Kirsten Van Landuyt

Environmental Scanning Electron Microscopy Connected with Energy Dispersive X-ray Analysis and Raman Techniques to Study ProRoot Mineral Trioxide Aggregate and Calcium Silicate Cements in Wet Conditions and in Real Time

INTRODUCTION ProRoot mineral trioxide aggregate (MTA) and calcium silicate cements are able to set in a moist environment. The aim of the study was to examine the surface structure and composition of a cement paste under wet conditions and in real time during setting by environmental scanning electron microscopy connected with energy dispersive x-ray analysis (ESEM-EDX) and micro-Raman techniques. METHODS White ProRoot MTA and experimental white tetrasilicate cement (wTC) and wTC containing bismuth oxide (wTC-Bi) were studied. Cement disks were analyzed 10 minutes after powder-liquid mixing (freshly prepared samples) and after immersion in Dulbecco phosphate-buffered saline at 37 degrees C for 24 hours (24-hour-aged samples). RESULTS Freshly prepared wet cements at ESEM-EDX exposed an irregular surface (displaying calcium, silicon, aluminum, chlorine reflexes, and bismuth traces in MTA and wTC-Bi) with needle-like and cubic-hexagonal shaped crystals. Aggregates of spheroidal Ca-P-rich crystals (spherulites) appeared on the surface of 24-hour-aged samples. The starting unhydrated powders displayed the typical Raman bands of Portland cement components: alite, belite, and calcium sulfate (only as anhydrite in MTA and as both anhydrite and gypsum in wTC and wTC-Bi). MTA powder showed higher amount of calcium carbonate and lower quantities of anhydrite and higher crystallinity of the silicate component, leading to a slower hydration reaction. Products/markers of hydration reactions were present on fresh samples; ettringite formed on the surface of all the cements; calcium hydroxide (portlandite) was detected only on the surface of wTC, but no conclusion can be drawn on wTC-Bi and MTA because of the interference of bismuth oxide. Calcium phosphate and calcite/aragonite bands were detected on all 24-hour-aged cements; portlandite was no longer detected on wTC. CONCLUSIONS ESEM and micro-Raman are powerful and suitable techniques to investigate endodontic calcium silicate hydrated cements in real time and in their humid state without inducing artifacts by sample preparation. The formation of apatite spherulites on calcium silicate cements might have clinical relevance.

Bulk-filling of high C-factor posterior cavities: Effect on adhesion to cavity-bottom dentin

OBJECTIVES To evaluate the effect of bulk-filling high C-factor posterior cavities on adhesion to cavity-bottom dentin. METHODS A universal flowable composite (G-ænial Universal Flo, GC), a bulk-fill flowable base composite (SDR Posterior Bulk Fill Flowable Base, Dentsply) and a conventional paste-like composite (Z100, 3M ESPE) were bonded (G-ænial Bond, GC) into standardized cavities with different cavity configurations (C-factors), namely C=3.86 (Class-I cavity of 2.5mm deep, bulk-filled), C=5.57 (Class-I cavity of 4mm deep, bulk-filled), C=1.95 (Class-I cavity of 2.5mm deep, filled in three equal layers) and C=0.26 (flat surface). After one-week water storage, the restorations were sectioned in 4 rectangular micro-specimens and subjected to a micro-tensile bond strength (μTBS) test. RESULTS Highly significant differences were found between pairs of means of the experimental groups (Kruskal-Wallis, p<0.0001). Using the bulk-fill flowable base composite SDR (Dentsply), no significant differences in μTBS were measured among all cavity configurations (p>0.05). Using the universal flowable composite G-ænial Universal Flo (GC) and the conventional paste-like composite Z100 (3M ESPE), the μTBS to cavity-bottom dentin was not significantly different from that of SDR (Dentsply) when the cavities were layer-filled or the flat surface was build up in layers; it was however significantly lower when the Class-I cavities were filled in bulk, irrespective of cavity depth. SIGNIFICANCE The filling technique and composite type may have a great impact on the adhesion of the composite, in particular in high C-factor cavities. While the bulk-fill flowable base composite provided satisfactory bond strengths regardless of filling technique and cavity depth, adhesion failed when conventional composites were used in bulk.

Enzymatic degradation of adhesive-dentin interfaces produced by mild self-etch adhesives

Endogenous matrix metalloproteinases (MMPs) released by adhesive procedures may degrade collagen in the hybrid layer and so compromise the bonding effectiveness of etch-and-rinse adhesives. In this study, endogenous enzymatic degradation was evaluated for several simplified self-etch adhesives. In addition, primers were modified by adding two MMP inhibitors: chlorhexidine, a commonly used disinfectant, but also a non-specific MMP inhibitor; and SB-3CT, a specific inhibitor of MMP-2 and MMP-9. Gelatin zymography of fresh human dentin powder was used to identify the enzymes released by the adhesives. Micro-tensile bond strength (μTBS) testing was used to assess the mechanical properties of resin-dentin interfaces over time. In none of the experimental groups treated with the mild self-etch adhesives was MMP-2 and/or MMP-9 identified. Also, no difference in the μTBS was measured for the inhibitor-modified and the control inhibitor-free adhesives after 6 months of water storage. It is concluded that in contrast to etch-and-rinse adhesives, the involvement of endogenous MMP-2 and MMP-9 in the bond-degradation process is minimal for mild self-etch adhesives.

Bond strength of self-etch adhesives to dentin prepared with three different diamond burs

OBJECTIVES To evaluate the micro-tensile bond strength (mu TBS) of a control etch-and-rinse and three self-etch (strong, mild, ultra-mild) adhesive systems to dentin prepared with three different grit size of diamond burs. METHODS Dentin surfaces were created from mid-coronal sound dentin in extracted, human third molars. The teeth were ground with high-speed medium grit (100 microm), fine grit (30 microm), or extra-fine grit (15 microm) diamond bur. Resin composite (Z100) was bonded to the surfaces using Optibond FL, Adper Prompt L-Pop, Clearfil SE Bond, or Clearfil S3 Bond. Rectangular micro-specimens were prepared using the slow-speed diamond saw and tested in tensile to determine the mu TBS. Failure analysis was performed using a stereo-microscope and Fe-SEM. Data were analyzed by two-way analysis of variance and Tukeys HSD test (p<0.05). RESULTS The etch-and-rinse adhesive yielded high micro-tensile values (58-69 MPa), irrespective of the diamond burs used. The bond strength values were comparable for Adper Prompt L-Pop and Clearfil SE Bond irrespective of the burs used. The mu TBS values were significantly higher as the particle size of diamond burs is smaller for Clearfil S3 Bond. Most failures were recorded as interfacial failure when the fine and extra-fine diamond burs were used. SIGNIFICANCE Overall, different grit-sized diamond burs did not affect the mechanical properties of the interface, except for the ultra-mild one step self-etch adhesive. This adhesive performed significantly better when a smaller grit size was used to prepare dentin surface.

Contamination of nanoparticles by endotoxin: evaluation of different test methods

BackgroundNanomaterials can be contaminated with endotoxin (lipopolysaccharides, LPS) during production or handling. In this study, we searched for a convenient in vitro method to evaluate endotoxin contamination in nanoparticle samples. We assessed the reliability of the commonly used limulus amebocyte lysate (LAL) assay and an alternative method based on toll-like receptor (TLR) 4 reporter cells when applied with particles (TiO2, Ag, CaCO3 and SiO2), or after extraction of the endotoxin as described in the ISO norm 29701.ResultsOur results indicate that the gel clot LAL assay is easily disturbed in the presence of nanoparticles; and that the endotoxin extraction protocol is not suitable at high particle concentrations. The chromogenic-based LAL endotoxin detection systems (chromogenic LAL assay and Endosafe-PTS), and the TLR4 reporter cells were not significantly perturbed.ConclusionWe demonstrated that nanoparticles can interfere with endotoxin detection systems indicating that a convenient test method must be chosen before assessing endotoxin contamination in nanoparticle samples.

Is secondary caries with composites a material-based problem?

