A MEMS nanoindenter with an integrated AFM cantilever gripper for nanomechanical characterization of compliant materials.

This work presents the development of a MEMS nanoindenter that uses exchangeable AFM probes for quasi-static nanomechanical characterization of compliant and ultra-compliant materials. While the electrostatic micro-force transducer of the MEMS nanoindenter provides a maximum indentation depth up to 9.5 µm with a maximum output force of 600 µN, experimental investigations reveal that it can achieve a depth and force resolution better than 4 pm Hz-1/2 and 0.3 nN Hz-1/2, in air for f≥ 1 Hz. A passive AFM probe gripper is integrated into the MEMS nanoindenter, allowing the nanoindenter to utilize various AFM probes as an indenter for material testing. A proof-of-principle experimental setup has been built to investigate the performance of the MEMS nanoindenter prototype. In proof-of-principle experiments, the prototype with a clamped diamond AFM probe successfully identified an atomic step (∼0.31 nm) within a Si < 111 > ultraflat sample using the scanning probe microscopy mode. The nanomechanical measurement capability of the MEMS nanoindenter prototype has been verified by means of measurements of reference polymer samples using a silicon AFM probe and by means of measurements of the elastic properties of a PDMS sample using a spherical diamond-coated AFM probe. Owing to its compact and low-cost but high-resolution capacitive readout system, this MEMS nanoindenter head can be further applied for in-situ quantitative nanomechanical measurements in AFMs and SEMs.

Penetration depth of irrigants into root dentine after sonic, ultrasonic and photoacoustic activation.

AIM: To compare penetration depths of endodontic irrigants into the dentinal tubules of extracted teeth when using several activation methods. METHODOLOGY: The root canals of 90 extracted human teeth were prepared to size 40, .06 taper. The straight and round-shaped root canals were distributed randomly into six groups, and final irrigation was performed with EDTA and sodium hypochlorite as follows: (I) manual dynamic activation, (II) Ultrasonic, (III) Sonic, (IV) PIPS (photon-induced photoacoustic streaming, (V) SWEEPS (shock-wave enhanced emission photoacoustic streaming) and (0) control without final irrigation or activation. Subsequently, methylene blue was inserted into the canals and activated according to the groups (I-V). Teeth were sectioned horizontally, imaged under a light microscope, and dye penetration depths were measured in six sections per tooth and 24 points on a virtual clock-face per section. Data were analysed statistically by nonparametric tests for whole teeth and separately for coronal, middle and apical thirds. RESULTS: Penetration of dye into the dentinal tubules was lowest for the controls. Median penetration depths amounted to 700-900 µm for groups I-V with differences in the apical thirds between group I and the other test groups. Minimum penetration depths were significantly greater for PIPS in the apical thirds (P ≤ 0.046). CONCLUSIONS: Greater penetration depths occurred in the apical thirds for ultrasonic, sonic and laser-induced activation compared to manual dynamic activation. PIPS was associated with deeper penetration of irrigants. The novel SWEEPS mode did not increase irrigant penetration.

Detection of pulsed blood flow through a molar pulp chamber and surrounding tissue in vitro.

OBJECTIVES: Due to severe limitations of dental pulp sensitivity tests, the direct recording of pulsed blood flow, using photoplethysmography (PPG), has been proposed. In vivo evaluation is methodologically difficult and in vitro models have hitherto been adversely influenced by shortcomings in emulating the in vivo situation. Consequently, the aim of this study was to test an improved data acquisition system and to use this configuration for recording pulsed blood in a new model. MATERIALS AND METHODS: We introduced a PPG signal detection system by recording signals under different blood flow conditions at two wavelengths (625 and 940 nm). Pulsed blood flow signals were measured using an in vitro model, containing a molar with a glass pulp and a resin socket, which closely resembled in vivo conditions with regard to volumetric blood flow, pulp anatomy, and surrounding tissue. RESULTS: The detection system showed improved signal strength without stronger blanketing of noise. On the tooth surface, it was possible to detect signals emanating from pulsed blood flow from the glass pulp and from surrounding tissue at 625 nm. At 940 nm, pulp derived signals were recorded, without interference signals from surrounding tissue. CONCLUSION: The PPG-based method has the potential to detect pulsed blood flow in small volumes in the pulp and (at 625 nm) also in adjacent tissues. CLINICAL RELEVANCE: The results show the need for clear differentiation of the spatial origins of blood flow signals of any vitality test method to be applied to teeth.

Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells.

AIM: To investigate the combinatorial effects of lipopolysaccharide (LPS) and extracted dentine matrix proteins (eDMP) on regenerative and inflammatory responses in human dental pulp stem cells (DPSCs). METHODOLOGY: Culture media were supplemented with several concentrations of LPS, eDMP and combinations of both. Cell viability was assessed over 1 week by MTT assay; cell survival was evaluated after 24 h and 7 days by flow cytometry. The expression of mineralization-associated marker genes was determined by real-time quantitative polymerase chain reaction (RT-qPCR). To analyse the inflammatory response, secretion of interleukin 6 (IL-6) was quantified in the initial and the late phase of cell culture by enzyme-linked immunosorbent assay (ELISA). Data were treated nonparametrically and Mann-Whitney U-tests were performed to compare all experimental groups (α = 0.05). RESULTS: Whereas LPS had no impact on viability, eDMP led to a concentration-dependent decrease, which was significant after 7 days (P ≤ 0.024). A moderate decline of cell survival induced by LPS was detected after 48 h (P ≤ 0.026), whereas eDMP was able to reverse this effect. eDMP alone caused increased expression of tested marker genes, LPS had no regulatory effect. Combined eDMP and LPS induced an upregulation of collagen type I and osteocalcin, whereas expression levels of dentine matrix acidic phosphoprotein and dentine sialophosphoprotein were similar to the control. IL-6-secretion was increased by LPS over time. eDMP markedly elevated initial production of IL-6 (P ≤ 0.002), but suppressed LPS-induced cytokine production in the later phase. CONCLUSIONS: Lipopolysaccharide did not affect cell viability but interfered with odontoblast-like cell differentiation of DPSCs. Proteins from the dentine matrix may have a protective effect, attenuate the detrimental impact of LPS and thus play an important role during pulp repair.

Dentine matrix proteins: isolation and effects on human pulp cells.

AIM: To establish a simplified and efficient protocol for the isolation and concentration of matrix proteins from human dentine, and to assess the effects of extracted dentine matrix proteins (eDMP) on the behaviour of human pulp cells. METHODOLOGY: Matrix proteins were isolated from human dentine, purified, concentrated and characterized with protein and enzyme-linked immunosorbent assays (ELISA). Culture media were supplemented with eDMP in different concentrations, referred to as eDMP 1-10 000, to assess viability and proliferation of human pulp cells by DNA and MTT assays; apoptotic events were quantified by flow cytometry. Chemotactic effects of eDMP were assessed in a modified Boyden chamber assay. Expression levels of odontoblastic marker genes in pulp cells cultured with eDMPs were determined by real-time quantitative PCR, and the ability to induce mineralization was demonstrated by alizarin red staining. Nonparametric statistical analysis was performed to pairwise compare different groups at all time-points (Mann-Whitney U-test, α = 0.05). RESULTS: High concentrations of eDMP exhibited significant antiproliferative effects (P ≤ 0.023) after 5 (eDMP 1000) and 7 days (eDMP 500) without affecting cell viability. Apoptosis was barely influenced (P ≥ 0.089). eDMP exerted a concentration-dependent chemotactic stimulus on dental pulp cells with statistical significance already at low dosage (P = 0.006 at eDMP 10). Changes in gene expression indicated a differentiation into odontoblast-like cells, which was corroborated by findings of mineral nodule formation. CONCLUSIONS: A novel, effective and time-saving protocol for isolation and concentration of dentine matrix proteins is presented. As eDMP stimulates chemotaxis, differentiation and mineralization without affecting viability, endogenous dentine matrix proteins might be valuable for approaches to regenerate or engineer dental pulp.

