Mechanistic study of the stabilization of dentin-bonded restorative interfaces via collagen reinforcement by multi-acrylamides.

Hydrolytically and enzymatically-stable multi-acrylamides have been proposed to increase the long-term durability of dental adhesive interfaces as alternatives to methacrylates. The aim of this study was to investigate the mechanical and biochemical properties of experimental adhesives containing multi-functional acrylamides concerning collagen reinforcement and metalloproteinases (MMP) activity. Multi-functional acrylamides, TMAAEA (Tris[(2-methylaminoacryl) ethylamine) and DEBAAP (N,N-Diethyl-1,3-bis(acrylamido) propane), along with the commercially available DMAM (N,N-dimethylacrylamide) (monofunctional acrylamide) and HEMA (2-Hydroxyethyl methacrylate) (monofunctional methacrylate - control) were tested for stability against enzymatic hydrolysis by cholesterol esterase/pseudocholinesterase (PC/PCE) solutions for up to 30 days. Collagen-derived substrate and gelatin zymography were performed to examine the effect of the compounds on the biological activity of human recombinant and dentin-extracted gelatinases MMP-2 and MMP-9. In situ zymography was carried out by fluorescent collagen degradation combined with confocal microscopy analysis. Hydroxyproline content was measured in collagen derived from dentin extracts though reaction with Ehrlich's reagent p-dimethylaminobenzaldehyde (DMAB), generating a stable chromophore measured at 550 nm. Storage shear modulus of demineralized dentin discs treated with the tested compounds was measured by oscillatory rheometry, in order to investigate potential collagen reinforcement. FT-IR was performed to determine qualitative differences in collagen based on observed changes in amide bands. The results were analyzed by ANOVA/Tukey's test (α = 0.05). Multi-acrylamides survived 30 days of incubation in cholinesterase/pseudo-cholinesterase (PC/PCE) solutions, while HEMA showed approximately 70 % overall degradation. Incubation with multi-acrylamides reduced collagen degradation as evidenced by the reduced hydroxyproline levels and by the 30 % increase inshear storage modulus. Biochemical and zymography assays showed no noticeable inhibition of recombinant and extracted MMPs enzymatic activity. The infra-red spectroscopy results for multi-functional acrylamides treated samples demonstrated shifts of the amide II bonds and marked increase in intensity of the bands 1200 cm-1, which may indicate partial collagen denaturation and some degree of cross-linking of the compounds with collagen, respectively. The multi-acrylamides exhibited not only comparable mechanical properties but also demonstrated significantly enhanced biochemical stability when compared to the widely used methacrylate control. Clinical relevance: These findings highlight the potential of multi-acrylamides to increase the bonding stability to tissues and, ultimately, contribute to the longevity of dental restorations.

Triacrylamide-Based Adhesives Stabilize Bonds in Physiologic Conditions.

In this study, an acrylamide-based adhesive was combined with a thiourethane-based composite to improve bond stability and reduce polymerization stress, respectively, of simulated composite restorations. The stability testing was conducted under physiologic conditions, combining mechanical and bacterial challenges. Urethane dimethacrylate was combined with a newly synthesized triacrylamide (TMAAEA) or HEMA (2-hydroxyethyl-methacrylate; control) to produce a 2-step total-etch adhesive system. Methacrylate-based composites (70 wt% silanized filler) were formulated, containing thiourethane oligomers at 0 (control) or 20 wt%. Standardized preparations in human third molars were restored; then, epoxy replicas were obtained from the occlusal surfaces before and after 7-d storage in water or with Streptococcus mutans biofilm, which was tested after storage in an incubator (static) or the bioreactor (mechanical challenge). Images were obtained from the replicas (scanning electron microscopy) and cross sections of the samples (confocal laser scanning microscopy) and then analyzed to obtain measurements of gap, bacterial infiltration, and demineralization. Microtensile bond strength of specimens stored in water or biofilm was assessed in 1-mm2 stick specimens. Data were analyzed with analysis of variance and Tukey's test (α = 0.05). HEMA-based materials had greater initial gap measurements, indicating more efficient bonding for the acrylamide materials. When tested in water, the triacrylamide-based adhesive had smaller gaps in the incubator or bioreactor. In the presence of biofilm, there was less difference among materials, but the acrylamide/thiourethane combination led to statistically lower gap formation in the bioreactor. HEMA and TMAAEA-based adhesives produced statistically similar microtensile bond strengths after being stored in water for 7 d, but after the same period with biofilm-challenged specimens, the TMAAEA-based adhesives were the only ones to retain the initial bond strength values. The use of a stable multiacrylamide-based adhesive led to the preservation of the resin-dentin bonded interface after a physiologically relevant challenge. Future studies will include a multispecies biofilm model.

