Biological niches within human calcified aortic valves. Towards understanding of the pathological biomineralization process

Despite recent advances, mineralization site, its microarchitecture, and composition in calcific heart valve remain poorly understood. A multiscale investigation, using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectrometry (EDS), from micrometre up to nanometre, was conducted on human severely calcified aortic and mitral valves, to provide new insights into calcificationp rocess. Our aim was to evaluate the spatial relationship existing between bioapatite crystals, their local growing microenvironment, and the presence of a hierarchical architecture. Here we detected the presence of bioapatite crystals in two different mineralization sites that suggest the action of two different growth processes:a pathological crystallization process that occurs in biological niches and is ascribed to a purely physicochemical process and a matrix- mediated mineralized process in which the extracellular matrix acts as the template for a site-directed nanocrystals nucleation. Different shapes of bioapatite crystallization were observed at micrometer scale in each microenvironment but at the nanoscale level crystals appear to be made up by the same subunit

Paracrystalline and crystalline inclusions of the human testis in cases of subfertility

Testicular crystalline inclusions, namely Charcot-Böttcher and Spangaro crystals of Sertoli cells, Lubarsch crystals of spermatogonia and Reinke crystals of Leydig cells, have been considered normal ultrastructural features of the post-pubertal human testis (Reinke, 1896; Chemes et al., 1977; Kaya et al., 1985). Nevertheless, their significance is not known. We have noted that these structures are dynamic and may disappear and reappear in some cases of subfertility. In particular, patients showing spermatogenesis arrest or germ cell aplasia usually do not show any crystalline inclusions. We study herein using transmission electron microscopy testicular biopsies of young infertile men (idiopathic infertility or varicocele) showing intracytoplasmic crystalline and/or paracrystalline inclusions. Sertoli cells’ paracrystalline inclusions consist of closely packed electron-dense longitudinal fibrils, sometimes including a granular and light core. Coarse ca. 5-25 μm long and 2-3 μm thick bundles composed of 5 or 10 nm thick filaments mainly locate in the basal cytoplasm near the nucleus. Reinke’s crystals, in turn, appear as variable-sized (2-5 μm long) polyhedral crystals with a honeycomb lattice and sharp edges consisting of 5-10 nm filaments filling wide areas of the cytoplasm. Alternatively, they may appear as filamentous/tubular, electron dense (0,5 μm long) units. In both cases they associate to mitochondria and dilated smooth endoplasmic reticulum. Due to their structure, whether these inclusions arise or not within the nucleus and are somehow transported to the cytoplasm is not yet clear. Although their exact molecular composition remains to be discovered, they are not likely to be associated to steroidogenesis but rather they may represent a kind of protein deposit

Can pulsed electromagnetic fields trigger on-demand drug release from high-tm magnetoliposomes?

Recently, magnetic nanoparticles (MNPs) have been used to trigger drug release from magnetoliposomes through a magneto-nanomechanical approach, where the mechanical actuation of the MNPs is used to enhance the membrane permeability. This result can be effectively achieved with low intensity non-thermal alternating magnetic field (AMF), which, however, found rare clinic application. Therefore, a different modality of generating non-thermal magnetic fields has now been investigated. Specifically, the ability of the intermittent signals generated by non-thermal pulsed electromagnetic fields (PEMFS) were used to verify if, once applied to high-transition temperature magnetoliposomes (high-Tm MLs), they could be able to efficiently trigger the release of a hydrophilic model drug. To this end, hydrophilic MNPs were combined with hydrogenated soybean phosphatidylcholine and cholesterol to design high-Tm MLs. The release of a dye was evaluated under the effect of PEMFs for different times. The MNPs motions produced by PEMF could effectively increase the bilayer permeability, without affecting the liposomes integrity and resulted in nearly 20% of release after 3 h exposure. Therefore, the current contribution provides an exciting proof-of-concept for the ability of PEMFS to trigger drug release, considering that PEMFS find already application in therapy due to their anti-inflammatory effects

