On the biocompatibility of endodontic sealers

Periapical tissue may be exposed to root canal filling materials in consequence of root canal therapy. There is scant scientific data about the biocompatibility of root canal filling materials of various chemistry on the periapical area. This study aimed to investigate the effects of different root canal sealers and their eluates on human alveolar osteoblasts in terms of cell proliferation, adhesion, morphology and gene expression in vitro. Five endodontic sealers (AH Plus®, Apexit®, Tubli-Seal®, Real Seal SE®, EndoRez®) and one gutta-percha obturation material (BeeFill®) were tested. Human alveolar osteoblasts derived from 3 different donors following incubation with sealer eluates after 24 h and 72 h were investigated by means of qPCR (gene expression). Morphological reactions of the alveolar osteoblasts were measured by culturing the cells for 3 d, and 7 d and 14 d, respectively, followed by scanning electron microscopy (morphology, adhesion) and fluorescence imaging of the actin cytoskeleton (morphology, proliferation). A repeated measures analysis was performed and p-values were adjusted by Tukey. While all sealers influenced the cell morphology and the expression of genes associated with apoptosis (Casp3), proliferation (histone H3), and inflammation (interleukin-6 and matrix metalloproteinases 1 and 3), mainly AH Plus® and Apexit® yielded a regular actin cytoskeleton and beneficial gene expression patterns. Regarding cell adhesion, only AH Plus® supported proper anchorage for alveolar osteoblasts. Our results provide evidence for the biocompatibility of epoxy resin-based endodontic sealers, i.e. AH Plus®, while other sealers proved cytotoxic for alveolar osteoblasts. Further studies are needed for understanding the bone cell reactions after endodontic treatment and the clinical decision-making regarding the sealer of choice for root canal fillings

The Skeletal Amino Acid Composition of the Marine Demosponge Aplysina cavernicola

It has been discovered during the past few years that demosponges of the order Verongida such as Aplysina cavernicola exhibit chitin-based skeletons. Verongida sponges are well known to produce bioactive brominated tyrosine derivatives. We could recently demonstrate that brominated compounds do not exclusively occur in the cellular matrix but also in the skeletons of the marine sponges Aplysina cavernicola and Ianthella basta. Our measurements imply that these yet unknown compounds are strongly, possibly covalently bound to the sponge skeletons. In the present work, we determined the skeletal amino acid composition of the demosponge A. cavernicola especially with respect to the presence of halogenated amino acids. The investigations of the skeletons before and after MeOH extraction confirmed that only a small amount of the brominated skeleton-bound compounds dissolves in MeOH. The main part of the brominated compounds is strongly attached to the skeletons but can be extracted for example by using Ba(OH)2. Various halogenated tyrosine derivatives were identified by GC-MS and LC-MS in these Ba(OH)2 extracts of the skeletons

Effect of high glucose levels and lipopolysaccharides‐induced inflammation on osteoblast mineralization over sandblasted/acid‐etched titanium surface

Background and Purpose Poorly controlled diabetes mellitus has been related to higher risk of implant treatment complications due to increased susceptibility to infection and delayed wound healing. Lipopolysaccharides (LPS) stimulate cytokine production leading to chronic inflammation and immunological host response that accentuates the destruction of periodontal tissues. This study aimed to evaluate the effect of different glycemic conditions on secretion and mineralization of bone matrix under sterile inflammation induced by LPS on osteoblasts seeded over sandblasted/acid‐etched (SLA) titanium surface. Materials and Methods Osteoblast cell viability was performed to determine the influence of different glucose concentrations (5.5, 8, 12, and 24 mM), which were chosen to reflect normal, postprandial, and high glucose values, similar to those typically seen in Diabetes mellitus under clinical conditions. Cells were seeded on titanium SLA discs (Straumann AG, Waldenburg, Switzerland) and exposed to glucose concentrations and LPS (1μg/mL) in order to test inflammatory response (qPCR) and mineralization (Alizarin Red staining). Results Osteoblast viability was severely decreased when exposed to higher glucose levels (≥12 mM) and LPS (P  .05). Osteoblasts exposed to LPS produced sparse extracellular matrix mineralization, especially combined with higher glucose values (≥12 mM), together with decreased calcium deposition compared to control (P < .05). Conclusions High glucose levels combined with LPS inflammatory stimulation elicited an adverse effect on the volume and quality of mineralized hard tissue formation on SLA titanium surfaces in vitro. Hence, both normal glucose levels and infection control including low levels of circulating LPS during initial osseointegration period may be required to increase the success rate of dental implants

