MMP-2 assay within the hybrid layer created by a two-step etch-and-rinse adhesive: biochemical and immunohistochemical analysis.

OBJECTIVE: Degradation of hybrid layers (HLs) within resin-infiltrated dentine results from multiple degradation factors, including collagenolytic activity of specific matrix metalloproteinases (MMPs). Inhibition of host-derived MMPs may, therefore, slow the degradation of HL. The null hypothesis tested is that the presence of MMP-2 is similar regardless of chlorhexidine (CHX) pre-treatment or the use of an adhesive. METHODS: Powdered dentine prepared from extracted human teeth was divided into 4 groups: (G1) mineralised powder (control group); (G2) dentine powder treated with 1% phosphoric acid for 1 min; (G3) 1% phosphoric acid-etched dentine treated with Adper Scotchbond 1 XT (SB1XT; 3M ESPE); (G4) 1% phosphoric acid-etched dentine treated with 0.2% CHX followed by SB1XT. The concentration of detectable pro-MMP-2 and MMP-2 was assayed using a colorimetric assay system (QuantiSir). In addition, the presence of MMP-2 in the HL was assessed in 1 year-aged adhesive-dentine interfaces using an immunohistochemical approach under FEI-SEM/TEM. RESULTS: In dentine powder treated with 1% phosphoric acid (G2), MMP-2 level decreased compared to controls (G1); the application of SB1XT (G3) resulted in an increase of MMP-2, whilst 0.2% CHX before SB1XT application (G4), reduced MMP-2. The FEI-SEM/TEM analysis revealed MMP-2 distribution within the HL of aged interfaces showing increase MMP-2 patterns in the control group and minor labelling in the CHX-pretreated specimens. CONCLUSION: The results of this study support the use of non-toxic MMPs inhibitors, such as CHX, as an appropriate additional step in bonding procedures in order to increase the longevity of the adhesive restorations.

Immunohistochemical and biochemical assay of MMP-3 in human dentine.

OBJECTIVE: The function of endogenous MMP-3 and its distribution within the human dentine is unclear. Thus, the aim of the present study was to assay the presence and distribution of MMP-3 within human sound dentine by means of biochemical and immunohistochemical assays. METHODS: Powdered dentine from extracted human teeth was prepared and (1) partially demineralised with 1% H(3)PO(4) for 10min or (2) untreated (control). The presence of MMP-3 was measured using a colorimetric assay system (QuantiSir™, Epigentek, USA). Additional cryo-fractured dentine fragments were processed for immunohistochemical identification of MMP-3 under FEI-SEM. Casein-zymography was used to investigate MMP-3 activity. RESULTS: MMP-3 detected level was 2.732ng/µL in partially demineralised dentine powder, whilst it increased to 3.280ng/µL in mineralised dentine. The FEI-SEM analysis revealed positive immunolabelling patterns for MMP-3, predominantly localized on the intertubular collagen fibrillar network showing MMP-3 directly or indirectly bound to the collagen fibrils. Casein-zymograms showed positive proteolytic activity for MMP-3 in demineralised dentine powder. CONCLUSION: The results of the study clearly revealed the presence and distribution of MMP3 in human sound dentine. Whilst the presence was verified, its role is still unclear. Future studies are needed to investigate the possible involvement of MMP-3 in physiological and pathological condition of the dentine-pulp complex.

Cytotoxic activity of guaiazulene on gingival fibroblasts and the influence of light exposure on guaiazulene-induced cell death.

Guaiazulene (GA) is widely used as a natural ingredient in many health care products and solutions. Although it has been reported to have interesting biological effects, GA and azulene derivatives have been proven to be cytotoxic against normal human cells and human tumor cells; moreover, guaiazulene has shown photomutagenic properties on bacterial strains. Therefore, we evaluated and compared the cytotoxicity of GA at different concentrations on human gingival fibroblast (HGF) cell cultures under normal conditions and under UV irradiation (UV-A dose: 6.4 J/cm(2)). The compound tested was found to significantly reduce cell viability (dose-dependent trend, IC(50) 72.1 µM), decrease protein procollagen α1 type I synthesis, a marker for HGF protein, and COL1A1 mRNA expression. The cytotoxic effects were accompanied by activation of an intrinsic apoptotic pathway, studied using transmission electron microscopy (TEM) and caspase-3 activation. The light exposure of the cell culture treated decreased GA-induced cell death (IC(50) 128.9 µM), suggesting a photoprotective effect due to the photodegradation of the toxic agent, guaiazulene. Furthermore, the products of the photodegradation reaction of GA proved not to be toxic against HGFs.

pPKCδ activates SC35 splicing factor during H9c2 myoblastic differentiation.

Although Protein Kinase C (PKC) isoforms' role in the neonatal and adult cardiac tissue development and ageing has been widely described "in vivo", the interaction of such enzymes with specific nuclear substrates needs to be investigated. The aim of our research has been the study of the expression, localization and interaction with the splicing factor SC35 of PKC isoforms (α, δ, ε, ζ) and their potential role in modulating the transcription machinery. H9c2 cells induced to myoblast differentiation in the presence of 1% Horse Serum (HS) have represented our experimental model. The expression of PKC isoforms, their distribution and interaction with SC35 have been evaluated by western blotting, co-immunoprecipitation and double gold immunolabeling for transmission and scanning electron microscopy. Our results show PKCδ as the most expressed isoform in differentiated cells. Surprisingly, the distribution of PKCδ and SC35 does not show any significant modification between 10%FBS and 1%HS treated samples and no co-localization is observed. Moreover the interaction between the phosphorylated form of PKCδ (pPKCδ) and SC35 increases, is distributed and co-localizes within the nucleus of differentiated H9c2. These data represent reasonable evidence of pPKCδ mediated SC35 splicing factor activation, suggesting its direct effect on transcription via interaction with the transcription machinery. Furthermore, this co-localization represents a crucial event resulting in downstream changes in transcription of components which determine the morphological modifications related to cardiomyoblast differentiated phenotype.

Suppression of procollagen {alpha}1 type 1 by long-term low-dose exposure to 2-hydroxyethylmethacrylate in human gingival fibroblasts in vitro.

The purpose of this study was to evaluate the cytotoxicity of low doses and long-term exposure to 2-hydroxyethylmethacrylate (HEMA) on the protein expression of human gingival fibroblasts (HGFs). Human gingival fibroblasts were exposed to different concentrations of HEMA ranging from 0.5 mmol/L to 3 mmol/L for periods of time from 72 hours to 2 weeks. A significant decrease in the expression of procollagen α1 type I protein was observed 72 hours after treatment of cells with 3 mmol/L HEMA. Although low concentrations of the monomer after 2 weeks of exposure to HEMA did not appear to induce any marked changes in the morphology or viability of cells, the expression of procollagen α1 type I protein and its messenger RNA (mRNA) markedly decreased. In conclusion, our data demonstrated that cell viability and morphology assays could be deficient parameters in evaluating the biocompatibility of dental resin materials.

The diagnosis of the cause of the death of Venerina.

Venerina (little Venus) is the name given to a wax model representing a pregnant young woman that was created in Florence (Italy) by Clemente Susini (1754-1814) in 1782. It is currently located in the historic Science Museum of the University of Bologna. The model was constructed so as to enable removal of the thoracic and abdominal walls and various organs, exposing the heart, diaphragm and an opened uterus with a well-developed fetus. The woman is small, about 145 cm (4' 9') tall and of delicate build; she looks like a teenage girl. We know that Clemente Susini worked directly with the cadaver and copied the anatomical preparation exactly. This artist often represented the true structure using a wax mould; the existence of two other versions of this specimen suggests that this model was made in this way. Therefore, Venerina's body may be a faithful representation of a young woman who died while pregnant. Observation of the body confirms that the organs are normal, except for the heart and great vessels. The walls of both ventricles are of equal thickness and the ventricles themselves of approximately equal size. The arch of the aorta and the enlarged pulmonary trunk are connected by a short duct about 3.5 mm in diameter. If this structure represents an open arterial duct, we can deduce that the two ventricles worked under the same conditions of blood pressure, hence their equal wall thickness. If the young woman died from this congenital disease, the cause of death has been diagnosed on a wax model of her body after more than two centuries.

Analysis of differential artificial ageing of the adhesive interface produced by a two-step etch-and-rinse adhesive.

This study was performed to evaluate the effects of different in vitro ageing techniques on the dentine-bonded interface produced by a two-step etch-and-rinse adhesive. Composite build-ups were bonded to sectioned human molars using XP BOND and cut into non-trimmed dentine-composite beams for microtensile testing. Beams were assigned to one of the following storage conditions: (i) artificial saliva, 24 h (control); (ii) 10% sodium hypochlorite (NaOCl), 1 h; (iii) 10% NaOCl, 3 h; (iv) 60,000 thermal cycles, 2 months; (v) artificial saliva, 2 months; (vi) 60,000 thermal cycles, 6 months; and (vii) artificial saliva, 6 months. Beams were then pulled until failure and bond strength was calculated. Additional specimens were examined to investigate interfacial nanoleakage expression. NaOCl solution significantly reduced bonding compared with the control (group 2 = group 3 < group 1); and thermocycling reduced the bond strength in comparison to specimens stored for the same time-period in artificial saliva (group 4 < group 5; group 6 < group 7). Artificial ageing affected bond strength only after 6 months of storage (group 7 < group 5 = group 1). Increased nanoleakage was found under all ageing conditions in comparison with controls. NaOCl solution is a rapid and reliable in vitro ageing method for examining the durability of the adhesive interface produced by two-step etch-and-rinse adhesive systems.

Push-out stress for fibre posts luted using different adhesive strategies.

The influence of thermocycling on the bond strength of fibre posts cemented with different luting approaches was investigated. A total of 84 human incisors were selected for the study. Sixty teeth were assigned to one of the following adhesive/cement combinations for push-out bond-strength evaluation: group 1, XP Bond/CoreXFlow + DT Light-Post; group 2, Panavia F 2.0 + Tech 21; or group 3, RelyX Unicem + RelyX. Bonded specimens were cut into 1-mm-thick slabs and either thermocycled (40,000 cycles) or stored in artificial saliva (control specimens) before push-out bond-strength testing. Additional specimens were processed for quantitative interfacial nanoleakage analysis. Thermocycling decreased the bond strength in specimens of groups 2 and 3, but did not affect the specimens from group 1. No difference was observed among luting approaches in control specimens. Thermocycling resulted in increased silver nitrate deposition (i.e. interfacial nanoleakage) in all groups. Within the limitations of the study, the use of an etch-and-rinse adhesive in combination with a dual-cure cement to lute fiber posts is the most stable luting procedure if compared with a self-etch resin-based cement or a self-adhesive cement, as assayed by thermocycling of the bonded specimens.

Influence of a commercial tattoo ink on protein production in human fibroblasts.

Tattooing is an ancient art and is still widely practiced all over the world. Since the biocompatibility of tattoo dyes has not been well researched, we studied the toxicity of a commercial tattoo ink, commonly used in tattoo lab and esthetic centers, on human fibroblasts. To test cell viability, MTT assays were carried out and scanning electron microscopy to visualize changes in the cell surface after the dye exposure was performed. A possible influence of the pigment on the expression of procollagen alpha1 type I protein was visualized by western blotting analysis. The results showed a reduction in cell viability, and electron microscopy demonstrated an unmodified cell surface completely covered by pigment particles. Western blotting analysis demonstrated a clear interference of the pigment on the expression of procollagen alpha1 type I protein. These data demonstrated that the commercial tattoo dye has a time-dependent effect on protein expression. A possible connection of the influence of the tattoo ink with clinical effects is discussed.

HEMA down-regulates procollagen alpha1 type I in human gingival fibroblasts.