OBJECTIVE Secondary caries (SC) is one of the most important reasons for the failure of composite restorations, and thus has wide-reaching implications for the longevity of affected teeth and the health expenditure. Yet, it is currently not known whether secondary caries with composites is a material-based problem. The objective was to review literature with regard to SC around composite restorations to obtain better insights in the mechanisms behind SC with composites. METHODS Using Pubmed and Medline, international literature was searched for all articles about the clinical diagnosis, incidence and prevalence, histopathology and factors involved in the onset and development of SC around composite restorations. Additional studies were included after checking the reference lists of included papers. RESULTS SC with composites is to some extent associated to the restorative material, as significantly more caries occurred with composites than with amalgam. On the other hand, the class of the composite restoration (class V versus others and class I versus class II) was also determining for the development of SC, suggesting also other influencing factors than the material itself. The mechanisms behind the development of SC are much less clear and are most probably multifactorial. Even though the role of gaps an microleakage is questioned by some researchers, there are also indications that interfacial failure may play a role. Interfacial gaps larger than 60 μm seem to predispose interfacial demineralization, and may thus lead to caries. The question is therefore whether such interfacial gaps occur clinically? Initially, a gap may originate through polymerization shrinkage and through failure to obtain a good bond. Higher incidences of SC are observed in practice-based than in university-based studies, which may be attributed to different caries risk profiles of the included patients, or to the technique-sensitive placement procedure of composites. More research is necessary to investigate whether large gaps may arise through degradation processes. Apart from these factors, composites also seem to favor the growth of cariogenic bacteria on their surface, which has been associated with specific surface properties, release of components and lack of antibacterial properties. SIGNIFICANCE Current literature suggests that the restorative material might influence the development of secondary caries in different ways. However, it should be emphasized that patient-related factors remain the most important determinant of secondary caries.

Global Methylation and Hydroxymethylation in DNA from Blood and Saliva in Healthy Volunteers

Aims. We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify and compare simultaneously global methylation and hydroxymethylation in human DNA of different tissues. Materials and Methods. Blood and saliva DNA from fourteen volunteers was processed for epigenetic endpoints using LC-MS/MS and PCR-pyrosequencing technology. Results. Global DNA methylation was significantly lower in saliva (mean 4.61% ±  0.80%), compared to blood samples (5.70% ± 0.22%). In contrast, saliva (0.036% ± 0.011%) revealed significantly higher hydroxymethylation compared to blood samples (mean 0.027% ± 0.004%). Whereas we did not find significant correlations for both epigenetic measures between the tissues, a significant association was observed between global methylation and global hydroxymethylation in saliva DNA. Neither LINE-1 nor Alu elements of blood and saliva correlated, nor were they correlated with the DNA hydroxymethylation of blood or saliva, respectively. Conclusion. Global DNA methylation and hydroxymethylation of cytosine can be quantified simultaneously by LC-MS/MS. Saliva DNA cannot be considered as a surrogate for blood DNA to study epigenetic endpoints.

Toxicity of Nanoparticles Embedded in Paints Compared with Pristine Nanoparticles in Mice

The unique physical and chemical properties of nanomaterials have led to their increased use in many industrial applications, including as a paint additive. For example, titanium dioxide (TiO2) engineered nanoparticles (ENPs) have well-established anti-UV, self-cleaning, and air purification effects. Silver (Ag) ENPs are renowned for their anti-microbial capabilities and silicon dioxide (SiO2) ENPs are used as fire retardants and anti-scratch coatings. In this study, the toxic effects and biodistribution of three pristine ENPs (TiO2, Ag, and SiO2), three aged paints containing ENPs (TiO2, Ag, and SiO2) along with control paints without ENPs were compared. BALB/c mice were oropharyngeally aspirated with ENPs or paint particles (20 μg/aspiration) once a week for 5 weeks and sacrificed either 2 or 28 days post final aspiration treatment. A bronchoalveolar lavage was performed and systemic blood toxicity was evaluated to ascertain cell counts, induction of inflammatory cytokines, and key blood parameters. In addition, the lung, liver, kidney, spleen, and heart were harvested and metal concentrations were determined. Exposure to pristine ENPs caused subtle effects in the lungs and negligible alterations in the blood. The most pronounced toxic effects were observed after Ag ENPs exposure; an increased neutrophil count and a twofold increase in pro-inflammatory cytokine secretion (keratinocyte chemoattractant (KC) and interleukin-1ß (IL-1ß)) were identified. The paint containing TiO2 ENPs did not modify macrophage and neutrophil counts, but mildly induced KC and IL-1ß. The paints containing Ag or SiO2 did not show significant toxicity. Biodistribution experiments showed distribution of Ag and Si outside the lung after aspiration to respectively pristine Ag or SiO2 ENPs. In conclusion, we demonstrated that even though direct exposure to ENPs induced some toxic effects, once they were embedded in a complex paint matrix little to no adverse toxicological effects were identified.

