all the compounds are efficient peroxyl radical scavengers. Comparison of the GPx-like activity of these compounds, by NADPH–GSH reductase coupled assay, indicated that CysSeSeCys exhibits the highest activity. Based on these results, it is concluded that among the compounds examined, CysSeSeCys, possessing the ability to reduce peroxyl radicals and hydroperoxides showed efficient anti-hemolytic activity.     

Biosynthesis of Threonine from Homoserine by Mixed Rumen Microorganisms: An In Vitro Study

The biosynthesis of threonine (Thr) by using the main biosynthetic pathway involving homoserine (Hser) was quantitatively investigated by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system. Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were incubated anaerobically at 39°C for 12 h with or without Hser (2 mm) as a substrate. Thr and other related compounds produced in both the supernatants and hydrolysates of the incubation were analyzed by HPLC. During a 12-h incubation period, 84.2%, 58.1%, and 92.0% of Hser disappeared in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were demonstrated for the first time to produce Thr from Hser, and the production of Thr from Hser in BP (371.9 and 297.2 μmol/g MN) (MN, microbial nitrogen) was about 13.0% and 9.1% higher than that in B alone (329.2 and 272.5 μmol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to synthesize Thr from Hser. Other metabolites produced from Hser were found to be glycine (Gly) and 2-aminobutyric acid (2AB) in B and BP. In P, Gly and 2AB were not found. The results mentioned above indicated the abilities of rumen bacteria and the mixture of rumen bacteria and protozoa to synthesize Thr de novo from Hser and appeared as first-time report. Received: 24 May 2000/Accepted: 4 August 2000     

Differential Suppressive Effects of Testosterone on Immune Function in Fresh Water Snake,Natrix piscator: An In Vitro Study

Reptiles represent the crucial phylogenetic group as they were the ancestors of both birds and mammals hence very important to study. The objectives of the present study were to investigate the potential roles of testosterone in the innate immune responses and splenic lymphocyte proliferation in fresh water snake, Natrix piscator. Animals were mildly anesthetized and spleens were taken out to study the splenic macrophage phagocytosis, super oxide production and nitrite release using in vitro testosterone. Splenic lymphocytes were isolated by density gradient centrifugation and were studied for mitogen induced proliferation in presence of in vitro testosterone. Testosterone suppressed the phagocytosis and nitrite release in a concentration dependent manner. Biphasic suppressive effect of testosterone was observed in superoxide production as judged by reduction of nitroblue tetrazolium salt where salt reduction was suppressed at lower and higher concentrations of testosterone. Mitogen induced splenic lymphocyte proliferation was also suppressed by testosterone. By suppressing immune responses, testosterone may, therefore, act as a physiological mechanism regulating the relative amount of energy invested into either reproductive effort or immunocompetence.     

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca²⁺-independent, as inhibition of Ca²⁺ channels or culture in the absence of Ca²⁺ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.     

Antioxidant Capacity of Ugni molinae Fruit Extract on Human Erythrocytes: An In Vitro Study

Ugni molinae is an important source of molecules with strong antioxidant activity widely used as a medicinal plant in Southern Chile–Argentina. Total phenol concentration from its fruit extract was 10.64 ± 0.04 mM gallic acid equivalents. Analysis by means of HPLC/MS indicated the presence of the anthocyanins cyanidin and peonidin, and the flavonol quercitin, all in glycosylated forms. Its antioxidant properties were assessed in human erythrocytes in vitro exposed to HClO oxidative stress. Scanning electron microscopy showed that HClO induced an alteration in erythrocytes from a normal shape to echinocytes; however, this change was highly attenuated in samples containing U. molinae extracts. It also had a tendency in order to reduce the hemolytic effect of HClO. In addition, X-ray diffraction experiments were performed in dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine bilayers, classes of lipids preferentially located in the outer and inner monolayers, respectively, of the human erythrocyte membrane. It was observed that U. molinae only interacted with DMPC. Results by fluorescence spectroscopy on DMPC large unilamellar vesicles and isolated unsealed human erythrocyte membranes also showed that it interacted with the erythrocyte membrane and DMPC. It is possible that the location of U. molinae components into the membrane outer monolayer might hinder the diffusion of HClO and of free radicals into cell membranes and the consequent decrease of the kinetics of free radical reactions.     

Deep Sea Water Prevents Balloon Angioplasty-Induced Hyperplasia through MMP-2: An In Vitro and In Vivo Study

Major facts about the development of restenosis include vascular smooth muscle cells (VSMCs) proliferation and migration. A previous study showed that in vitro treatment with magnesium chloride has the potential to affect the proliferation and migration of VSMCs. Magnesium is the major element in deep sea water (DSW) and is a biologically active mineral. It is unclear whether DSW intake can prevent abnormal proliferation and migration of VSMCs as well as balloon angioplasty-induced neointimal hyperplasia. Thus, we attempted to evaluate the anti-restenotic effects of DSW and its possible molecular mechanisms. Several concentrations of DSW, based on the dietary recommendations (RDA) for magnesium, were applied to a model of balloon angioplasty in SD rats. The results showed that DSW intake markedly increased magnesium content within the vascular wall and reduced the development of neointimal hyperplasia. The immunohistochemical analysis also showed that the expression of proteins associated with cell proliferation and migration were decreased in the balloon angioplasty groups with DSW supplement. Furthermore, in vitro treatment with DSW has a dose-dependent inhibitory effect on serum-stimulated proliferation and migration of VSMCs, whose effects might be mediated by modulation of mitogen-activated protein kinase (MAPK) signaling and of the activity of matrix metalloproteinase-2 (MMP-2). Our study suggested that DSW intake can help prevent neointimal hyperplasia (or restenosis), whose effects may be partially regulated by magnesium and other minerals.     

The Expression of CD90/Thy-1 in Hepatocellular Carcinoma: An In Vivo and In Vitro Study

Although the CD90 (Thy-1) was proposed as biomarker of several tumors and cancer stem cells, the involvement of this molecule in the progression of hepatocellular carcinoma (HCC) and other less frequent hepatic neoplasms is still undefined. The distribution of CD90 was investigated both in in vivo (human tissues samples) and in vitro (human HCC cell line JHH-6). A total of 67 liver tumors were analyzed: 51 HCC, 6 cholangiocarcinoma and 10 hepatoblastoma. In all cases, paired tissue sample of both the tumor and cirrhotic liver was available. Hepatic tissue obtained in 12 healthy livers was used as control. CD90 gene expression was studied by RT-qPCR, protein expression was assessed by quantitative Western Blot, immunofluorescence and flow cytometry. The CD90 expression analysis showed a significant increment in tumor compared to both its paired cirrhotic tissue and normal liver (p<0.05 and p<0.001, respectively). This increase was accompanied by the up-regulation of stromal component in the cancer, as demonstrated by alpha smooth muscle actin staining. In vitro analysis of JHH-6 cell line showed a higher proliferation capacity of CD90⁺ compared to CD90^- cells (p<0.001), also noticed in 3D clonogenic assay (p<0.05), associated by a significant higher expression of the promoting factors (hepatocyte growth factor, fibroblast associated protein and alpha smooth muscle actin 2). A higher expression of the breast cancer resistance protein was found in CD90⁺ subpopulation while the multidrug resistance protein 1 showed an opposite behavior. Collectively, these results point to the importance of CD90 in the HCC.