2-Hydroxyethyl methacrylate (HEMA) can be released from restorative materials and diffused into the tooth pulp over long periods of time. Although cytotoxicity due to high concentrations of monomers has been well studied, little is known about the risk of chronic toxicity resulting from low concentrations. The purpose of the study was to evaluate the effects of a minor toxic concentration of HEMA in the synthesis and expression of procollagen alpha1 type I produced by human gingival fibroblasts (HGF). HGF were exposed to 3 mM HEMA from 24 to 96 h. An MTT assay was performed to evaluate cell viability while reverse-transcriptase polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR), and Western-blot analysis were carried out to evaluate the variability in the expression and synthesis of procollagen alpha1. Immunofluorescence was performed to detect the protein inside the cells. The results showed that there was a strong reduction of procollagen alpha 1 type I expression at 72 and 96 h. These findings demonstrate that, even if it does not reduce cell viability, 3 mM HEMA interferes both with the synthesis of the procollagen alpha 1 type I protein and its mRNA expression, suggesting that normal cell production and activity are modified by HEMA at concentrations below those which cause acute cytotoxicity.

Involvement of nuclear PLCbeta1 in lamin B1 phosphorylation and G2/M cell cycle progression.

Inositide-specific phospholipase Cbeta1 (PLCbeta1) signaling in cell proliferation has been investigated thoroughly in the G(1) cell cycle phase. However, little is known about its involvement in G(2)/M progression. We used murine erythroleukemia cells to investigate the role of PLCbeta1 in G(2)/M cell cycle progression and screened a number of candidate intermediate players, particularly mitogen-activated protein kinase (MAPK) and protein kinase C (PKC), which can, potentially, transduce serum mitogenic stimulus and induce lamin B1 phosphorylation, leading to G(2)/M progression. We report that PLCbeta1 colocalizes and physically interacts with lamin B1. Studies of the effects of inhibitors and selective si-RNA mediated silencing showed a role of JNK, PKCalpha, PKCbetaI, and the beta1 isoform of PI-PLC in cell accumulation in G(2)/M [as observed by fluorescence-activated cell sorter (FACS)]. To shed light on the mechanism, we considered that the final signaling target was lamin B1 phosphorylation. When JNK, PKCalpha, or PLCbeta1 were silenced, lamin B1 exhibited a lower extent of phosphorylation, as compared to control. The salient features to emerge from these studies are a common pathway in which JNK is likely to represent a link between mitogenic stimulus and activation of PLCbeta1, and, foremost, the finding that the PLCbeta1-mediated pathway represents a functional nuclear inositide signaling in the G(2)/M transition.

Expression of procollagen alpha1 type I and tenascin proteins induced by HEMA in human pulp fibroblasts.

In the dental pulp extracellular matrix, the main macromolecules are collagenous proteins, non-collagenous proteins and proteoglycans. Regulated synthesis of the interstitial collagens, in particular, type I collagen, is important during development and wound healing but also in a number of pathological conditions. Tenascin is also a matrix protein highly expressed during development while it decreases in mature organs. Under pathological conditions such as infections and inflammation, during tumorigenesis and mechanical stress applied to cells in culture or tissue in vivo, the expression of tenascin is increased. In this study, HEMA, widely used in dentistry, ophthalmology and drug delivery, has been used to study its influence on the expression of procollagen alpha1 type I and tenascin proteins in the primary cultures of human pulp fibroblasts. Different concentrations of the resin monomer and different times of exposition were tested. The influence of HEMA on the cell viability was evaluated by means of an MTT assay while immunofluorescence and western blotting analysis were performed to detect possible interference with the presence and the synthesis of these proteins. We observed a strong reduction in cell viability in specimens treated for 96 h and 168 h, especially at concentrations of 1 and 3 mmol/L HEMA. Both immunofluorescence and western blotting analysis demonstrated a reduction of procollagen alpha1 type I protein and an overexpression of tenascin protein. Our results showed that long-term exposure and low concentrations of HEMA influence normal cell activity, such as the synthesis of some of the dental pulp extracellular matrix proteins.

Effect of fixative on chromatin structure and DNA detection.

In this study, we analyzed the chromatin ultrastructure in interphase cells after different chemical fixations. In light of the fact that there is little information regarding the fixation of biological samples in combination with molecular biology methods (such as DNA extraction and in situ hybridization methods) we analyzed the ultrastructure of chromatin in interphase cells fixed with different fixatives and tested under the same conditions for both DNA extraction and in situ hybridization. The results showed that, among the different combinations and concentrations we analyzed, the solution of 4% paraformaldehyde/0.1% glutaraldehyde was the best compromise in order to achieve a well-preserved morphology, successful DNA extraction, and specific signaling of in situ hybridization, suggesting a low interference of this fixative with the chromatin organization.