Ultrasonic activation of irrigants increases growth factor release from human dentine.

OBJECTIVES: Bioactive proteins are sequestered in human dentine and play a decisive role in dental pulp regeneration and repair. They can be released and exposed on the dentine surface by acids, but also chelators, such as ethylenediaminetetraacetic acid (EDTA). The objectives of this study were (i) to evaluate whether ultrasonic activation of irrigants in the root canal will promote growth factor release from dentine and (ii) to collect bioactive proteins in a physiological solution. MATERIALS AND METHODS: Human dentine disks underwent irrigation with and without ultrasonic activation. The protocols included treatment by either a single or two consecutive steps with 10 % EDTA and phosphate-buffered saline (PBS), where each sample was treated three times. To mimic clinical conditions, selected irrigation regimens were applied to root canals of extracted human teeth after preparation. Amounts of transforming growth factor ß1 (TGF-ß1) in solution were quantified using enzyme-linked immunosorbent assays. Nonparametric statistical analysis was performed to compare different groups as well as repetitions within a group (Mann-Whitney U test, α = 0.05). Additionally, morphological changes of dentine surfaces were visualized by scanning electron microscopy (SEM). RESULTS: TGF-ß1 was not detectable after irrigation of dentine with PBS, neither with nor without ultrasonic activation. Irrigation with EDTA released TGF-ß1, and ultrasonic activation of EDTA enhanced this effect. However, preceding EDTA conditioning enabled the release of bioactive proteins into PBS solution. Similar results were observed in dentine disks and root canals. Visualization of dentine surfaces after different treatment revealed superficial erosion after ultrasonic activation irrespective of the irrigant solution, but different degrees of exposure of organic substance. CONCLUSIONS: Ultrasonic activation enhances growth factor release from human dentine. Bioactive proteins can be isolated in physiological solvents and may act as autologous supplements for regenerative endodontic treatment or pulp tissue engineering. CLINICAL RELEVANCE: Autologous growth factors from human dentine can advance treatment strategies in dental pulp tissue engineering.

Loss of DJ-1 impairs antioxidant response by altered glutamine and serine metabolism.

The oncogene DJ-1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 has an important function in cellular antioxidant responses, but its role in central metabolism of neurons is still elusive. We applied stable isotope assisted metabolic profiling to investigate the effect of a functional loss of DJ-1 and show that DJ-1 deficient neuronal cells exhibit decreased glutamine influx and reduced serine biosynthesis. By providing precursors for GSH synthesis, these two metabolic pathways are important contributors to cellular antioxidant response. Down-regulation of these pathways, as a result of loss of DJ-1 leads to an impaired antioxidant response. Furthermore, DJ-1 deficient mouse microglia showed a weak but constitutive pro-inflammatory activation. The combined effects of altered central metabolism and constitutive activation of glia cells raise the susceptibility of dopaminergic neurons towards degeneration in patients harboring mutated DJ-1. Our work reveals metabolic alterations leading to increased cellular instability and identifies potential new intervention points that can further be studied in the light of novel translational medicine approaches.

EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells.

