Morpho-functional effects of different universal dental adhesives on human gingival fibroblasts: an in vitro study.

To analyze the effects of four universal adhesives (Optibond Solo Plus-OB, Universal Bond-UB, Prime&Bond Active-PBA, FuturaBond M + -FB) on human gingival fibroblasts in terms of cytotoxicity, morphology and function. After in vitro exposure for up to 48 h, fibroblast viability was determined by the MTT assay determined, morphology by phase-contrast microscopy and migration by the scratch wound assay. Expression levels of IL1ß, IL6, IL8, IL10, TNFα and VEGF genes were assessed by RT-PCR and their protein production by Western blot analysis. Apoptosis and cell cycle were analyzed by flow cytometry. OB and UB induced early morphological changes on fibroblasts (3 h) with extended cell death at 24 h/48 h. Gene expression of collagen type I and fibronectin increased fivefold compared with controls, elastin disappeared and elastase increased threefold, indicating gingival tissue tended to become fibrotic. Only UB and OB increased gene expression of inflammatory markers: IL1ß at 3 and 48 h (up to about three times), IL6 and IL8 at 3 h (up to almost four times) which corresponded to the increase of the activated form NF-kB. All adhesives showed an effect on the functionality of fibroblasts with cytotoxic effect time and concentration dependent. Among all the OB and UB adhesives, they showed the greatest cell damage. The in-depth analysis of the effects of universal adhesives and possible functional effects represents an important information for the clinician towards choosing the most suitable adhesive system.

Effects of nicotine on porcine pre-pupertal sertoli cells: An in vitro study.

Smoke components, such as nicotine and its major metabolites, cross the blood-testis barrier and are detectable in the seminal plasma of both active smokers and individuals exposed to cigarette smoke. In vivo studies in a rat model have further demonstrated that nicotine exposure reduces the weight of the testis, as well as the number of spermatocytes and spermatids, and affects the ultrastructure of Sertoli cells (SC) - which serve as sentinels of spermatogenesis - causing intense germ cell sloughing in the tubular lumen that compromises offspring fertility. This study sought to determine the effects of nicotine on the viability and function of purified pig pre-pubertal SC. Nicotine exposure reduced the mRNA expression and protein levels of anti-Mullerian hormone (AMH) and inhibin B and impaired FSH-r sensitivity via the downregulation of FSH-r and aromatase gene expression compared to untreated SC. Overall, our study suggests that nicotine can significantly alter extracellular matrix and tight junction protein gene expression (e.g., laminin, integrin, and occludin), thus compromising cross-talk between the interstitial and tubular compartments and enhancing blood-testis barrier (BTB) permeability via downregulation of the mitogen-activated protein kinase (MAPK) pathway. These findings further elucidate a potential mechanism of action underlying nicotine exposure's detrimental effects on SC function in vivo.

In Vitro Anti-Inflammatory Effects of Phenolic Compounds from Moraiolo Virgin Olive Oil (MVOO) in Brain Cells via Regulating the TLR4/NLRP3 Axis.

Neuroinflammation is a feature of many classic neurodegenerative diseases. In the healthy brain, microglia cells are distributed throughout the brain and are constantly surveilling the central nervous system (CNS). In response to CNS injury, microglia quickly react by secreting a wide array of apoptotic molecules. Virgin olive oil (VOO) is universally recognized as a symbol of the Mediterranean diet. In the current study, using lipopolysaccharide (LPS)-stimulated BV2 microglia, the anti-inflammatory effects of VOO phenolic extracts from Moraiolo cultivar (MVOO-PE) were investigated. The results showed that low concentration of MVOO-PE prevented microglia cell death and attenuated the LPS-induced activation of toll-like receptor 4 (TLR4)/NOD-like receptor pyrin domain-containing-3 (NLRP3) signaling cascade. The levels of TLR4 and NF-kB were diminished, as well as NLRP3 inflammasome and interleukin-1ß (IL-1ß) production. Cyclooxygenase-2 (COX-2) isoenzyme and ionized calcium binding adaptor molecule 1 (Iba-1) inflammatory mediator were also reduced. By modulating the TLR4/NLRP3 axis, MVOO-PE pretreatment was able to significantly down-regulate the mRNA expression of inflammatory mediators and suppress the cytokine secretion. Finally, we showed protective effect of MVOO-PE in a transwell neuron-microglia co-culture system. In conclusion, these results suggest that MVOO-PE could exerts anti-inflammatory activity on brain cells and become a promising candidate for preventing several neuroinflammatory diseases.