Bonding effectiveness and interfacial characterization of a HEMA/TEGDMA-free three-step etch&rinse adhesive.

OBJECTIVES Small and hydrophilic monomers as HEMA and TEGDMA can easily penetrate human tissues. For biocompatibility issues it is therefore better to avoid such monomers in dental adhesive formulations. The purpose of this study was (1) to determine the micro-tensile bond strength (muTBS) to enamel/dentin of a HEMA/TEGDMA-free three-step etch&rinse adhesive (cmf Adhesive System, Saremco), and (2) to characterize its interfacial interaction with enamel/dentin using transmission electron microscopy (TEM). METHODS Twelve intact freshly extracted human third molars were used for muTBS. The three-step etch&rinse adhesive OptiBond FL (Kerr) served as control adhesive. After bonding procedures and subsequent composite build-up (Z100, 3M ESPE), specimens were sectioned to sticks and trimmed at the interface to a cylindrical hour-glass shape. TEM section of non-demineralized and demineralized sections were prepared by ultra-microtomy and examined. RESULTS The muTBS of the cmf Adhesive System to enamel was not statistically significantly different from that of the control (OptiBond FL) adhesive (p=0.3512); the muTBS to dentin was however significantly lower (p<0.0001). TEM showed a tight interface at enamel for both adhesives. A relatively thick, completely demineralized and acid-resistant hybrid layer was formed at dentin. Some degree of spot- and cluster-like nano-leakage was observed with cmf Adhesive System, but did not differ in extent and form in comparison to that observed with the OptiBond FL control. CONCLUSIONS The new three-step adhesive cmf Adhesive System showed indications of good bonding effectiveness, certainly to enamel and only significantly lower to dentin, when compared to the gold-standard adhesive. The latter may be related to the adapted HEMA/TEGDMA-free formulation and the somewhat lower mechanical properties.

Should we be concerned about composite (nano-)dust?

UNLABELLED Dental personnel often come into contact with composite dust upon polishing or grinding composites. Contemporary composites typically contain high amounts of (silica) nano-filler, but so far it has never been investigated whether composite dust may be hazardous. OBJECTIVES The objectives of this study were (1) to characterize composite dust in vitro and (2) to assess the clinical exposure. METHODS Polymerized blocks of contemporary composites were ground with a diamond bur according to a clinically relevant protocol, and aerosolized dust was collected on 1-μm pore-size filters and gravimetrically quantified. In addition, the dust was characterized by transmission electron microscopy. Respirable dust was quantified with a mass particle counter in a dental operatory. RESULTS All composites released respirable dust (<5μm) in vitro. These observations were corroborated by the clinical measurements; however only short episodes of high concentrations of respirable dust upon polishing composites could be observed. Electron microscopic analysis showed that the size of the dust varied widely with particles larger than 10μm, but submicron and even nano-sized particles could also be observed. The dust particles often consisted of multiple filler particles contained in resin, but single nano-filler particles could also frequently be distinguished. SIGNIFICANCE This study showed that inhalation of composite dust is better avoided. Therefore, it is recommended to always use water-cooling upon polishing or removing composites, to use good aspiration, to frequently ventilate the dental operatory and to wear masks with high particle-filtration efficiency for small particle sizes.