Diffusion of monomer compounds through dentin in conditions of simulated pulpal pressure

Dental biomaterials are not inert in the environment of the oral cavity. Some of their constituents are able to leach from the matrix of the material and cause adverse effects to the surrounding tissues and to remote body organs. From this point of view, some of the constituents of the dental biomaterials are able to penetrate through the remaining dentin, reach the pulp tissue and cause biologic responses of the pulp tissue.Aim: The aim of this in vitro study is the identification and the quantitative analysis of the compounds HEMA (2-Hydroxyethyl methacrylate), ΒΡΑ (bisphenol A), TEGDMA (Triethylene glycol dimethacrylate), UDMA (Urethane dimethacrylate) and BisGMA (Bisphenol A glycerolate dimethacrylate), in the pulp chamber of human teeth, through the remaining dentin.Materials and Methods: For the determination of the compounds HEMA, ΒΡΑ, TEGDMA, UDMA and BisGMA two suitable HPLC methods were developed. Four pilot studies were also conducted to study: i) The ability of the compounds HEMA, ΒΡΑ, TEGDMA, UDMA and BisGMA to cross through the remaining dentin. ii) The elution of the compounds HEMA, ΒΡΑ, TEGDMA, UDMA and BisGMA from two commercial composite resin cements and iii) the identification of these compounds in the pulp chamber. Methanol and Ringer’s solution were used as elution media. iv) The effect of three different treatments of the dentin disk, on the permeability of the examined compounds.Four kinds of specimens were prepared and finally, considering the information that was gathered from the pilot studies, a new in vitro model, suitable for permeability studies was developed. The new model simulates the positive pulpal pressure of natural teeth, utilizes a dentin disk of uniform thickness, resembles the clinical conditions of cementation and allows the calculation of the exact quantity of the resin cement that it is placed on each specimen. The model is also suitable for long term studies and requires no sophisticated equipment. v) The effect of time on the concentrations of the compounds that were identified through dentin was also examined. Twenty specimens were constructed and were randomly divided in two groups. The specimens were filled with Ringer’s solution and then the resin cements Multilink Automix (Ivoclar, Vivadent) and Variolink II (Ivoclar, Vivadent) were applied on group A (N=10) and group B (N=10) respectively. At 5 min, 20 min, 1 h, 2 h, 21 h, 3 d, 7 d, 10 d and 21 d time intervals, the whole eluate was retrieved and replaced with fresh Ringer’s solution. Each eluate was analyzed by High Performance Liquid Chromatography (HPLC).Results: The results from the pilot studies were: i) All the examined compounds are able to cross the remaining dentin. However the compounds HEMA, BPA and TEGDMA can cross dentin faster than UDMA and BisGMA, as they were identified in the pulp chamber eluate at the end of the 1 h time interval, after the application of a 100 mg/L driving concentration. On the other hand, UDMA and BisGMA, were identified in the pulp chamber eluate at the end of the 3 h time interval and after the serial application of 100 mg/L, 250 mg/L and 500 mg/L high concentrated solutions. ii) The composite resin cement Multilink Automix elutes HEMA, BPA, UDMA and BisGMA and the composite resin cement Variolink II elutes TEGDMA, BPA, UDMA, BisGMA, in Ringer’s solution elution media. The same compounds were also detected in