Cytotoxicity of universal dental adhesive systems: Assessment in vitro assays on human gingival fibroblasts.

Universal adhesives are the most important innovation in restorative dentistry. They are composed of different monomers, solvents and fillers. The potential cytotoxic effect of these materials is an important scientific aspect in recent literature. The aim of this study was to determine, using different in vitro techniques, the cytotoxicity evaluation of seven universal enamel-dental adhesives on human gingival fibroblasts. For this purpose, seven universal dental enamel adhesives have been evaluated by in vitro cytotoxicity tests using direct contact tests (an unpolymerized and a polymerized method) and an indirect contact test: preparation of extracts. The polymerized method showed a cytotoxicity range from 36% (G-PremioBond, GPB) to 79% (FuturaBond M+, FB). With the unpolymerized direct methods the range was from 4% (Prime&Bond Active, PBA) to 40% (Ibond Universal, IB) for undiluted adhesives; generally passing to the major dilutions the test showed a strong inhibitory activity by all the adhesives. Whereas with the indirect method by diluting the extracts of all dental adhesives the cell viability increased. The data obtained from the work has shown a lower cytotoxic effect of Optibond Solo Plus (OB) and Adhesive Universal (AU) with more reliable results with the extracts technique. The choice of reliable in vitro cytotoxic technique could represent, in dental practice, an important aid for clinical procedures in the use of adhesive systems.

Biological, thermal and mechanical characterization of modified glass ionomer cements: The role of nanohydroxyapatite, ciprofloxacin and zinc l-carnosine.

The study evaluated the effects of 4 wt% nanohydroxyapatite (HA), 6 wt% zinc l-carnosine (MDA) and 1.5 wt% Ciprofloxacin (AB) on the mechanical, thermal and biological properties of glass ionomer cements (GIC). Filler and additive concentrations were selected after a previous study had tested single components and different percentages. Specimens included five silicon molds of each GIC cement for all tests. They were stored at room temperature for 24 h from specimen collection to analysis. Mechanical tests, calorimetric analysis, morphological investigation, antibacterial and cell viability assays were conducted. One-way analysis of variance (ANOVA) was used for data analysis with significance set at p < 0.05. Adding HA, MDA and AB to GICs modified their thermal, mechanical and microbiological properties. Polymerization increased. A slight decrease in the compressive strength of modified GICs was observed in dry condition (p < 0.05). Cement extracts affected cell viability in relation to extract dilution. Mechanical behavior improved in modified glass ionomer cements, especially with the powder formulated antibiotic. Overall cytotoxicity was reduced. Therefore adding nanohydroxyapatite, antibiotic and a mucosal defensive agent to conventional glass ionomer cement in special need patients could improve the clinical, preventive and therapeutic performance of the cements, without altering their mechanical properties.

Nicotine induces apoptosis in human osteoblasts via a novel mechanism driven by H2O2 and entailing Glyoxalase 1-dependent MG-H1 accumulation leading to TG2-mediated NF-kB desensitization: Implication for smokers-related osteoporosis.

Nicotine contained in cigarette smoke contributes to the onset of several diseases, including osteoporosis, whose emerging pathogenic mechanism is associated with osteoblasts apoptosis. Scanty information is available on the molecular mechanisms of nicotine on osteoblasts apoptosis and, consequently, on an important aspect of the pathogenesis of smokers-related osteoporosis. Glyoxalase 1 (Glo1) is the detoxification enzyme of methylglyoxal (MG), a major precursor of advanced glycation end products (AGEs), potent pro-apoptotic agents. Hydroimidazolone (MG-H1) is the major AGE derived from the spontaneous MG adduction of arginine residues. The aim of this study was to investigate whether, and by means of which mechanism, the antiglycation defence Glo1 was involved in the apoptosis induced by 0.1 and 1µM nicotine in human primary osteoblasts chronically exposed for 11 and 21 days. By using gene overexpression/silencing and scavenging/inhibitory agents, we demonstrated that nicotine induces a significant intracellular accumulation of hydrogen peroxide (H2O2) that, by inhibiting Glo1, drives MG-H1 accumulation/release. MG-H1, in turn, triggers H2O2 overproduction via receptor for AGEs (RAGE) and, in parallel, an apoptotic mitochondrial pathway by inducing Transglutaminase 2 (TG2) downregulation-dependent NF-kB desensitization. Measurements of H2O2, Glo1 and MG-H1 circulating levels in smokers compared with non-smokers or in smokers with osteoporosis compared with those without this bone-related disease supported the results obtained in vitro. Our findings newly pose the antiglycation enzymatic defense Glo1 and MG-H1 among the molecular events involved in nicotine-induced reactive oxygen species-mediated osteoblasts apoptosis, a crucial event in smoker-related osteoporosis, and suggest novel exposure markers in health surveillance programmes related to smokers-associated osteoporosis.

Variations in gene expression of lung macromolecules after induction chemotherapy for lung cancer.

OBJECTIVES: Preoperative chemotherapy may play a role in postoperative respiratory complications due to subclinical parenchymal damage. We investigated the gene expression of lung tissue components after neoadjuvant chemotherapy of alveolar-capillary membrane, extracellular matrix and membrane proteins. METHODS: The study group included 14 patients submitted to pulmonary resection for lung cancer after 3 cycles of gemcitabine-cisplatin, while the control group included 14 naive-treatment patients. RNA was extracted from frozen tissue obtained by healthy lung specimens using EZ1 RNA Universal Tissue kit and automatically purified by BioRobot EZ1 instrument. Three hundred nanograms of total RNA was reverse transcribed to complementary DNA and used to evaluate the gene expression of type I and III collagen, elastin, syndecan, metalloproteinase 13 and aquaporins (AQPs) in real-time polymerase chain reaction. Results were expressed as the mean ± standard deviation of 3 independent experiments. Analysis of variance followed by Sheffe's F-test was performed. RESULTS: Among the alveolar-capillary membrane and extracellular matrix genes, type I-III collagens and syndecan were significantly up-regulated (+645%, +327% and +261%, respectively), while elastin and metalloproteinase 13 were down-regulated in the study group versus control group (-46% and -77%, respectively). Furthermore, chemotherapy was associated with a significant up-regulation of AQP expressions (AQP1:+51% and AQP5:+36%). CONCLUSIONS: We observed, in the treated group, increases in the mean values of gene expressions for macromolecules involved in the remodelling of both the alveolar septa and parenchyma scaffold, thereby supporting the hypothesis that induction chemotherapy may foster a fibrosing effect on the pulmonary parenchyma and lead to altering the alveolar-capillary membrane.

Cytotoxicity of three commercial mouthrinses on extracellular matrix metabolism and human gingival cell behaviour.

This study evaluated the effects of commercially available antiseptic mouthrinses on human gingival fibroblast and keratinocyte behaviour and metabolism. Three mouthrinses containing essential oil (EO), chlorhexidine (CHX) and amine fluoride/stannous fluoride (AFSF), were tested in an in vitro study. Human gingival fibroblasts and keratinocytes were washed with 10% or 30% concentration of the commercial mouthrinses and their effects on cell adhesion and proliferation were investigated as well as the specific gene expression of markers involved in oral mucosa metabolism. As markers of cell metabolism, type I and IV collagens, laminin, fibronectin, fibromodulin and integrins were studied with real-time PCR. Moreover, interleukin-1 secretion, one of the major pro-inflammatory cytokines, was evaluated. The results showed that CHX significantly reduced fibroblast and keratinocyte substrate adhesion capacities and CHX and EO inhibited cell proliferation better than AFSF rinse. The gene expression of several matrix components and cell adhesion receptors was downregulated in cells washed with CHX and EO compared with those washed with AFSF rinse. In conclusion, the AFSF mouthrinse does not induce or induces to a lesser extent the onset of irritation and/or cytotoxicity than CHX or EO. These findings and those of future studies will enable us to gain further insight into the clinical significance and effects of commercial mouthrinses. Pending further investigations, clinicians should be aware of the potentially adverse effects of mouthrinses and warn their patients against making improper use of these products.

Sub-toxic nicotine concentrations affect extracellular matrix and growth factor signaling gene expressions in human osteoblasts.

Exposure to nicotine and other compounds contained in cigarette smoking affects human health. This study examined the effects of exposure to a single or multiple sub-toxic nicotine concentrations on human osteoblasts. Cell growth and expression of genes involved in bone differentiation, extracellular matrix (ECM) metabolism, and growth factor signaling pathways were investigated in nicotine-treated cells compared to untreated cells. Depending on osteoblast concentration and maturation stages, nicotine differently regulated cell growth. Real-time PCR showed regulated expressions of genes expressed by nicotine-treated osteoblasts compared to untreated cells. Among ECM genes, type I collagen was down-regulated and osteonectin was up-regulated in nicotine-treated osteoblasts; similarly, fibroblast growth factor-1 (FGF1) and fibroblast growth factor-2 (FGF2), two members of FGF signaling system, were discordantly modulated; genes involved in osteoblast maturation and differentiation such as alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2), and bone sialoprotein (BSP) were over-expressed after drug treatment. Our results show a positive association between nicotine exposure and osteoblast phenotype and illustrate for the first time a mechanism whereby acute or chronic exposure to sub-toxic nicotine concentrations may affect bone formation through the impairment of growth factor signaling system and ECM metabolism.

Diazepam effects on non-syndromic cleft lip with or without palate: epidemiological studies, clinical findings, genes and extracellular matrix.

IMPORTANCE OF THE FIELD: This review analyses international studies investigating the combined genetic and environmental causes of cleft lip with or without cleft palate (CL/P) and describes successes and limitations in identifying underlying genetic and environmental factors. CL/P, the most common congenital facial malformation, is a major public health burden in terms of medical costs and emotional stress to patients and families. Because genetic and environmental factors determine risk of occurrence, CL/P has a complex, multifactor aetiology. AREAS COVERED IN THIS REVIEW: English language reports from 1980 to 2010 were searched for in Medline, PubMed, Science Citation Index, textbooks and review articles on drugs and pregnancy. Key words were diazepam or benzodiazepine(s) combined with cleft lip, cleft palate, oral malformations, prenatal exposure, GABA, gene expression and extracellular matrix. WHAT THE READER WILL GAIN: This review presents an updated assessment of the mutagenic and genotoxic effects of diazepam (DZ), one of the most commonly used benzodiazepines, on CL/P occurrence. TAKE HOME MESSAGE: Data are divergent; more studies are needed for an in-depth picture of the effects of DZ during gestation on the child's development, particularly on orofacial clefts.

Effects of sub-toxic Cadmium concentrations on bone gene expression program: results of an in vitro study.

Since occupational and environmental exposure to the heavy metal Cadmium (Cd) affects human health this study investigated the effects of exposure to a single, or multiple, sub-toxic Cd concentrations on sub-confluent and confluent human osteoblast growth and expression of specific bone differentiation markers. RT-PCR quantified gene expression of type I collagen, metalloprotease (MMP13), runt-related transcription factor-2 (RUNX2), osterix, osteocalcin, osteonectin, alkaline phosphatase, integrins and bone sialoprotein (BSP). Expression of fibroblast growth factors 1 and 2 (FGF1, FGF2), transforming growth factor-beta(3) (TGFbeta(3)) and bone morphogenetic protein-2 (BMP2) were also evaluated to determine whether Cd-related effects were mediated by an imbalance in expression. Depending on osteoblast concentration and maturation stages, Cd inhibited or stimulated cell growth, decreased type I collagen, increased MMP13, FGF1 and BMP2 gene expression and stimulated the mineralization process only in continuously exposed cultures. These results suggest that in vivo, acute or chronic exposure to sub-toxic Cd concentrations may affect bone formation differently and support the hypothesis that Cd-induced bone disorders may involve downstream changes in growth factor expression. The results are of interest in forensic and occupational medicine in establishing preventive measures to reduce professional exposure risks.

Effects of hydroxyapatite and Biostite on osteogenic induction of hMSC.

When isolated from the iliac crest human mesenchymal stem cells (hMSC) differentiate into osteoblast-like cells with appropriate stimulation in culture. This in vitro study tested the hypothesis that Biostite and hydroxyapatite (HA) affect proliferation and differentiation of hMSC into osteoblastic cells. Cell proliferation was determined by measuring 3H-thymidine incorporation into DNA and typical markers of osteoblastic phenotype were determined by RT-PCR assay. No differences emerged in cell proliferation cultures with Biostite or hydroxyapatite (HA), but gene expression analysis revealed higher expression of collagen,alkaline phosphatase (ALP), osteopontin and bone sialoprotein (BSP) in the presence of Biostite. TGFb2 production, as assessed by an Elisa kit, and Runx2 expression by RT-PCR, were greater in Biostite cultures, suggesting Biostite provides a better environment for hMSC differentiation into osteoblasts and is, potentially, a more promising bone-filling material than HA.

Effects of titanium surface roughness on mesenchymal stem cell commitment and differentiation signaling.

PURPOSE: Human mesenchymal stem cells (hMSCs) are primary cells capable of differentiating to osteocytic lineage when stimulated under appropriate conditions. This study examined changes in hMSC morphology, proliferation, and gene expression after growth on machined or dual acid-etched (AE) titanium surfaces. MATERIALS AND METHODS: hMSCs, isolated from adult human bone marrow, were cultured on titanium surfaces. The two specimens of titanium surfaces in this study included machined and AE titanium disks. Cell morphology was evaluated by scanning electron microscopy, and cell proliferation and collagen synthesis were estimated by measuring the amount of 3H-thymidine incorporation into DNA and 3H-proline incorporation into collagen fibers. Alkaline phosphatase (ALP) activity was determined by measuring the release of p-nitrophenol from disodium p-nitrophenyl phosphate. Changes in gene expression for bone morphogenetic protein-2 (BMP-2), Runx2 type II, Osterix (Osx), osteopontin, type I collagen, ALP, osteocalcin, and bone sialoprotein were determined by reverse-transcriptase polymerase chain reaction after 22 days of in vitro culture in osteogenic medium. RESULTS: The two substrates had no significant effects on cell adhesion and proliferation. Morphologic characteristics were observed by scanning electron microscopy. hMSCs on the machined surface spread more and were flatter than cells cultured on the AE surface. Osteopontin mRNA expression was similar on all surfaces, and the other mRNA transcripts were increased in hMSC cultured on AE surface. In particular, BMP-2, Runx2, and Osx, three osteogenic factors that induce the progressive differentiation of multipotent mesenchymal cells into osteoblasts, were expressed more on AE titanium than on machined titanium. Collagen and ALP assays confirmed the highest level of mRNA transcripts correlated with increases in these proteins. CONCLUSION: These results showed that an AE titanium surface stimulated the expression of markers of osteoblastic phenotype more than a machined titanium surface.

Patterns of some extracellular matrix gene expression are similar in cells from cleft lip-palate patients and in human palatal fibroblasts exposed to diazepam in culture.

Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFbeta(3)) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences.

Differences in extracellular matrix production and basic fibroblast growth factor response in skin fibroblasts from sporadic and familial Alzheimer's disease.

Extracellular matrix (ECM) molecules and growth factors, such as fibroblast growth factor (FGF), play a crucial role in Alzheimer's disease (AD). The purpose of this investigation was to determine whether phenotypic alterations in ECM production are present in non-neuronal AD cells associated with different FGF expression and response. Synthesis of glycosaminoglycans (GAG) and collagen were measured in skin fibroblasts from patients with familial, sporadic AD (FAD and SAD respectively), and from age-matched controls by radiolabeled precursors. Proteoglycans (PG), metalloprotease (MMP)-1, and FGF gene expressions were measured by reverse transcription-polymerase chain reaction. The results showed different ECM neosynthesis and mRNA levels in the two AD fibroblast populations. FAD accumulated more collagen and secreted less GAG than SAD. Biglycan PG was upregulated in FAD while betaglycan, syndecan, and decorin were markedly downregulated in SAD fibroblasts. We found a significant decrease of MMP1, more marked in FAD than in SAD fibroblasts. Constitutive FGF expression was greatly reduced in both pathological conditions (SAD>FAD). Moreover, an inverse high affinity/low affinity FGF receptor ratio between SAD and FAD fibroblasts was observed. FGF treatment differently modulated ECM molecule production and gene expression in the two cell populations. These observations in association with the changes in FGF gene expression and in the FGF receptor number, suggest that cellular mechanisms downstream from FGF receptor binding are involved in the two different forms of AD.

Effect of titanium surface roughness on human osteoblast proliferation and gene expression in vitro.

PURPOSE: Cell proliferation and extracellular matrix formation are primary events in bone formation. At the dental implant-tissue interface, implant surface roughness modulates osteoblast functions. The aim of the present in vitro study was to investigate the effect of varying surface roughness of titanium implant material on cell proliferation and mRNA expression of specific markers of osteoblast phenotype. MATERIALS AND METHODS: Primary cultures of osteoblasts derived from human mandibular bone were cultured on titanium surfaces. Three titanium surfaces were studied: machined titanium, microsandblasted titanium, and macro-sandblasted titanium (average surface roughnesses of 0.5 and 3 microm, respectively). Cell morphology was estimated by scanning electron microscope analysis and cell proliferation by measuring the amount of 3H-thymidine incorporation into DNA. mRNA expression of osteonectin, osteopontin, bone sialoprotein (BSP), and Runx2, which are markers of osteoblastic phenotype, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Human osteoblasts cultured on machined titanium spread more and were flatter than cells cultured on rough titanium. All blasted surfaces showed significantly higher DNA synthesis than the machined surfaces. Osteonectin mRNA expression was similar on all surfaces. Other mRNA transcripts were increased in osteoblasts cultured on rough titanium surfaces, particularly the macrosandblasted surface. CONCLUSIONS: An average surface roughness of 3 microm (macro-sandblasted titanium) is more suitable than an average surface roughness of 0.5 microm (micro-sandblasted titanium) in favoring osteoblast differentiation in vitro.

Silica, hyaluronate, and alveolar macrophage functional differentiation.

BACKGROUND: Silicosis is mediated by macrophages, their soluble mediators, and extracellular matrix molecules. In this study, we investigated the effects of silica and/or hyaluronate (HA) on several alveolar macrophage responses. METHODS: We evaluated glycosaminoglycan (GAG) production by radiolabeled precursors, nitric oxide (NO) release by its oxidation product, phagocytic activity by Candida albicans internalization, and the secretion of two fibrogenic cytokines, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta, by specific assays. RESULTS: Silica significantly reduced GAG secretion, particularly HA secretion. Alone, it decreased Candida uptake; associated with HA, it enhanced the reduction. Silica and Candida reduced NO release, which was not significantly affected when silica- or Candida-exposed cells were also treated with HA. TNF-alpha and TGF-beta activities were stimulated by silica but reduced by HA. CONCLUSIONS: The results suggest that silica and HA modify alveolar macrophage functional differentiation. Silica- and HA-induced modifications of the microenvironment could determine whether the response proceeds toward healing and repair or toward lung chronic pathology.