Characterization of a Stemness-Optimized Purification Method for Human Dental-Pulp Stem Cells: An Approach to Standardization

Human dental pulp stem cells (hDPSCs) are promising for oral/craniofacial regeneration, but their purification and characterization is not yet standardized. hDPSCs from three donors were purified by magnetic activated cell sorting (MACS)-assisted STRO-1-positive cell enrichment (+), colony derivation (c), or a combination of both (c/+). Immunophenotype, clonogenicity, stemness marker expression, senescence, and proliferation were analyzed. Multilineage differentiation was assessed by qPCR, immunohistochemistry, and extracellular matrix mineralization. To confirm the credibility of the results, repeated measures analysis and post hoc p-value adjustment were applied. All hDPSC fractions expressed STRO-1 and were similar for several surface markers, while their clonogenicity and expression of CD10/44/105/146, and 166 varied with the purification method. (+) cells proliferated significantly faster than (c/+), while (c) showed the highest increase in metabolic activity. Colony formation was most efficient in (+) cells, which also exhibited the lowest cellular senescence. All hDPSCs produced mineralized extracellular matrix. Regarding osteogenic induction, (c/+) revealed a significant increase in mRNA expression of COL5A1 and COL6A1, while osteogenic marker genes were detected at varying levels. (c/+) were the only population missing BDNF gene transcription increase during neurogenic induction. All hDPSCs were able to differentiate into chondrocytes. In summary, the three hDPSCs populations showed differences in phenotype, stemness, proliferation, and differentiation capacity. The data suggest that STRO-1-positive cell enrichment is the optimal choice for hDPSCs purification to maintain hDPSCs stemness. Furthermore, an (immuno) phenotypic characterization is the minimum requirement for quality control in hDPSCs studies

Brominated Skeletal Components of the Marine Demosponges, Aplysina cavernicola and Ianthella basta: Analytical and Biochemical Investigations

Demosponges possess a skeleton made of a composite material with various organic constituents and/or siliceous spicules. Chitin is an integral part of the skeleton of different sponges of the order Verongida. Moreover, sponges of the order Verongida, such as Aplysina cavernicola or Ianthella basta, are well-known for the biosynthesis of brominated tyrosine derivates, characteristic bioactive natural products. It has been unknown so far whether these compounds are exclusively present in the cellular matrix or whether they may also be incorporated into the chitin-based skeletons. In the present study, we therefore examined the skeletons of A. cavernicola and I. basta with respect to the presence of bromotyrosine metabolites. The chitin-based-skeletons isolated from these sponges indeed contain significant amounts of brominated compounds, which are not easily extractable from the skeletons by common solvents, such as MeOH, as shown by HPLC analyses in combination with NMR and IR spectroscopic measurements. Quantitative potentiometric analyses confirm that the skeleton-associated bromine mainly withstands the MeOH-based extraction. This observation suggests that the respective, but yet unidentified, brominated compounds are strongly bound to the sponge skeletons, possibly by covalent bonding. Moreover, gene fragments of halogenases suggested to be responsible for the incorporation of bromine into organic molecules could be amplified from DNA isolated from sponge samples enriched for sponge-associated bacteria

Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation

International audienceAims: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF.Methods and results: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected.Conclusions: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins