Human iPSC-Derived Neurons as A Platform for Deciphering the Mechanisms behind Brain Aging

With an increased life expectancy among humans, aging has recently emerged as a major focus in biomedical research. The lack of in vitro aging models—especially for neurological disorders, where access to human brain tissues is limited—has hampered the progress in studies on human brain aging and various age-associated neurodegenerative diseases at the cellular and molecular level. In this review, we provide an overview of age-related changes in the transcriptome, in signaling pathways, and in relation to epigenetic factors that occur in senescent neurons. Moreover, we explore the current cell models used to study neuronal aging in vitro, including immortalized cell lines, primary neuronal culture, neurons directly converted from fibroblasts (Fib-iNs), and iPSC-derived neurons (iPSC-iNs); we also discuss the advantages and limitations of these models. In addition, the key phenotypes associated with cellular senescence that have been observed by these models are compared. Finally, we focus on the potential of combining human iPSC-iNs with genome editing technology in order to further our understanding of brain aging and neurodegenerative diseases, and discuss the future directions and challenges in the field.

Is Surface Metastability of Today’s Ceramic Bearings a Clinical Issue?

Recent studies on zirconia-toughened alumina (ZTA) evidenced that in vivo aged implants display a much higher monoclinic zirconia content than expected from in vitro simulations by autoclaving. At the moment, there is no agreement on the source of this discrepancy: Some research groups ascribe it to the effect of mechanical impact shocks, which are generally not implemented in standard in vitro aging or hip walking simulators. Others invoke the effect of metal transfer, which should trigger an autocatalytic reaction in the body fluid environment, accelerating the kinetics of tetragonal-to-monoclinic transformation in vivo. Extrapolations of the aging kinetics from high (autoclave) to in vivo temperature are also often disputed. Last, Raman spectroscopy is by far the preferred method to quantify the amount of monoclinically transformed zirconia. There are, however, many sources of errors that may negatively affect Raman results, meaning that the final interpretation might be flawed. In this work, we applied Raman spectroscopy to determine the monoclinic content in as-received and in vitro aged ZTA hip joint implants, and in one long-term retrieval study. We calculated the monoclinic content with the most used equations in the literature and compared it with the results of X-ray diffraction obtained on a similar probe depth. Our results show, contrary to many previous studies, that the long-term surface stability of ZTA ceramics is preserved. This suggests that the Raman technique does not offer consistent and unique results for the analysis of surface degradation. Moreover, we discuss here that tetragonal-to-monoclinic transformation is also necessary to limit contact damage and wear stripe extension. Thus, the surface metastability of zirconia-containing ceramics may be a non-issue.

Physical and chemical mechanisms involved in adhesion of orthodontic bonding composites: in vitro evaluations

Background Bond strength of orthodontic composite is strongly influenced by molecular and structural mechanisms. Aim of this in vitro study was to compare bond strength of light-cure orthodontic composites by measuring debonding forces and evaluating locations of bond failure. Investigations on chemical compositions clarified adhesive behaviors and abilities, exploring effects of ageing processes in this junction materials. Methods Twelve enamel discs, from human premolars, were randomly coupled to one orthodontic adhesive system (Transbond XT™ 3 M UNITEK, USA, Light-Cure Orthodontic Paste, LEONE, Italy and Bisco Ortho Bracket Paste LC, BISCO, Illinois) and underwent to Shear Bond Strength test. Metallic brackets were bonded to twenty-seven human premolar, with one of the adhesive systems, to quantify, at FE-SEM magnifications, after debonding, the residual material on enamel and bracket base surfaces. Raman Spectroscopy analysis was performed on eight discs of each composites to investigate on chemical compositions, before and after accelerated aging procedures in human saliva and sugary drink. Results Orthodontic adhesive systems showed similar strength of adhesion to enamel. The breakage of adhesive-adherent bond occurs in TXT at enamel-adhesive interface while in Bisco and Leone at adhesive-bracket interface. Accelerated in vitro aging demonstrated good physical–chemical stability for all composites, Bisco only, was weakly contaminated with respect to the other materials. Conclusion A similar, clinically adequate and acceptable bond strength to enamel for debonding maneuvers was recorded in all orthodontic adhesive systems under examination. No significant chemical alterations are recorded, even in highly critical situations, not altering the initial mechanical properties of materials.

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

ATR-FTIR Analysis of Orthodontic Invisalign® Aligners Subjected to Various In Vitro Aging Treatments

Clear and removable tooth aligners for orthodontics treatments have become an increasingly popular alternative to fixed appliances. Even if protocols suggest removing aligners before eating or drinking, most patients retain them when they drink beverages. Alterations in the material during the daily use could determine a reduction in the application forces, affecting the desired orthodontic movement; the knowledge of how this material reacts when subjected to different aging processes is mandatory to establish the predictability of the orthodontic treatment. According to this, the aim of the present study was to assess a new objective approach, coupling spectroscopic and chemometric tools, to evaluate the changes occurring in Invisalign® aligners, the most widely used brand, exposed in vitro to coffee, tea, Coca Cola® and UV radiation for 24 and 48 h. In particular, ATR-FTIR spectroscopy was utilized to characterize, at the molecular level, the chemical and color modifications in the surfaces of the appliances; the obtained data were submitted to PCA and one-way ANOVA and Tukey’s multiple comparison test. Moreover, a colorimetry analysis was carried out to evaluate any changes in color and transparency. Coffee and tea samples displayed the major color changes between the tested groups. The differences highlighted in the spectral features of coffee, tea and UV-treated samples were mainly ascribable to color and transparency changes, because the chemical properties remained unaltered.

Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells

Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.

Effects of Carbonated Drinks on Mechanical Properties of Three Types of Thermoplastic Aligner Materials: An In vitro Study

Objectives: The aim was to assess the mechanical properties of the three types of thermoplastic aligner materials before and after in vitro aging with carbonated drinks. Materials and methods: Twelve samples of thermoplastic aligner materials produced by three different manufacturers (Leone S.P.A, Florence, Italy; Duran, SCHEU-dental GmbH, Iserlohn, Germany; Essix ACE, Dentsply Raintree Essix, United States) were selected. Samples were thermoformed and later aged in vitro at a constant temperature in artificial saliva along with carbonated drinks (10 min each day) for 2 weeks. The mechanical properties were characterized using universal testing machine such as instron (MultiTest 10-i) and the results were compared with the control groups (before exposure to carbonated drinks). Results: All the above-mentioned thermoplastic materials tested showed an insignificant ( p > 0.05) decrease in stiffness, yield strength, and elastic modulus after aging. The stiffness of the thermoplastic materials increased with an increase in thickness. The flexure modulus was higher for the thinner materials, whereas it was lower for the thicker materials. Conclusion: Experimental results indicate that the aligner material will remain stable during and following exposure to carbonated drinks, which suggests that the orthodontic force from thermoplastic appliances does not decrease with clinical usage of carbonated drinks.

In Vitro Aging of Human Skin Fibroblasts: Age-Dependent Changes in 4-Hydroxynonenal Metabolism

Evidence suggests that the increased production of free radicals and reactive oxygen species lead to cellular aging. One of the consequences is lipid peroxidation generating reactive aldehydic products, such as 4-hydroxynonenal (HNE) that modify proteins and form adducts with DNA bases. To prevent damage by HNE, it is metabolized. The primary metabolic products are the glutathione conjugate (GSH-HNE), the corresponding 4-hydroxynonenoic acid (HNA), and the alcohol 1,4-dihydroxynonene (DHN). Since HNE metabolism can potentially change during in vitro aging, cell cultures of primary human dermal fibroblasts from several donors were cultured until senescence. After different time points up to 30 min of incubation with 5 µM HNE, the extracellular medium was analyzed for metabolites via liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS). The metabolites appeared in the extracellular medium 5 min after incubation followed by a time-dependent increase. But, the formation of GSH-HNL and GSH-DHN decreased with increasing in vitro age. As a consequence, the HNE levels in the cells increase and there is more protein modification observed. Furthermore, after 3 h of incubation with 5 µM HNE, younger cells showed less proliferative capacity, while in older cells slight increase in the mitotic index was noticed.