The galectin-3/RAGE dyad modulates vascular osteogenesis in atherosclerosis

Vascular calcification correlates with inflammation and plaque instability in a dual manner, depending on the spotty/granular (micro) or sheet-like/lamellated (macro) pattern of calcification. Modified lipoproteins trigger both inflammation and calcification via receptors for advanced lipoxidation/glycation endproducts (ALEs/AGEs). This study compared the roles of galectin-3 and receptor for AGEs (RAGE), two ALEs/AGEs-receptors with diverging effects on inflammation and bone metabolism, in the process of vascular calcification. We evaluated galectin-3 and RAGE expression/localization in 62 human carotid plaques and its relation to calcification pattern, plaque phenotype, and markers of inflammation and vascular osteogenesis; and the effect of galectin-3 ablation and/or exposure to an ALE/AGE on vascular smooth muscle cell (VSMC) osteogenic differentiation. While RAGE co-localized with inflammatory cells in unstable regions with microcalcification, galectin-3 was expressed also by VSMCs, especially in macrocalcified areas, where it co-localized with alkaline phosphatase. Expression of galectin-3 and osteogenic markers was higher in macrocalcified plaques, whereas the opposite occurred for RAGE and inflammatory markers. Galectin-3-deficient VSMCs exhibited defective osteogenic differentiation, as shown by altered expression of osteogenic transcription factors and proteins, blunted activation of pro-osteoblastogenic Wnt/β-catenin signalling and proliferation, enhanced apoptosis, and disorganized mineralization. These abnormalities were associated with RAGE up-regulation, but were only in part prevented by RAGE silencing, and were partially mimicked or exacerbated by treatment with an AGE/ALE. These data indicate a novel molecular mechanism by which galectin-3 and RAGE modulate in divergent ways, not only inflammation, but also vascular osteogenesis, by modulating Wnt/β-catenin signalling, and independently of ALEs/AGEs

Introducing medical students to scientific research: an early electron-microscopy laboratory attendance experience

In the light of importance that “evidence-based medicine” has assumed in recent years (Snelgrove et al. 2009), we offer to the students of the first-year medical-degree the chance of an early exposure to the work in the ultrastructural research laboratory “Pietro M. Motta”. On an elective basis, students attended the laboratory in small groups. They were guided and supported by a qualified researcher, a post-graduate student, a graduate student and a technical-staff unit. During the week of attendance students performed several activities: at first they have visited the laboratory where the technicalities of the equipment were illustrated, than they have taken part to a lecture on the methods used to prepare the biological samples for Scanning and Transmission Electron Microscopy. In the following days preparation of samples for Scanning and Transmission Electron Microscopy was carried on and a guided discussion on scientific articles concerning the samples used in the experiments was conducted (Familiari et al. 2006). Later, samples were observed using a light microscope and both transmission and scanning electron microscope. At the end of the week students had taken hands-on in various stages of preparation, observation and analysis of the samples. The discussion with the researcher and the post-graduate doctors/ students provided the attendant students with key concepts regarding scientific work that are the basis of theory and practice of biomedical research projects, not only of ultrastructural type. The students’ early exposure to the work and methodology characteristic of ultrastructural research may prove useful, not only when promoting indepth understanding of microscopic anatomy, but also as a motivational base upon which to instil a correct approach to scientific research in future doctors

Enamel remineralization and repair results of Biomimetic Hydroxyapatite toothpaste on deciduous teeth: an effective option to fluoride toothpaste

Background: Dental caries is a recognized worldwide public health problem. Despite being one of the most efective strategies against dental caries, the excessive use of fuorine may result in a potential risk of developing dental fuorosis especially in children under age of six. The purpose of this work is to analyze a fuorine-free toothpaste containing Biomimetic Hydroxyapatite to assess enamel re-mineralizing and repairing properties. Results: The study was performed in vitro and in vivo, comparing the hydroxyapatite toothpaste with two others toothpaste containing diferent fuorine concentrations. The coating efect of the micro-structured Hydroxyapatite nanoparticles reintegrates the enamel with a biomimetic flm reproducing the structure and the morphology of the biologic Hydroxyapatite of the enamel. As demonstrated, the coating is due to the deposit of a new layer of apatite, which presents fewer particles than the natural enamel, not based on the chemical—physical changes occurring in fuorinated toothpastes. Moreover, it shows resistance to brushing as a consequence of chemical bonds between the synthetic and natural crystals of the enamel. Conclusions: The use of Biomimetic Hydroxyapatite toothpastes has proven to be a valuable prevention measure against dental caries in primary dentition since it prevents the risk of fuorosis

The application of a fluoride-and-vitamin D solution to deciduous teeth promotes formation of persistent mineral crystals: a morphological ex-vivo study

Background: The use of effective, low-cost, and easy-to-use products for early caries management will avoid loss of dental vitality and impairment in oral function. The ability of fluoride to re-mineralize dental surfaces has been widely reported as well as vitamin D demonstrated to have significant potential in improving the remineralization of early lesions on enamel surfaces. The aim of the present ex vivo study was to evaluate the effect of a fluoride and vitamin D solution in terms of formation of mineral crystals on the enamel of primary teeth, and their permanence over time on dental surfaces. Methods: Sixteen extracted deciduous teeth were cut to obtain 64 specimens that were divided into two groups. The first consisted of immersion of specimens for 4 days in a fluoride solution (T1); in the second group, the specimens were immersed for 4 days (T1) in fluoride and Vitamin D solution, and for a further 2 (T2) and 4 days (T3) in saline solution. Then, samples were morphologically analyzed by using Variable Pressure Scanning Electron Microscope (VPSEM) and underwent 3D surface reconstruction. Results: After a 4-day immersion in both solutions, octahedral-shaped crystals were formed on the enamel surface of primary teeth, demonstrating any statistically significant differences in terms of number, size, and shape. Moreover, the binding of the same crystals seemed to be strong enough to be maintained until 4 days in saline solution. However, a partial dissolution was observed in a time-dependent manner. Conclusions: A topical application of fluoride and Vitamin D promoted the formation of persistent mineral crystals on enamel surfaces of deciduous teeth and should be further studied to be potentially used as an alternative strategy in preventive dentistry

Bioactive cements: from biological properties to clinical applications

Aim: Calcium silicate-based cements represent safe and predictable materials widely used in different fields of endodontics. They can be applied as pulp dressing agents during vital pulp therapy (VPT) of carious-affected deciduous or permanent teeth with immature roots as well as endodontic cements in case of root perforation or regenerative endodontic procedures. Therefore, it’s crucial to demonstrate biocompatible and antibiofilm properties of bioactive cements (i.e. MTA and Biodentine) in order to support their successful use in the clinical field. Materials and Methods: Biocompatibility of ProRootMTA and Biodentine specimens was assessed through cell culture of Saos-2 cells and both cement extracts by viability assay, oxidative stress analysis and immunofluorescence evaluation; on the other hand, antibiofilm efficacy was assessed by evaluating the biofilm forming ability of Streptococcus mutans onProRootMTA and Biodentine disks using Crystal Violet assay. Results: Cells exposed to ProRootMTA and Biodentine showed a good cell viability, slightly better in presence of the first; moreover, cells seeded on ProRootMTA presented a higher degree of biocompatibility compared to Biodentine. Accordingly, Biodentine demonstrated lightly fewer promising outcomes in terms of oxidative stress and focal adhesions of cells than ProRoot MTA, although the differences were not statistically significant. Inhibition of superficial colonization as well as biofilm forming ability of S. mutants were successfully obtained with both evaluated cements, even though ProRootMTA demonstrated a more efficient time-dependent antibiofilm effect than Biodentine. Conclusion: Bioactive cements proved to be biocompatible and to possess antibiofilm properties. When compared, MTA would seem to perform slightly better and could be considered as the gold standard material in the endodontic procedures

Epstein-barr virus-encoded microRNA-BART18-3p promotes colorectal cancer progression by targeting de novo lipogenesis

The Epstein-Barr virus (EBV) genome encodes a cluster of 22 viral microRNAs, called miR-BamHI-A rightward transcripts (miR-BARTs), which are shown to promote the development of cancer. Here, this study reports that EBV-miR-BART18-3p is highly expressed in colorectal cancer (CRC) and is closely associated with the pathological and advanced clinical stages of CRC. Ectopic expression of EBV-miR-BART18-3p leads to increased migration and invasion capacities of CRC cells in vitro and causes tumor metastasis in vivo. Mechanistically, EBV-miR-BART18-3p activates the hypoxia inducible factor 1 subunit alpha/lactate dehydrogenase A axis by targeting Sirtuin, which promotes lactate accumulation and acetyl-CoA production in CRC cells under hypoxic condition. Increased acetyl-CoA utilization subsequently leads to histone acetylation of fatty acid synthase and fatty acid synthase-dependent fat synthesis, which in turn drives de novo lipogenesis. The oncogenic role of EBV-miR-BART18-3p is confirmed in the patient-derived tumor xenograft mouse model. Altogether, the findings define a novel mechanism of EBV-miR-BART18-3p in CRC development through the lipogenesis pathway and provide a potential clinical intervention target for CRC