methanol elution media, but several unknown compounds were also detected. iii) Through the remaining dentin, after the treatment of the specimens with Multilink Automix cement, ΗΕΜΑ and BisGMA were detected when the elution media was methanol, but when the elution media was Ringer’s solution, only HEMA was detected, in lower concentrations. After the treatment of the specimens with Variolink II cement, TEGDMA, UDMA and BisGMA were detected when the elution media was methanol, but when the elution media was Ringer’s solution TEGDMA and UDMA were detected in lower concentrations. iv)The different ways of treatment of the dentin disk didn’t alter the diffusion pattern of the permeability of the compounds through the remaining dentin. Higher concentrations of the compounds were detected when the dentin disk was treated with EDTA. v) Finally, time had a significant effect on the concentrations of the examined compounds: The higher concentrations of HEMA were detected on 21 h, 5 min, 20 min, 1 h and 3 d time intervals, the higher concentrations of TEGDMA on 3 d, 7 d and 21 h time intervals and the higher concentrations of UDMA were detected on 3 d, 7 d and 21 h time intervals. Conclusions: All the examined compounds are able to penetrate through the remaining dentin in the pulp chamber, but when commercial resin cements were applied, only some of them were detected through dentin. The concentrations of the compounds that were detected from two resin cements through a 0.85 mm dentin disk, under a pulpal pressure of 14.1 cmH2O, did not reach toxic levels for the pulp. Time had a statistical significant effect on the concentrations of HEMA, TEGDMA and UDMA compounds.Οδοντιατρικά βιοϋλικά που εκλύουν ενώσεις σε σημαντικές συγκεντρώσεις είναι πιθανόν να προκαλέσουν βιολογικές αντιδράσεις. Ορισμένες από τις ενώσεις αυτές είναι ικανές να διέλθουν στον πολφικό θάλαμο των δοντιών, διαμέσου της οδοντίνης και να προκαλέσουν βιολογικές αντιδράσεις από τους πολφικούς ιστούς.Σκοπός: Ο σκοπός της συγκεκριμένης μελέτης είναι ο ποιοτικός και ποσοτικός προσδιορισμός των ενώσεων HEMA (2-Hydroxyethyl methacrylate), ΒΡΑ (bisphenol A), TEGDMA (Triethylene glycol dimethacrylate), UDMA (Urethane dimethacrylate) και BisGMA (Bisphenol A glycerolate dimethacrylate), στον πολφικό θάλαμο ανθρώπινων δοντιών διαμέσου της οδοντίνης σε συνθήκες in vitro.Υλικά και Μέθοδοι: Αναπτύχθηκαν δύο χρωματογραφικές μέθοδοι κατάλληλες για τον προσδιορισμό των εξεταζόμενων ενώσεων. Διεξήχθησαν τέσσερις προκαταρκτικές μελέτες στις οποίες εξετάστηκαν: i) Η ικανότητα των ενώσεων HEMA, ΒΡΑ, TEGDMA, UDMA και BisGMA να διέρχονται διαμέσου της οδοντίνης. ii) Η έκλουση των ενώσεων HEMA, ΒΡΑ, TEGDMA, UDMA και BisGMA από δύο εμπορικά σκευάσματα σύνθετων ρητινωδών κονιών και iii) η διέλευσή τους μέσω της υπολειμματικής οδοντίνης στον πολφικό θάλαμο. Ως μέσο έκλουσης χρησιμοποιήθηκε η μεθανόλη και το διάλυμα Ringer. iv) Εξετάστηκε επίσης η επίδραση που μπορεί να έχουν τρεις διαφορετικές κατεργασίες της οδοντίνης στη διέλευση των ενώσεων HEMA, ΒΡΑ, TEGDMA, UDMA και BisGMA. Για τη διεξαγωγή των παραπάνω μελετών χρησιμοποιήθηκαν τέσσερα διαφορετικά είδη δοκιμίων. Συνοψίζοντας τα αποτελέσματα των προκαταρκτικών μελετών, κατασκευάστηκε ένα νέο πειραματικό, in vitro μοντέλο, κατάλληλο για μελέτες διαπερατότητας της οδοντίνης. Το μοντέλο αυτό εξομοιώνει την πολφική πίεση, χρησιμοποιεί συγκεκριμένο πάχος δίσκου οδοντίνης, παρομοιάζει αδρά τις συνθήκες συγκόλλησης με αυτές που επικρατούν στη στοματική κοιλότητα