Effects of citric acid and EDTA conditioning on exposed root dentin: An immunohistochemical analysis of collagen and proteoglycans.

OBJECTIVE: Preservation of structural and biochemical properties of the root dentin matrix is crucial to favor healing and regenerative periodontal processes. Aim of this study was to evaluate the biochemical characteristics of collagen and chondroitin sulphate of root dentin surfaces exposed by periodontal disease after acid conditioning by means of an immunohistochemical technique. DESIGN: Human teeth scheduled for extraction due to periodontal reason were submitted to: (A) scaling and root planning; (B) ultrasonic instrumentation; (C) no instrumentation. Teeth were then exposed to: (1) 10% citric acid; (2) 17% EDTA; (3) no etching. A double immunolabeling technique was performed to identify type-I collagen and proteoglycans and analyzed under FEI-SEM. RESULTS: Use of 10% citric acid revealed intense labeling for collagen fibrils and proteoglycans; lower labeling was found after EDTA conditioning. Unetched specimens showed residual smear layer on the dentin surface resulting in no evident surface labeling. CONCLUSIONS: This study supports the hypothesis that manual or ultrasonic instrumentation alone is not able to expose the sound dentin matrix, whereas a subsequent acidic conditioning exposes collagen fibrils and associated proteoglycans. The immunohistochemical technique revealed that despite their acidity, both citric acid and EDTA were able to preserve the structural and biochemical properties of the exposed dentin matrix.

Nanoleakage within the hybrid layer: a correlative FEISEM/TEM investigation.

The aim of this study was to compare the nanoleakage patterns of the resin-dentin interfaces of three dentin bonding systems at both TEM and field emission in lens SEM (FEI-SEM) levels. A standardized smear layer was created with 180-grit silicon carbide paper (SiC) on dentin disks obtained from 18 noncarious human third molars. Specimens were randomly divided into three groups and bonded with a two-step total etching adhesive (Single Bond, SB), a two-step, self-etching adhesive (Clearfil SE BOND, SEB), and a one-step, self-etching adhesive (XENO III, XEIII). Nanoleakage was evaluated by using an ammoniacal silver-nitrate solution. Specimens were processed for TEM and FEI-SEM observation. The TEM of SB revealed silver deposits in adhesive and hybrid layers (HL). High-magnification FEI-SEM micrographs clearly identified these deposits as spherical clusters mainly associated with nonembedded collagen fibrils. TEM and FEI-SEM examination of SEB revealed some clusters of silver deposits within porosities and small channels of the HL. Additional silver deposits were observed between the peritubular dentin walls and the resin tags. XEIII revealed very fine and diffuse silver grains throughout the entire HL. SEM visualization of nanoleakage at a high level of resolution has not been previously described. FEI-SEM technology supported the TEM visualization with three-dimensional morphological data of the relations between the HL constituents and nanoleakage. The results of the present study confirm the hypothesis that both total- and self-etch adhesives are not able to fully infiltrate the dentin substrate.

Immunohistochemical analysis of collagen fibrils within the hybrid layer: a FEISEM study.

This study evaluated the immunohistochemical labeling pattern of dentin collagen fibrils within hybrid layers created by different bonding systems using high resolution SEM. Four different adhesive materials, including self-etching and total-etching systems, were examined: Prime & Bond NT, OptiBond SOLO Plus, Single Bond and Clearfil Protect Bond. All materials were applied to the dentin of extracted human third molars. After cutting the bonded specimens transversely, an anti-collagen type I antibody was incubated on the surface of the dentin-adhesive interface and gold-conjugated secondary antibody was applied to reveal collagen labeling under high resolution SEM. The hybrid layers showed a significant number of collagen fibrils embedded in the resin matrix. The presence of exposed dentin collagen fibrils, as determined by positive labeling with an anti-type I collagen monoclonal antibody, is considered an indication of the presence of incompletely embedded fibrils and, thus, the quality of dentin matrix hybridization. The hybrid layers produced by total-etching systems showed higher labeling compared to those produced by the self-etching system. Positive collagen labeling was also found along the resin tags produced by total-etching adhesive systems.