AIM: To evaluate the effect of dentine conditioning on migration, adhesion and differentiation of dental pulp stem cells. METHODOLOGY: Dentine discs prepared from extracted human molars were pre-treated with EDTA (10%), NaOCl (5.25%) or H2 O. Migration of dental pulp stem cells towards pre-treated dentine after 24 and 48 h was assessed in a modified Boyden chamber assay. Cell adhesion was evaluated indirectly by measuring cell viability. Expression of mineralization-associated genes (COL1A1, ALP, BSP, DSPP, RUNX2) in cells cultured on pre-treated dentine for 7 days was determined by RT-qPCR. Nonparametric statistical analysis was performed for cell migration and cell viability data to compare different groups and time-points (Mann-Whitney U-test, α = 0.05). RESULTS: Treatment of dentine with H2 O or EDTA allowed for cell attachment, which was prohibited by NaOCl with statistical significance (P = 0.000). Furthermore, EDTA conditioning induced cell migration towards dentine. The expression of mineralization-associated genes was increased in dental pulp cells cultured on dentine after EDTA conditioning compared to H2 O-pre-treated dentine discs. CONCLUSIONS: EDTA conditioning of dentine promoted the adhesion, migration and differentiation of dental pulp stem cells towards or onto dentine. A pre-treatment with EDTA as the final step of an irrigation protocol for regenerative endodontic procedures has the potential to act favourably on new tissue formation within the root canal.

Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements.

OBJECTIVES: Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine™ offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material. MATERIALS AND METHODS: Test materials included a new tricalcium silicate (Biodentine™, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac™ Molar Aplicap™, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α = 0.05). RESULTS: Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike. CONCLUSIONS: Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive. CLINICAL RELEVANCE: The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.

Individual growth detection of bacterial species in an in vitro oral polymicrobial biofilm model.

Most in vitro studies on the antibacterial effects of antiseptics have used planktonic bacteria in monocultures. However, this study design does not reflect the in vivo situation in oral cavities harboring different bacterial species that live in symbiotic relationships in biofilms. The aim of this study was to establish a simple in vitro polymicrobial model consisting of only three bacterial strains of different phases of oral biofilm formation to simulate in vivo oral conditions. Therefore, we studied the biofilm formation of Actinomyces naeslundii (An), Fusobacterium nucleatum (Fn), and Enterococcus faecalis (Ef) on 96-well tissue culture plates under static anaerobic conditions using artificial saliva according to the method established by Pratten et al. that was supplemented with 1 g l(-1) sucrose. Growth was separately determined for each bacterial strain after incubation periods of up to 72 h by means of quantitative real-time polymerase chain reaction and live/dead staining. Presence of an extracellular polymeric substance (EPS) was visualized by Concanavalin A staining. Increasing incubation times of up to 72 h showed adhesion and propagation of the bacterial strains with artificial saliva formulation. An and Ef had significantly higher growth rates than Fn. Live/dead staining showed a median of 49.9 % (range 46.0-53.0 %) of living bacteria after 72 h of incubation, and 3D fluorescence microscopy showed a three-dimensional structure containing EPS. An in vitro oral polymicrobial biofilm model was established to better simulate oral conditions and had the advantage of providing the well-controlled experimental conditions of in vitro testing.

In vitro optical detection of simulated blood pulse in a human tooth pulp model.

OBJECTIVE: Noninvasive optical methods such as photoplethysmography, established for blood pulse detection in organs, have been proposed for vitality testing of human dental pulp. However, no information is available on the mechanism of action in a closed pulp chamber and on the impairing influence of other than pulpal blood flow sources. Therefore, the aim of the present in vitro study was to develop a device for the optical detection of pulpal blood pulse and to investigate the influence of different parameters (including gingival blood flow [GBF] simulation) on the derived signals. MATERIALS AND METHODS: Air, Millipore water, human erythrocyte suspensions (HES), non-particulate hemoglobin suspension (NPHS), and lysed hemoglobin suspension (LHES) were pulsed through a flexible (silicone) or a rigid (glass) tube placed within an extracted human molar in a tooth-gingiva model. HES was additionally pulsed through a rigid tube around the tooth, simulating GBF alone or combined with the flow through the tooth by two separate peristaltic pumps. Light from high-power light-emitting diodes (625 nm (red) and 940 nm (infrared [IR]); Golden Dragon, Osram, Germany) was introduced to the coronal/buccal part of the tooth, and the signal amplitude [∆U, in volts] of transmitted light was detected by a sensor at the opposite side of the tooth. Signal processing was carried out by means of a newly developed blood pulse detector. Finally, experiments were repeated with the application of rubber dam (blue, purple, pink, and black), aluminum foil, and black antistatic plastic foil. Nonparametric statistical analysis was applied (n = 5; α = 0.05). RESULTS: Signals were obtained for HES and LHES, but not with air, Millipore water, or NPHS. Using a flexible tube, signals for HES were higher for IR compared to red light, whereas for the rigid tube, the signals were significantly higher for red light than for IR. In general, significantly less signal amplitude was recorded for HES with the rigid glass tube than with the flexible tube, but it was still enough to be detected. ∆U from gingiva compared to tooth was significantly lower for red light and higher for IR. Shielding the gingiva was effective for 940 nm light and negligible for 625 nm light. CONCLUSIONS: Pulpal blood pulse can be optically detected in a rigid environment such as a pulp chamber, but GBF may interfere with the signal and the shielding effect of the rubber dam depends on the light wavelength used. CLINICAL RELEVANCE: The optically based recording of blood pulse may be a suitable method for pulp vitality testing, if improvements in the differentiation between different sources of blood pulse are possible.

Establishment of an optimized ex vivo system for artificial root canal infection evaluated by use of sodium hypochlorite and the photodynamic therapy.

AIM: To establish a refined model of artificially infected root canals and confirm its suitability as a sensitive ex vivo method to assess the efficacy of disinfecting agents. Disinfection was evaluated using sodium hypochlorite (NaOCl), either blocked or unblocked by sodium thiosulphate, and a recently promoted method of disinfection, the antibacterial photodynamic therapy (PDT). METHODOLOGY: The roots of bovine incisors were sectioned into three parts, the canals of coronal and middle regions were filled with a suspension of Enterococcus faecalis and the apical region with culture medium. After 7 days, coronal sections were disinfected using NaOCl (0.5%, 1.0% and 3.0% for 30, 60 and 600 s) or a system for photoactivated chemotherapy (PACT; Cumdente, Tübingen, Germany) for antibacterial PDT. Apical sections served as sterile controls and middle sections as bacterial growth controls. In half of the NaOCl-treated specimens, disinfection was arrested. Dentine chips from biopsies at different depths from the central canal towards the periphery were plated and assessed for colony-forming units (CFU). Disinfection was considered biologically relevant if the reduction of CFU was at least three log10 orders of magnitude. RESULTS: Colony-forming units of 10³ - 104 in growth controls indicated effective artificial infection. A biologically relevant reduction of CFU was accomplished with unblocked NaOCl, but not after blocking with NaOCl nor with PDT. CONCLUSIONS: The system reliably detected disinfection of the root canal and dentinal tubules and proved suitable for ex vivo testing of root canal disinfection. The effect of NaOCl depended on the duration of impact. Under the present experimental conditions, the antibacterial PDT system did not achieve sufficient disinfection.

Efficacy of a Mouthwash Containing CHX and CPC in SARS-CoV-2-Positive Patients: A Randomized Controlled Clinical Trial.

