Development of Bi- and Tri-Layer Nanofibrous Membranes Based on the Sulfated Polysaccharide Carrageenan for Periodontal Tissue Regeneration.

Periodontitis is a microbially-induced inflammation of the periodontium that is characterized by the destruction of the periodontal ligament (PDL) and alveolar bone and constitutes the principal cause of teeth loss in adults. Periodontal tissue regeneration can be achieved through guided tissue/bone regeneration (GTR/GBR) membranes that act as a physical barrier preventing epithelial infiltration and providing adequate time and space for PDL cells and osteoblasts to proliferate into the affected area. Electrospun nanofibrous scaffolds, simulating the natural architecture of the extracellular matrix (ECM), have attracted increasing attention in periodontal tissue engineering. Carrageenans are ideal candidates for the development of novel nanofibrous GTR/GBR membranes, since previous studies have highlighted the potential of carrageenans for bone regeneration by promoting the attachment and proliferation of osteoblasts. Herein, we report the development of bi- and tri-layer nanofibrous GTR/GBR membranes based on carrageenans and other biocompatible polymers for the regeneration of periodontal tissue. The fabricated membranes were morphologically characterized, and their thermal and mechanical properties were determined. Their periodontal tissue regeneration potential was investigated through the evaluation of cell attachment, biocompatibility, and osteogenic differentiation of human PDL cells seeded on the prepared membranes.

A Novel Approach to Chemical Mixture Risk Assessment-Linking Data from Population-Based Epidemiology and Experimental Animal Tests.

Humans are continuously exposed to chemicals with suspected or proven endocrine disrupting chemicals (EDCs). Risk management of EDCs presents a major unmet challenge because the available data for adverse health effects are generated by examining one compound at a time, whereas real-life exposures are to mixtures of chemicals. In this work, we integrate epidemiological and experimental evidence toward a whole mixture strategy for risk assessment. To illustrate, we conduct the following four steps in a case study: (1) identification of single EDCs ("bad actors")-measured in prenatal blood/urine in the SELMA study-that are associated with a shorter anogenital distance (AGD) in baby boys; (2) definition and construction of a "typical" mixture consisting of the "bad actors" identified in Step 1; (3) experimentally testing this mixture in an in vivo animal model to estimate a dose-response relationship and determine a point of departure (i.e., reference dose [RfD]) associated with an adverse health outcome; and (4) use a statistical measure of "sufficient similarity" to compare the experimental RfD (from Step 3) to the exposure measured in the human population and generate a "similar mixture risk indicator" (SMRI). The objective of this exercise is to generate a proof of concept for the systematic integration of epidemiological and experimental evidence with mixture risk assessment strategies. Using a whole mixture approach, we could find a higher rate of pregnant women under risk (13%) when comparing with the data from more traditional models of additivity (3%), or a compound-by-compound strategy (1.6%).

Psychometric and biohormonal indices of dental anxiety in children. A prospective cohort study.

The stress of dental treatment often elicits negative emotions in children, expressed as dental fear or anxiety. Highly anxious children obstruct treatment and avoid therapy, further amplifying oral health problems. The aim of this study was to examine the neuroendocrine and autonomic nervous system responses to dental treatment and their possible interactions and associations with psychometric indices of anxiety, caries, previous dental experience, anesthesia, age and gender in school children. Upon informed consent, saliva was obtained from 97 children (59% males, mean age ± SD: 89.73 ± 15 months) in the Clinic of pediatric dentistry before treatment, immediately post-treatment and at the recall visit to determine cortisol and salivary alpha-amylase (sAA) levels. Dental and general anxiety was assessed through specific questionnaires completed by the children. Compared to pre-treatment, cortisol levels were increased following treatment, while sAA levels were higher at the recall. Pre- and post-treatment cortisol and sAA responses were positively correlated. Dental and general anxiety questionnaire scores were also significantly correlated with each other. The integrated autonomic and neuroendocrine responses prior to treatment were correlated with state anxiety and those following treatment with dental anxiety. However, univariable and multivariable linear regression analysis associated post-treatment cortisol, but not sAA, levels with dental anxiety. No associations of cortisol or sAA responses with caries, age, gender, previous dental experience or anesthesia were detected. These data provide some evidence that both sAA and cortisol levels are altered in children in anticipation or during dental treatment, but only cortisol levels are associated to dental anxiety.