με τη χρήση σταθερής δύναμης 25 Ν, και δίνει τη δυνατότητα υπολογισμού της ποσότητας της κονίας που τοποθετείται στο κάθε δοκίμιο. v) Η κυρίως μελέτη αφορούσε τον ποσοτικό προσδιορισμό και τη μελέτη της επίδρασης του χρόνου στις συγκεντρώσεις των εξεταζόμενων ενώσεων που ανιχνεύθηκαν διαμέσω της οδοντίνης. Κατασκευάστηκαν είκοσι δοκίμια που χωρίστηκαν τυχαία σε δύο ομάδες των δέκα. Στην ομάδα Α χρησιμοποιήθηκε η ρητινώδης κονία Multilink Automix (Ivoclar, Vivadent) και στη Β ομάδα χρησιμοποιήθηκε η κονία Variolink II (Ivoclar, Vivadent). Τα δοκίμια πληρώθηκαν με διάλυμα Ringer και το υγρό πλήρωσης των δοκιμίων αφαιρέθηκε εξ’ ολοκλήρου και αντικαταστάθηκε με καινούριο σε εννέα χρονικές στιγμές: 5 min, 20 min, 1 h, 2 h, 21 h, 3 d, 7 d, 10 d, 21 d. Τα δείγματα που προέκυψαν από τη μελέτη του καθενός από τα 20 δοκίμια στις 9 χρονικές στιγμές, αναλύθηκαν με τη τεχνική της HPLC. Η στατιστική ανάλυση έγινε με τη μέθοδο ANOVA repeated measures.Αποτελέσματα: i) Διαπιστώθηκε ότι όλες οι εξεταζόμενες ενώσεις μπορούν να διέλθουν διαμέσου της οδοντίνης. Ωστόσο, οι ενώσεις HEMA, BPA και TEGDMA φαίνεται να διέρχονται γρηγορότερα ενώ απαιτείται και μικρότερη αρχική συγκέντρωση, καθώς πέρασαν από την εναπομένουσα οδοντίνη στη μεθανόλη του πολφικού θαλάμου στο διάστημα της 1 h από την εφαρμογή του πυκνού διαλύματος των 100 mg/L. Οι ενώσεις UDMA και BisGMA ανιχνεύθηκαν μετά από 3 h και μετά τη διαδοχική εφαρμογή πυκνών διαλυμάτων έως 500 mg/L. ii) Διαπιστώθηκε ότι οι ρητινώδεις κονίες Multilink Automix και Variolink II εκλύουν HEMA, BPA, UDMA, BisGMA και TEGDMA, BPA, UDMA, BisGMA αντίστοιχα, όταν τοποθετηθούν σε διάλυμα Ringer. Οι ίδιες ενώσεις, εκτός του ΒΡΑ, εκλύονται και μετά την τοποθέτηση των κονιών σε μεθανόλη, όμως παράλληλα ανιχνεύονται και ορισμένες άγνωστες ενώσεις. iii) Όταν ως μέσο έκλουσης χρησιμοποιείται η μεθανόλη, μετά από την εφαρμογή της κονίας Multilink Automix διαμέσου του φραγμού οδοντίνης ανιχνεύεται ΗΕΜΑ και BisGMA ενώ όταν εφαρμόζεται η κονία Variolink II ανιχνεύονται TEGDMA, UDMA και BisGMA. Τα αποτελέσματα αλλάζουν όταν χρησιμοποιείται ως μέσο έκλουσης το διάλυμα Ringer. Τότε, διαμέσου του φραγμού οδοντίνης, ανιχνεύεται μόνο ΗΕΜΑ όταν χρησιμοποιείται η κονία Multilink Automix και TEGDMA και UDMA όταν χρησιμοποιείται η κονία Variolink II. iv) Οι κατεργασίες που εφαρμόστηκαν στο δίσκο οδοντίνης δεν είχαν κάποια καταλυτική επίδραση ως προς το γενικό μοντέλο διόδου των εξεταζόμενων συγκεντρώσεων. Ωστόσο, υψηλότερες συγκεντρώσεις παρατηρήθηκαν στην περίπτωση που ο δίσκος οδοντίνης κατεργάστηκε με EDTA. Τέλος, v) η μελέτη της επίδρασης του χρόνου έδειξε ότι υπάρχουν στατιστικά σημαντικές διαφορές στις τιμές των συγκεντρώσεων των ενώσεων HEMA, TEGDMA και UDMA εξαιτίας του χρόνου. Για το ΗΕΜΑ οι υψηλότερες τιμές παρατηρήθηκαν στις 21 h, 5 min, 20 min, 1 h και 3 d, για το TEGDMA στις 3 d, 7 d, 21 h και για το UDMA στις 3 d, 7 d, 21 h. Συμπεράσματα: Όλες οι εξεταζόμενες ενώσεις έχουν την ικανότητα να διέλθουν διαμέσου της οδοντίνης στον πολφικό θάλαμο, ωστόσο από τις συγκεκριμένες ρητινώδεις κονίες ανιχνεύθηκαν στον πολφικό θάλαμο ορισμένες μόνο απ’ αυτές. Οι συγκεντρώσεις των ενώσεων που ανιχνεύθηκαν στον πολφικό θάλαμο με το συγκεκριμένο πειραματικό μοντέλο, που χρησιμοποιεί πάχος δίσκου οδοντίνης 0,85 mm και πολφική πίεση 14,1 cmH2O, ήταν σε επίπεδα χαμηλότερα από αυτά που θεωρούνται τοξικά και παρουσιάζουν πτωτική πορεία σε σχέση με το χρόνο. Ο χρόνος έχει στατιστικά σημαντική επίδραση στις συγκεντρώσεις των ενώσεων HEMA, TEGDMA και UDMA

