Cytotoxicity and estrogenicity of a novel 3-dimensional printed orthodontic aligner

INTRODUCTION Orthodontic aligners printed with in-office 3-dimensional (3D) procedures have been described, but no data on their biocompatibility exist. This study investigates the cytotoxicity and estrogenicity of a 3D-printed orthodontic aligner by assessing its biological and behavioral effects. METHODS Ten sets of 1 type of aligner were immersed in sterile deionized water for 14 days, and the cytotoxicity and estrogenicity of released factors were assessed via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays on human gingival fibroblasts and the estrogen-sensitive MCF-7 and the estrogen-insensitive MDA-MB-231 breast cancer cell lines. 17β-Estradiol and bisphenol-A were used as positive controls. The statistical analysis of data was performed with generalized linear models at a 0.05 level of significance. RESULTS No signs of cytotoxicity were seen for the aligner samples for concentrations (v/v) of 20% (P = 0.32), 10% (P = 0.79), or 5% (P = 0.76). The antioxidant activity expressed as the capacity to reduce intracellular levels of reactive oxygen species was not affected in the aligner samples (P = 0.08). No significant estrogenicity was induced by the aligner samples compared with eluents from the negative control for both MCF-7 (P = 0.65) and MDA-MB-231 (P = 0.78). As expected, 17β-Estradiol and bisphenol-A stimulated MCF-7 cell proliferation, whereas no effect was observed on MDA-MB-231 cells. CONCLUSIONS In conclusion, if any factors were released during the 14-day aging of 3D-printed aligners in water, these were not found to be cytotoxic for human gingival fibroblasts and did not affect their intracellular reactive oxygen species levels. Moreover, no estrogenic effects of these putative eluates were observed based on an E-screen assay

Organotypic Cultures of Intervertebral Disc Cells: Responses to Growth Factors and Signaling Pathways Involved

Intervertebral disc (IVD) degeneration is strongly associated with low back pain, a major cause of disability worldwide. An in-depth understanding of IVD cell physiology is required for the design of novel regenerative therapies. Accordingly, aim of this work was the study of IVD cell responses to mitogenic growth factors in a three-dimensional (3D) organotypic milieu, comprising characteristic molecules of IVD’s extracellular matrix. In particular, annulus fibrosus (AF) cells were cultured inside collagen type-I gels, while nucleus pulposus (NP) cells in chondroitin sulfate A (CSA) supplemented collagen gels, and the effects of Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-Like Growth Factor-I (IGF-I) were assessed. All three growth factors stimulated DNA synthesis in both AF and NP 3D cell cultures, with potencies similar to those observed previously in monolayers. CSA supplementation inhibited basal DNA synthesis rates, without affecting the response to growth factors. ERK and Akt were found to be phosphorylated following growth factor stimulation. Blockade of these two signaling pathways using pharmacologic inhibitors significantly, though not completely, inhibited growth factor-induced DNA synthesis. The proposed culture systems may prove useful for further in vitro studies aiming at future interventions for IVD regeneration

Special Issue “Anti-Aging Properties of Natural Compounds”

Aging is defined as the progressive loss of an organism’s homeostatic balance [...

The Unexpected Anabolic Phenotype and Extended Longevity of Skin Fibroblasts after Chronic Glucocorticoid Excess

Intense stress can challenge tissue homeostasis and accelerate the ageing process. However, several lines of evidence indicate that repeated mild stresses can have beneficial and even life-prolonging effects. Hypersecretion of glucocorticoids (GC) represents the major hormonal response to stress. Besides its life-sustaining role, GC excess, usually due to several side-effects that promote a “catabolic” phenotype, can be detrimental for several tissues. Cushing's syndrome patients are characterized by chronic endogenous GC excess and consequently at the time of diagnosis they have an atrophic elderly-like skin. Interestingly, when Cushing's syndrome fibroblasts were removed from the high-GC milieu in vivo and cultured in vitro under standard conditions they express an “anabolic” phenotype, i.e. they restore their ability for collagen synthesis, they secrete reduced levels of metalloproteases (MMP-1 and MMP-2) and have an increased proliferative capacity and contractility. Furthermore, these cells exhibit a significant extension of their proliferative lifespan, while they respond better to exogenous stress by producing significantly higher levels of heat-shock protein-70 (HSP70). These results imply that long-term hypercortisolism in vivo can have beneficial consequences on fibroblast physiology in vitro