Ultra-morphology of self-etching adhesives on ground enamel: a high resolution SEM study.

PURPOSE: To evaluate the effect of different commercial self-etching agents on enamel morphology using high-resolution in-lens scanning electron microscopy (FEISEM). METHODS: The bonding systems selected for the study were: Prime & Bond NT (no-etch technique), Prime & Bond 2.1 (no-etch technique), NRC/Prime and Bond NT, Syntac Single Component, Prompt L-Pop, F2000, and Clearfil SE Bond. The positive control group was prepared with Single Bond upon etching enamel with the proprietary 35% phosphoric acid gel. 24 extracted human molars were equally and randomly assigned to the experimental and control groups. All bonding materials were applied on enamel following the manufacturers' instructions. A thin layer of composite was applied on the polymerized adhesive agent, after which the enamel was dissolved to obtain replicas that were observed under a FEISEM. RESULTS: Observations revealed different morphological features brought about by the adhesive systems. A relationship between the morphological appearance and the pH of the adhesive solutions was found. Three different groups of self etching were identified: Group 1 showed no or little evidence of modifications on the enamel surface (Prime & Bond NT and Prime & Bond 2.1, no-etch technique), Group 2 revealed major aggressive properties that were able to disclose the prism morphology (Syntac Single Component, F2000, Clearfil SE Bond), and Group 3 (NRC/Prime & Bond NT and Prompt L-Pop) revealed morphological features similar to those obtained with Single Bond after etching with phosphoric acid (control group).

Effects of different acid and sodium hypochlorite treatments on dentin collagen: a FEISEM analysis.

PURPOSE: To evaluate if different acid treatments of dentin surface might remove different amount of mineralized dentin, thus exposing and modifying the CF network. METHODS: Dentin disks prepared from human third molars with a low-speed diamond saw were etched for 15 seconds with the tested acids: citric acid 10%, maleic acid 10%, 2.5% oxalic acid, 35% phosphoric acid and 24% EDTA gel. Specimens were then submitted to a 5% solution of NaOCl for 2 minutes or 5 minutes, fixed, and observed utilizing a field emission in lens SEM (FEISEM). Control specimens were also prepared by omitting the etching agent and/or the NaOCl solution. RESULTS: The different acid treatments created specific dentin morphological pattern. CF exposure was in relationship with the acid used. The NaOCl solution greatly affects the acid etched dentin by removing the CF, nevertheless the effect of NaOCl was greatly influenced by the previous acid treatment.

Morphological study of resin-dentin bonding with TEM and in-lens FESEM.

PURPOSE: To evaluate the morphological characteristics of the dentin-resin interface using high-magnification electron microscopy and correlate the TEM findings of resin-dentin interfaces with the corresponding in-lens Field Emission SEM (FEISEM) observation. METHODS: Twelve 800 microm-thick dentin disks were obtained from middle dentin and assigned to four groups: (1) a self-etching adhesive material, Clearfil SE Bond; (2) a solvent-free total-etch simplified adhesive, One Coat Bond; (3) an acetone-based total-etch simplified adhesive, Prime&Bond NT; (4) an acetone-based total-etch simplified adhesive, Prime&Bond NT, upon a short dentin deproteinization with 10% NaOCl gel (AD Gel) for 15 seconds. Disks were restored with a 1 mm thick layer of a low-viscosity composite. Four sticks with a cross section of 1 mm2 were taken from each restored dentin disk, decalcified in buffered 10% EDTA, fixed, and stained with lead citrate followed by uranyl acetate, or kept in water. The sticks were sectioned in 85 nm-thick slices, and mounted on Ni grids. After TEM observation, the same grids were coated with a 1.5 nm Pt-C film and observed under the FEISEM. RESULTS: The TEM analysis revealed an intimate adaptation of all adhesive systems to dentin, with penetrating resin tags observed in transverse sections. The FEISEM revealed novel morphological features usually observed in the resin-dentin interface with distinctive arrangement for each of the adhesive systems. FEISEM and TEM appear as mutually complementary tools for the fine observation of the ultra-structure and the spatial relationship between the resin components and the collagen fibers of the hybrid layer.