Fermentation Supernatants of Pleurotus eryngii Mushroom Ameliorate Intestinal Epithelial Barrier Dysfunction in Lipopolysaccharide-Induced Caco-2 Cells via Upregulation of Tight Junctions

In recent years, modulation of gut microbiota through prebiotics has garnered interest as a potential to ameliorate intestinal barrier dysfunction. The aim of the study was to examine the in vitro effect of fermentation supernatants (FSs) from rich in β-glucan Pleurotus eryngii mushrooms on the expression levels of tight junctions (TJs) genes in Caco-2 cells stimulated by bacterial lipopolysaccharides (LPS). Mushrooms were fermented using fecal inocula in an in vitro batch culture model. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre-incubation with FS, co- and post-incubation. Reverse transcription PCR was applied to measure the expression levels of zonulin-1, occludin and claudin-1 genes. FSs from P. eryngii mushrooms led to a significant upregulation of the TJs gene expression in pre-incubation state, indicating potential preventive action. Down-regulation of all TJs gene expression levels was observed when the cells were challenged with LPS. The FS negative control (gut microbiota of each donor with no carbohydrate source) exhibited a significant upregulation of TJs expression levels compared to the cells that were challenged with LPS, for all three conditions. Overall, our data highlighted the positive and potential protective effects of P. eryngii mushrooms in upregulation of TJs’ genes.Funding agency:Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation under the call RESEARCH-CREATE-INNOVATE T1EDK-03404 </p

In Vitro Fermentation of Edible Mushrooms: Effects on Faecal Microbiota Characteristics of Autistic and Neurotypical Children

Children with autism spectrum disorder (ASD) often suffer gastrointestinal disturbances consistent with gut microbiota (GM) alterations. Treatment with pro/prebiotics may potentially alleviate gut symptoms, but the evidence for prebiotics is scarce. This study aims to evaluate the effects of edible mushrooms (Pleurotus, Basidiomycota) and prebiotic compounds on GM composition and metabolite production in vitro, using faecal samples from autistic and non-autistic children. Specific microbial populations were enumerated after 24 h of fermentation by quantitative PCR, and the metabolic production was determined by gas chromatography. Higher levels of Prevotella spp. and Bifidobacterium spp. were measured in neurotypical children compared to ASD children. A total of 24 h fermentation of Pleurotus eryngii and P. ostreatus mushroom powder increased the levels of Bifidobacterium, while known prebiotics increased the levels of total bacteria and Bacteroides in both groups. Only P. eryngii mushrooms resulted in significantly elevated levels of total bacteria Bacteroides and Feacalibacterium prausnitzii compared to the negative control (NC) in the ASD group. Both mushrooms induced elevated levels of butyrate after 24 h of fermentation, while short-chain fructooligosaccharides induced increased levels of acetate in the ASD group, compared to NC. Overall, this study highlights the positive effect of edible mushrooms on the GM and metabolic activity of children with ASD

In Vitro Fermentation of Edible Mushrooms: Effects on Faecal Microbiota Characteristics of Autistic and Neurotypical Children

Children with autism spectrum disorder (ASD) often suffer gastrointestinal disturbances consistent with gut microbiota (GM) alterations. Treatment with pro/prebiotics may potentially alleviate gut symptoms, but the evidence for prebiotics is scarce. This study aims to evaluate the effects of edible mushrooms (Pleurotus, Basidiomycota) and prebiotic compounds on GM composition and metabolite production in vitro, using faecal samples from autistic and non-autistic children. Specific microbial populations were enumerated after 24 h of fermentation by quantitative PCR, and the metabolic production was determined by gas chromatography. Higher levels of Prevotella spp. and Bifidobacterium spp. were measured in neurotypical children compared to ASD children. A total of 24 h fermentation of Pleurotus eryngii and P. ostreatus mushroom powder increased the levels of Bifidobacterium, while known prebiotics increased the levels of total bacteria and Bacteroides in both groups. Only P. eryngii mushrooms resulted in significantly elevated levels of total bacteria Bacteroides and Feacalibacterium prausnitzii compared to the negative control (NC) in the ASD group. Both mushrooms induced elevated levels of butyrate after 24 h of fermentation, while short-chain fructooligosaccharides induced increased levels of acetate in the ASD group, compared to NC. Overall, this study highlights the positive effect of edible mushrooms on the GM and metabolic activity of children with ASD