A human-relevant mixture of endocrine disrupting chemicals induces changes in hippocampal DNA methylation correlating with hyperactive behavior in male mice.

Humans are ubiquitously exposed to endocrine disrupting chemicals (EDCs), substances that interfere with endogenous hormonal signaling. Exposure during early development is of particular concern due to the programming role of hormones during this period. A previous epidemiological study has shown association between prenatal co-exposure to 8 EDCs (Mixture N1) and language delay in children, suggesting an effect of this mixture on neurodevelopment. Furthermore, in utero exposure to Mixture N1 altered gene expression and behavior in adult mice. In this study, we investigated whether epigenetic mechanisms could underlie the long term effects of Mixture N1 on gene expression and behavior. To this end, we analyzed DNA methylation at regulatory regions of genes whose expression was affected by Mixture N1 in the hippocampus of in utero exposed mice using bisulfite-pyrosequencing. We show that Mixture N1 decreases DNA methylation in males at three genes that are part of the hypothalamus-pituitary-adrenal (HPA) axis: Nr3c1, Nr3c2, and Crhr1, coding for the glucocorticoid receptor, the mineralocorticoid receptor, and the corticotropin releasing hormone receptor 1, respectively. Furthermore, we show that the decrease in Nr3c1 methylation correlates with increased gene expression, and that Nr3c1, Nr3c2, and Crhr1 methylation correlates with hyperactivity and reduction in social behavior. These findings indicate that an EDC mixture corresponding to a human exposure scenario induces epigenetic changes, and thus programming effects, on the HPA axis that are reflected in the behavioral phenotypes of the adult male offspring.

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

Endocrine-disrupting chemicals and behaviour: A high risk to take?

Early life exposure to endocrine-disrupting chemicals (EDCs) is considered a potential risk factor for aberrant brain development and the emergence of behavioral deficits. The purpose of this review is to summarize the toxic effects of bisphenol-A (BPA) and phthalate exposure during pre-, -post- or perinatal life on different types of behaviour in male and female rodents. Despite results not being always consistent, most probably due to methodological issues, it is highly probable that early life exposure to BPA or/and phthalates, affects various aspects of behaviour in the offspring. Adverse effects include: Increased levels of anxiety, altered exploratory behaviour, reduced social interaction or increased aggression and deficits in spatial or recognition learning and memory. These effects have been observed with a wide range of doses, in some cases even below the currently employed Tolerable Daily Intake dose for either BPA or phthalates.

In utero exposure to phthalates and reproductive toxicity in rodents.

Phthalates, widely used as plasticizers, are contained in many everyday products. Human biomonitoring studies detect their presence in biological fluids of a large part of the population worldwide. Maternal exposure during pregnancy has been related with aberrations in the reproductive growth of male infants. Rodent studies show that gestational exposure to single phthalates elicits reproductive toxicity in both sexes. Early aberrations include inhibition of gonadal sex determining gene expression and steroidogenesis, histopathology, and disturbed gametogenesis, leading later in life to dysfunctions in sperm production and oocyte reserves. Animal studies of in utero exposure to mixtures of phthalates, better mimicking human exposures, revealed analogous reproductive dysfunctions with the single compounds, but also indicated the combined actions and cumulative effects exerted by these chemicals. Further understanding the underlying mechanisms and the species differences in phthalate-induced reproductive toxicity will help to improve the risk assessment for human exposure to these toxicants.

Long term transcriptional and behavioral effects in mice developmentally exposed to a mixture of endocrine disruptors associated with delayed human neurodevelopment.

Accumulating evidence suggests that gestational exposure to endocrine disrupting chemicals (EDCs) may interfere with normal brain development and predispose for later dysfunctions. The current study focuses on the exposure impact of mixtures of EDCs that better mimics the real-life situation. We herein describe a mixture of phthalates, pesticides and bisphenol A (mixture N1) detected in pregnant women of the SELMA cohort and associated with language delay in their children. To study the long-term impact of developmental exposure to N1 on brain physiology and behavior we administered this mixture to mice throughout gestation at doses 0×, 0.5×, 10×, 100× and 500× the geometric mean of SELMA mothers' concentrations, and examined their offspring in adulthood. Mixture N1 exposure increased active coping during swimming stress in both sexes, increased locomotion and reduced social interaction in male progeny. The expression of corticosterone receptors, their regulator Fkbp5, corticotropin releasing hormone and its receptor, oxytocin and its receptor, estrogen receptor beta, serotonin receptors (Htr1a, Htr2a) and glutamate receptor subunit Grin2b, were modified in the limbic system of adult animals, in a region-specific, sexually-dimorphic and experience-dependent manner. Principal component analysis revealed gene clusters associated with the observed behavioral responses, mostly related to the stress axis. This integration of epidemiology-based data with an experimental model increases the evidence that prenatal exposure to EDC mixtures impacts later life brain functions.

Gestational exposure to an epidemiologically defined mixture of phthalates leads to gonadal dysfunction in mouse offspring of both sexes.

The increasing concern for the reproductive toxicity of abundantly used phthalates requires reliable tools for exposure risk assessment to mixtures of chemicals, based on real life human exposure and disorder-associated epidemiological evidence. We herein used a mixture of four phthalate monoesters (33% mono-butyl phthalate, 16% mono-benzyl phthalate, 21% mono-ethyl hexyl phthalate, and 30% mono-isononyl phthalate), detected in 1st trimester urine of 194 pregnant women and identified as bad actors for a shorter anogenital distance (AGD) in their baby boys. Mice were treated with 0, 0.26, 2.6 and 13 mg/kg/d of the mixture, corresponding to 0x, 10x, 100x, 500x levels detected in the pregnant women. Adverse outcomes detected in the reproductive system of the offspring in pre-puberty and adulthood included reduced AGD index and gonadal weight, changes in gonadal histology and altered expression of key regulators of gonadal growth and steroidogenesis. Most aberrations were apparent in both sexes, though more pronounced in males, and exhibited a non-monotonic pattern. The phthalate mixture directly affected expression of steroidogenesis as demonstrated in a relevant in vitro model. The detected adversities at exposures close to the levels detected in pregnant women, raise concern on the existing safety limits for early-life human exposures and emphasizes the need for re-evaluation of the exposure risk.

Early responses of human pulp to direct capping with resin adhesive systems and calcium hydroxide.

OBJECTIVE: Early responses of human pulp to Prime&Bond/phosphoric acid, Clearfil SE Bond, Clearfil S3 Bond and Dycal were investigated ex vivo. MATERIALS AND METHODS: The three adhesives, Dycal or buffer (DPBS) were applied directly onto the pulp of human teeth slices that were placed in culture for 4 days. Cell viability was monitored by the MTT assay during the culture period. After 4 days, tissue integrity was examined by hematoxylin-eosin staining. Vimentin levels were assessed by Western blotting. TUNEL assay was applied for apoptotic cell detection at specific pulp areas. RESULTS: Profound reduction of cell viability and tissue integrity was observed in adhesive-treated groups, while the impact of Dycal was found to be less harmful. Extended apoptosis was caused mostly by the Clearfil SE and Prime&Bond. All adhesives reduced Vimentin levels. SIGNIFICANCE: The study provides evidence that early pulp responses to direct capping with different adhesive systems or calcium hydroxide may vary significantly and underline the need for further studies in relevant ex vivo systems.

Developmental exposure to bisphenol A alters expression and DNA methylation of Fkbp5, an important regulator of the stress response.

Bisphenol A (BPA), an abundant endocrine disruptor, affects stress-responsiveness and related behaviors in children. In rats, perinatal BPA exposure modifies stress response in pubertal offspring via unknown mechanisms. Here we examined possible epigenetic modifications in the glucocorticoid receptor gene and its regulator Fkbp5 in hypothalamus and hippocampus of exposed offspring. We found increased DNA methylation of Fkbp5 and reduced protein levels in the hippocampus of exposed male rats. Similar effects were obtained in a male hippocampal cell line when exposed to BPA during differentiation. The estrogen receptor (ER) antagonist ICI 182,780 or ERß knock-down affected Fkbp5 expression and methylation similarly to BPA. Further, BPA's effect on Fkbp5 was abolished upon knock-down of ERß, suggesting a role for this receptor in mediating BPA's effects on Fkbp5. These data demonstrate that developmental BPA exposure modifies Fkbp5 methylation and expression in male rats, which may be related to its impact on stress responsiveness.

Bone regeneration in critical-size calvarial defects using human dental pulp cells in an extracellular matrix-based scaffold.

The rat calvarial defect is an established model to evaluate craniofacial bone regeneration using cell-scaffold biocomplexes. Dental pulp harbors stem cells with significant osteogenic properties. Extracellular matrix (ECM)-like scaffolds simulate the environment that cells observe in vivo. In the present study, we evaluated the osteogenic effect of a biocomplex of human dental pulp cells and a hyaluronic-based hydrogel scaffold in calvarial defects of immunocompetent rats. Dental pulp cells at the 2nd passage were characterized by flow cytometry, osteodifferentiated ex vivo for 4 days and the whole population was encapsulated in the synthetic ECM matrix. Cell vitality was verified 24 h upon encapsulation. 5 mm calvarial defects were created in 30 male rats and filled with the biocomplex, the scaffold alone, or left untreated. Histological evaluation at 8 weeks showed incomplete bone regeneration in all groups. The scaffold was not fully degraded and entrapped cells were detected in it. Histomorphometry showed statistically significant superior new bone formation in the biocomplex-treated group, compared to the two other groups. The present study provides evidence that the whole population of human dental pulp cells can advance bone healing when transplanted in immunocompetent animals and highlights the importance of proper scaffold degradation in cell-driven bioengineering treatments.

Effect of cytosine arabinoside on cerebellar neurofilaments during development: A sexual dimorphism.

Previous reports suggest that the resistance of neuronal cytoskeleton to drug toxicity may vary with age and gender. The aim of the present study was to assess the impact of cytosine arabinoside (AraC) treatment on neurofilament (NF) levels and phosphorylation status in the developing cerebellum of male, female and testosterone propionate (1.25 mg/rat)-androgenized female rats. AraC (200 mg/kg bw) was administered from postnatal day (PND) 14-16 and changes in the level and phosphorylation of NFs were detected at PND 16 by Western blot analysis. The drug had no effect in male pups, while it increased the non-phosphorylated NF subunits of medium and low molecular weight in females. Androgenization of females prevented the AraC-induced increase in NF subunits. The levels of estrogen receptor beta (ER-ß), known to mediate neuroprotective actions of estrogens in the brain, were significantly higher in the developing female cerebellum, as compared to males and androgenized females. These data show that the neurofilament cytoskeleton in the developing rat cerebellum exhibits resistance to AraC that appears sexually dimorphic. In young males the resistance is exemplified by a lack of responsiveness, whereas in juvenile females it is presented by an androgenization-sensitive NF upregulation.

Perinatal exposure to low-dose bisphenol A affects the neuroendocrine stress response in rats.

Bisphenol A (BPA) is an estrogen-mimicking endocrine disruptor. Early-life exposures to low doses of BPA exert long-lasting effects on animals' reproductive and brain physiology. However, little is known about the effects of BPA on the stress-response system. Given the interaction of sex and stress hormones, we examined the effect of a low perinatal BPA exposure on the function of the hypothalamic-pituitary-adrenal (HPA) axis at rest and upon application of acute stress. Throughout pregnancy and lactation rats received daily 40 µg BPA/kg body weight orally via cornflakes. We studied the effect of this low but chronic exposure to BPA in the male and female offspring at puberty. BPA exposure led to abnormal adrenal histology including reduced zona reticularis especially in male offspring, hyperplasia of zona fasciculata in both sexes, and increased adrenal weight in female offspring. BPA-treated females had increased basal corticosterone and reduced hypothalamic glucocorticoid receptors (GR) levels. Stressed BPA-exposed females exhibited anxiety-like behavioral coping, a less rigorous corticosterone response, and did not downregulate GR in the hypothalamus, compared with control females. BPA-exposed males exhibited a heightened corticosterone stress response compared with females; they also displayed increased pro-opiomelanocortin mRNA levels and retained the prestress levels of pituitary corticotropin-releasing hormone-receptor 1, compared with control males. We found that perinatal chronic exposure to a low dose of BPA perturbs the basal and stress-induced activity of the HPA axis in a sexually dimorphic manner at adolescence. Exposure to BPA might contribute to increased susceptibility to stress-related disorders in later life.