Soon after the outbreak of the coronavirus disease 2019 (COVID-19) pandemic, preprocedural mouthwashes were recommended for temporarily reducing intraoral viral load and infectivity of individuals potentially infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in order to protect medical personnel. Particularly, the antiseptic cetylpyridinium chloride (CPC) has shown virucidal effects against SARS-CoV-2 in vitro. Therefore, the aim of this randomized controlled clinical trial was to investigate the efficacy of a commercially available mouthwash containing CPC and chlorhexidine digluconate (CHX) at 0.05% each in SARS-CoV-2-positive patients as compared to a placebo mouthwash. Sixty-one patients who tested positive for SARS-CoV-2 with onset of symptoms within the last 72 h were included in this study. Oropharyngeal specimens were taken at baseline, whereupon patients had to gargle mouth and throat with 20 mL test or placebo (0.9% NaCl) mouthwash for 60 s. After 30 min, further oropharyngeal specimens were collected. Viral load was analyzed by quantitative reverse transcriptase polymerase chain reaction, and infectivity of oropharyngeal specimens was analyzed by virus rescue in cell culture and quantified via determination of tissue culture infectious doses 50% (TCID50). Data were analyzed nonparametrically (α = 0.05). Viral load slightly but significantly decreased upon gargling in the test group (P = 0.0435) but not in the placebo group. Viral infectivity as measured by TCID50 also significantly decreased in the test group (P = 0.0313), whereas there was no significant effect but a trend in the placebo group. Furthermore, it was found that the specimens from patients with a vaccine booster exhibited significantly lower infectivity at baseline as compared to those without vaccine booster (P = 0.0231). This study indicates that a preprocedural mouthwash containing CPC and CHX could slightly but significantly reduce the viral load and infectivity in SARS-CoV-2-positive patients. Further studies are needed to corroborate these results and investigate whether the observed reductions in viral load and infectivity could translate into clinically useful effects in reducing COVID-19 transmission (German Clinical Trials Register DRKS00027812).

Combination of 10% EDTA, Photosan, and a blue light hand-held photopolymerizer to inactivate leading oral bacteria in dentistry in vitro.

AIMS: The goal of this study was to investigate the phototoxicity of Photosan in combination with EDTA and a hand-held photopolymerizer used in dentistry for light-curing resins against leading key pathogens in caries, endodontic treatment failures, and periodontitis respectively. METHODS AND RESULTS: Cellular uptake of Photosan was detected by fluorescence spectroscopy for Streptococcus mutans and Enterococcus faecalis but not for Aggregatibacter actinomycetemcomitans. Addition of 10% EDTA enabled the uptake of Photosan by A. actinomycetemcomitans. Killing of S. mutans and E. faecalis mediated by Photosan and blue light was concentration and light dose dependent, achieving a >or=99.9% (>or=3 log(10) reduction) efficacy of bacteria killing. In the presence of 10% EDTA, Photosan induced a reduction of >or=4 log(10) in the viability of A. actinomycetemcomitans at a concentration of 50 microg ml(-1), upon activation at a dose of 9.65 J cm(-2) for 60 s. EDTA alone, light alone, and Photosan alone were not able to kill bacteria. CONCLUSIONS: Ten per cent EDTA and Photosan cause a potent phototoxicity against oral bacteria upon illumination with a photopolymerizer. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing antibiotic resistance and insufficient drug concentrations within the sulcus fluid are responsible for lacking antimicrobial efficacy. This study provides useful information that combination of Photosan, EDTA, and a photopolymerizer may be a potentially powerful tool for the efficient destroying of key oral bacteria.

Cytotoxicity and genotoxicity of pulp capping materials in two cell lines.

AIM: The aim of this study was to evaluate the cytotoxicity and genotoxicity of the new castor oil bean cement (COB) material in comparison to commonly used pulp capping materials. METHODOLOGY: Specimens of COB, calcium hydroxide (Hydro C), and mineral trioxide aggregate (white and gray MTA) were extracted in culture medium (91.6 mm(2) sample surface mL(-1)). Transfected human pulp cells (tHPCs) were exposed to dilutions of the extracts for 1 h, and the generation of reactive oxygen species (ROS) was determined by flow cytometry (FACS) using H(2)DCF-DA as a dye. Survival of tHPCs was measured photometrically using a crystal violet assay after a 24-h exposure period. Genotoxicity as indicated by the formation of micronuclei in V79 cells, and the modification of the normal cell cycle by extracts of the materials was analysed by FACS. RESULTS: Clear cytotoxic effects were detected only with extracts of Hydro C under the current experimental conditions. The two MTA preparations induced an insignificant reduction in the number of cells. In contrast, the extracts of COB slightly induced cell proliferation. Extracts of Hydro C caused a twofold increase in ROS production, whilst the other tested materials were ineffective. An increase in the number of micronuclei was not detected with any material tested; Hydro C slightly increased the number of cells in G1 and G2. CONCLUSIONS: The COB and the two MTA preparations did not negatively influence cell survival or ROS production and may thus be further considered for pulp capping studies.

Rheological properties of fresh and reconstituted milk protein concentrates under standard and processing conditions.

As the processability of fresh and reconstituted milk protein concentrates crucially depends on their rheological properties, a considerable amount of studies focuses on this topic. By means of a direct comparison, we are the first to clearly show that distinct rheological differences can exist between fresh and reconstituted milk protein concentrates under standard and processing conditions. We show that reconstituted milk protein concentrates made from commercial milk protein powders exhibit higher viscosities than fresh ones. Furthermore, we found that during intense shearing, the reconstituted milk protein concentrates undergo a loss of structure, which manifests itself in a significant viscosity decrease. The inverse effect can be observed for fresh milk protein concentrates. Besides these differences, the reconstituted milk protein concentrates exhibit gel-like properties above a certain protein content. We attribute these observations to protein-protein interactions in the milk protein powder, which are induced by manufacturing and/or storing conditions. Our results demonstrate that rheological properties of fresh and reconstituted milk protein concentrates are quantitatively not invariably interchangeable. Thus, the purpose of this article is to emphasize the necessity for researchers and engineers to take into account the rheological particularities of different milk protein concentrates prior to usage.

The induction of oxidative stress, cytotoxicity, and genotoxicity by dental adhesives.

OBJECTIVES: Polymerized dental resin materials release residual monomers that may interact with pulp tissues. We hypothesized that dental adhesives might cause cytotoxicity in pulp cells via the generation of reactive oxygen species (ROS), which may also contribute to genotoxic effects in vitro. METHODS: For cytotoxicity testing, transformed human pulp-derived cells were exposed to extracts of primers and bonding agents of Clearfil SE bond, Clearfil Protect bond, AdheSE, Prompt L-Pop, and Excite for 24h. The cytotoxicity of the same materials was also analyzed in a dentin barrier test device using three-dimensional pulp cell cultures. The generation of ROS in monolayer cultures was measured after a 1h exposure period by flow cytometry (FACS), and genotoxicity as indicated by the formation of micronuclei was determined in V79 cells after a 24h exposure period. RESULTS: The dentin primers and bonding agents decrease cell survival in a dose-related manner. Cytotoxicity of bonding agents based on concentrations which caused 50% cell death (EC50) were ranked as follows: Excite (0.16 mg/ml)>AdheSE bond (0.30 mg/ml)>Clearfil Protect bond (0.35 mg/ml)>Clearfil SE bond (0.37 mg/ml), and Prompt L-Pop bond (0.68 mg/ml). Dentin primers were about 10-fold less effective. In contrast, no cytotoxic effects of the dental adhesives were observed in a dentin barrier test device. Yet, all dental adhesives increased the amounts of ROS about fivefold in pulp cells in a dose-related manner, and, again, the bonding agents were more efficient than the dentin primers. Finally, the number of micronuclei was increased about sixfold by extracts of the AdheSE primer. SIGNIFICANCE: Our results suggest that the cytotoxic potencies demonstrated by these materials might be of clinical relevance, since all dental adhesives disturbed the cellular redox state of pulp cells in monolayer cultures. As a result, the concentrations of biologically active ingredients of some of the agents may be high enough to modify pulp cell metabolism when the materials are used in deep cavities or directly contact pulp tissue.

Inhibition of TEGDMA and HEMA-induced genotoxicity and cell cycle arrest by N-acetylcysteine.

OBJECTIVES: Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) are able to cause an imbalance of the redox state in mammalian cells. The resulting oxidative stress originating from reactive oxygen species (ROS) has been associated with cytotoxicity. We hypothesized that ROS might contribute to the generation of genotoxicity by TEGDMA and HEMA as well. Therefore, we examined the formation of micronuclei in V79 cells by both resin monomers in the presence of the antioxidant N-acetylcysteine (NAC), which scavenges ROS. In addition, we analyzed the effects of TEGDMA and HEMA on the normal cell cycle in the presence of NAC. METHODS: V79 fibroblasts were exposed to increasing concentrations of TEGDMA and HEMA in the presence and absence of NAC for 24h. Genotoxicity was indicated by the formation of micronuclei. The modification of the normal cell cycle was analyzed by flow cytometry (FACS). RESULTS: A dose-related increase in the number of micronuclei in V79 cells-induced by TEGDMA and HEMA indicated genotoxicity of both chemicals. However, the formation of micronuclei was reduced in the presence of 10 mmol/L NAC, indicating its protective role. A cell cycle delay in G2 phase caused by TEGDMA was absent when cells were co-treated with NAC. Similarly, the presence of NAC led to a reversion of the cell cycle delay in HEMA-treated cell cultures. SIGNIFICANCE: Our results suggest that genotoxic effects and the modification of the cell cycle caused by TEGDMA and HEMA are mediated, at least in part, by oxidative stress.

Note: Nanomechanical characterization of soft materials using a micro-machined nanoforce transducer with an FIB-made pyramidal tip.

The quantitative nanomechanical characterization of soft materials using the nanoindentation tech-nique requires further improvements in the performances of instruments, including their force resolution in particular. A micro-machined silicon nanoforce transducer based upon electrostatic comb drives featuring the force and depth resolutions down to ∼1 nN and 0.2 nm, respectively, is described. At the end of the MEMS transducer's main shaft, a pyramidal tip is fabricated using a focused ion beam facility. A proof-of-principle setup with this MEMS nanoindenter has been established to measure the mechanical properties of soft polydimethylsiloxane. First measurement results demonstrate that the prototype measurement system is able to quantitatively characterize soft materials with elastic moduli down to a few MPa.

Critical role of superoxide anions and hydroxyl radicals in HEMA-induced apoptosis.

OBJECTIVE: Resin monomers such as 2-hydroxyethyl methacrylate (HEMA) induce apoptosis because of the excess formation of reactive oxygen species (ROS). The portion of ROS including superoxide anions, hydrogen peroxide or hydroxyl radicals in monomer-induced apoptosis is unknown. Here, the effectiveness of superoxide anions or hydroxyl radicals was analyzed using tempol or sodium formate as radical scavengers. METHODS: RAW264.7 mouse macrophages were exposed to HEMA (0-6-8mM) in the presence of tempol (0-0.05-0.5-5.0mM) or sodium formate (0-1-5-10mM). The formation of ROS using DCFH2-DA or dihydrorhodamine 123 (DHR123) as fluorescent dyes and the induction of apoptosis was determined by flow cytometry after 1h or 24h exposure periods. Expression of enzymes related to ROS metabolism was detected by Western blotting. RESULTS: DCF fluorescence significantly increased after short exposure (1h) while DHR123 fluorescence was enhanced after a long exposure period (24h) in cells treated with HEMA. Although no influence was detected on the formation of ROS, tempol or sodium formate protected cells from HEMA-induced apoptosis. The number of cells in late apoptosis or necrosis induced with 6 or 8mM HEMA was reduced in the presence of tempol or low concentrations of sodium formate. HEMA-induced expression of catalase, indicating oxidative stress, decreased in the presence of tempol. SIGNIFICANCE: Superoxide anions and hydroxyl radicals contribute to HEMA-induced apoptosis. The current findings support the development of strategies based on the pharmacological inhibition of enzymes producing superoxide anions finally converted to hydroxyl radicals to compensate for potential adverse tissue reactions associated with dental composites.