Immunomodulating Activity of Pleurotus eryngii Mushrooms Following Their In Vitro Fermentation by Human Fecal Microbiota

Recent studies have revealed the crucial role of several edible mushrooms and fungal compounds, mainly polysaccharides, in human health and disease. The investigation of the immunomodulating effects of mushroom polysaccharides, especially β-glucans, and the link between their anticancer and immunomodulatory properties with their possible prebiotic activity on gut micro-organisms has been the subject of intense research over the last decade. We investigated the immunomodulating effects of Pleurotus eryngii mushrooms, selected due to their high β-glucan content, strong lactogenic effect, and potent geno-protective properties, following in vitro fermentation by fecal inocula from healthy elderly volunteers (&gt;60 years old). The immunomodulating properties of the fermentation supernatants (FSs) were initially investigated in U937-derived human macrophages. Gene expression as well as pro-(TNF-α, IL-1β) and anti-inflammatory cytokines (IL-10, IL-1Rα) were assessed and correlated with the fermentation process. The presence of P. eryngii in the fermentation process led to modifications in immune response, as indicated by the altered gene expression and levels of the cytokines examined, a finding consistent for all volunteers. The FSs immunomodulating effect on the volunteers’ peripheral blood mononuclear cells (PBMCs) was verified through the use of cytometry by time of flight (CyTOF) analysis. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

In Vitro Fermentation of <i>Pleurotus eryngii</i> Mushrooms by Human Fecal Microbiota: Metataxonomic Analysis and Metabolomic Profiling of Fermentation Products

Edible mushrooms contain biologically active compounds with antioxidant, antimicrobial, immunomodulatory and anticancer properties. The link between their anticancer and immunomodulatory properties with their possible prebiotic activity on gut micro-organisms has been the subject of intense research over the last decade. Lyophilized Pleurotus eryngii (PE) mushrooms, selected due to their strong lactogenic effect and anti-genotoxic, immunomodulatory properties, underwent in vitro static batch fermentation for 24 h by fecal microbiota from eight elderly apparently healthy volunteers (>65 years old). The fermentation-induced changes in fecal microbiota communities were examined using Next Generation Sequencing of the hypervariable regions of the 16S rRNA gene. Primary processing and analysis were conducted using the Ion Reporter Suite. Changes in the global metabolic profile were assessed by 1H NMR spectroscopy, and metabolites were assigned by 2D NMR spectroscopy and the MetaboMiner platform. PLS-DA analysis of both metataxonomic and metabolomic data showed a significant cluster separation of PE fermented samples relative to controls. DEseq2 analysis showed that the abundance of families such as Lactobacillaceae and Bifidobacteriaceae were increased in PE samples. Accordingly, in metabolomics, more than twenty metabolites including SCFAs, essential amino acids, and neurotransmitters discriminate PE samples from the respective controls, further validating the metataxonomic findings

Global Sensory Impairment in Older Adults in the United States

OBJECTIVES: Age-related decline of the five classical senses (vision, smell, hearing, touch, and taste) poses significant burdens on older adults. The co-occurrence of multiple sensory deficits in older adults is not well characterized and may reflect a common mechanism resulting in global sensory impairment. DESIGN, SETTING, AND PARTICIPANTS: The National Social Life, Health, and Aging Project, a representative, population-based study of community dwelling older US adults (57-85 years of age), collected biomarkers, social and health history, and other physiological measures, including sensory function. MEASUREMENTS: We estimated the frequency with which impairment co-occurred across the five senses as an integrated measure of sensory aging. We hypothesized that multisensory deficits would be common and reflect global sensory impairment which would largely explain the effects of age, gender, and race on sensory dysfunction. RESULTS: Two thirds (67%) of the older US population suffer from two or more sensory deficits, 27% from just one, and only 6% had none. Impairment of the sense of taste was the most common (74%); 70% had a poor sense of touch; 22% had poor sense of smell; 20% had impaired corrected vision; and 18% had poor corrected hearing. Older adults, men, African Americans, and Hispanics had greater multisensory impairment (all P<0.01). Global sensory impairment largely accounted for the effects of age, gender, and race on the likelihood of impairment of each of the five senses. CONCLUSION: Multisensory impairment is prevalent in older US adults. These data support the concept of a common process that underlies sensory aging across the five senses. Clinicians assessing patients with a sensory deficit should consider further evaluation for additional cooccurring sensory deficits