In vitro estrogenicity of dental resin sealants

PURPOSE: This investigation assessed the estrogenic action of various types of sealants. METHODS: Three light-cured sealants (Denton Clear, Delton Opaque, Ultraseal XT Plus) were applied in polyethylene molds (Ø:10mm, h:2mm, n:8) and photopolymerized (40 seconds, halogen bulb unit, standard mode 650 mW/cm 2 intensity). All specimens were immersed in normal saline for 1 week at 37 °C. Samples of eluents at concentrations of 5% and 10% (volume per volume) were tested for estrogenicity by measuring their effect on the proliferation of the estrogen responsive MCF-7 breast cancer cells. In addition, an estrogen insensitive cell line was used as a control (MDA-MB-231) to exclude a possible cytostatic effect of the tested materials. All assays were repeated 4 times, and results analyzed with a 2-way analysis of variance and Tukey's test at the 0.05 level. RESULTS: Eluents of the sealants tested at concentrations of 5% and 10% did not possess estrogenicity, except for the eluent of one sealant (Delton Opaque) at concentration 10%, which caused an induction of the proliferation rate of the MCF-7 cells. CONCLUSIONS: Eluents of sealants tested at a concentration of 5% had no estrogenic activity. The eluent of Delton Opaque at a concentration of 10% possessed some estrogenic activity

Acid Tolerance of Streptococcus macedonicus as Assessed by Flow Cytometry and Single-Cell Sorting

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC(4)(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge

In Vitro Assessment of Cytotoxicity and Estrogenicity of Vivera® Retainers

AIM To investigate the cytotoxicity and estrogenicity of Vivera® retainers by assessing their biological behavioral effects as-received from the manufacturer and after retrieved from patients. MATERIALS AND METHODS In this, in vitro investigation six sets (maxillary and mandibular) of Vivera® retainers, three as received and three retrieved after four weeks of use by patients of an orthodontic postgraduate clinic, were immersed in the normal saline solution for 14 days following different modes of sterilization. The estrogenicity assays involved two cell lines, namely the estrogen-sensitive MCF-7 and the estrogen-insensitive MDA-MB-231. Following a 6 day incubation with the solutions to be tested, at concentrations varying from 5% to 20% v/v in medium supplemented with 2% fetal calf serum devoid of endogenous estrogens, estrogenicity was assessed by cell counting; p-Estradiol was used as positive control. The statistical analysis of data was performed with two-way analysis of variance (ANOVA) with appliance and concentration as predictors. Differences were further investigated with the Tukey multiple comparison tests at the 0.05 level of significance. RESULTS No significant MCF-7 proliferation was induced by the three samples compared either to the eluents from as-received retainers or to the negative control. As expected, p-estradiol induced a potent stimulation of MCF-7 cell proliferation, while no effect was observed on MDA-MB-231 cells. CONCLUSION Under the conditions of this experiment eluents of as-received and retrieved Vivera® retainers did not seem to exhibit xenoestrogenic activity. CLINICAL SIGNIFICANCE Vivera® retainers can be used as part-time removable oral appliances following the manufacturer's instructions

Structure-activity relationship investigation of methoxy substitution on anticancer pyrimido[4,5-c]quinolin-1(2H)-ones

Pyrimido[4,5-c]quinolin-1(2H)-one derivatives were shown to exert interesting biological activities including anticancer, antimicrobial, and cardiovascular. Substitution with methoxy groups played a crucial role in the anticancer activity of known anticancer agents. This study explores the contribution of diverse-positioned methoxy substituents to the antimigratory and cytotoxic activities of this class. Synthesized analogues were tested in the MTT, cell cycle, and wound-healing assays. Previous studies on this class reported weak to medium antimitotic activity. Therefore, all compounds were subjected to tubulin polymerization assay and in silico molecular docking study at the colchicine binding site of tubulin. The 2-methoxy and 2,4-dimethoxy substitutions at the 2-arylpyrimido functionality enhanced the antimigratory activity in the 9-methoxy-substituted series like 6 and 9. The 3,4,5-trimethoxy substitutions at the 2-arylpyrimido group also significantly improved the antimigratory activity in the presence or absence of the 9-methoxy substitution as represented by 13 and 22, respectively. Docking experiments showed two distinct orientations at the colchicine binding site of tubulin. The first, achieved by 7 and 16-20, coincides with that of colchicine and positions the methoxy-substituted 2-aryl ring deep in the highly hydrophobic narrow end of the funnel-shaped binding pocket. On the other hand, 5, 6, 9-14 and 21, were oriented towards the wider opening of the binding pocket. Pyrimido[4,5-c] quinolin-1(2H)-ones are promising antimigratory hits with potential for future use to control metastatic breast cancers. © 2013 Springer Science+Business Media New York