Probing stress relaxation behavior in glassy methacrylate networks containing thio-carbamate additives.

The incorporation of thiourethane prepolymer (TU) into either the organic phase or as a surface treatment for filler particles in composites reduces polymerization stress and improves fracture toughness. The aim of this study was to gain insight into the influence of the inclusion of thiourethanes on the resulting network of methacrylate-based materials polymerized via free-radical mechanisms. Dynamic mechanical analysis was used to elucidate network parameters and potential stress relaxation behavior of these networks. TU oligomers were synthesized using a combination of trimethylol-tris-3-mercaptopropionate and dicyclohexylmethane 4,4'-diisocyanate and added into composite formulations at 20 wt% replacing part of the organic matrix and/or as TU-silanes used to functionalize filler particles (TU-matrix, TU-Sil or TU-matrix/sil). Materials not containing any form of TU were used as the control (in those cases, 3-(trimethoxysilyl)propyl methacrylate was used as the silane agent). Filler was added at 50 wt%. Degree of conversion was evaluated by near-IR spectroscopy, mechanical properties by 3-point bending and rotational rheometry. Dynamic mechanical analysis was used to obtain network parameters (glass transition temperature (Tg), storage modulus, cross-link density, and breadth of tan delta a proxy for network homogeneity - temperature sweep experiments) and to evaluate the potential for network relaxation (stress relaxation). TU-containing formulations showed 10% higher DC than the control. The time to reach storage/loss modulus crossover in the rheometer experiments was significantly longer for TU-matrix and TU-matrix/sil in comparison with the control (21.6, 27.9, and 5.1 s, respectively). TU-matrix and TU-matrix/sil presented significant lower Tg than the control (151.5, 153.8, and 161.3 °C, respectively). There were no statistical differences among the groups in terms of shear modulus, cross-link density, breadth of tan delta, flexural strength/modulus, and toughness. For at least one group (TU-matrix/sil), the relaxation time was four times faster than for the control at 105 °C. The addition of TU additives into dental polymers resulted in a stark reduction in the stress relaxation time. This behavior, in tandem with the network characterization and mechanical properties seems to indicate the TU networks undergo a variety of reversible associative and dissociative chemical reactions which facilitate enhanced stress relief.

Thiourethane filler functionalization for dental resin composites: Concentration-dependent effects on toughening, stress reduction and depth of cure.

OBJECTIVES: The aim of this study was to modify the surface of fillers used in dental composites by the synthesis of two novel thiourethane oligomeric silanes, used to functionalize the silica-containing inorganic particles. Several thiourethane silane concentrations were tested during the silanization process to systematically assess the effect of silane coverage on experimental composite conversion, polymerization stress and fracture toughness. MATERIALS AND METHODS: Two different thiourethane silanes were synthesized based either on 1,6-hexanediol-diissocynate (HDDI), or 1,3-bis(1-isocyanato-1-methylethyl) benzene (BDI). Conventional 3-(Trimethoxysilyl)propyl methacrylate was used as the control. Glass fillers were silanized with 1, 2 or 4 wt% of each thiourethane silane, then evaluated by thermogravimetrical analysis. Photopolymerizable resin composites were prepared with Bis-GMA/UDMA/TEGDMA and 50 wt% silanized glass filler. Polymerization kinetics and degree of conversion were tested using Near-IR. Bioman was used to test polymerization stress. Data were analyzed with two-way ANOVA/Tukey's test (α = 5%). RESULTS: The mass of silane coupled to the filler increased with the concentrations of thiourethane in the silanizing solution, as expected. Thiourethane-containing groups exhibited significantly higher degree of conversion compared to control groups, except for BDI 4%. HDDI 4%, BDI 2% and BDI 4% showed significantly lower polymerization stress than control groups. HDDI 4% exhibited significantly higher fracture toughness. CONCLUSIONS AND CLINICAL SIGNIFICANCE: Novel filler functionalization with thiourethane silanes may be a promising alternative for improving dental composites properties by significantly increasing the degree of conversion, fracture toughness and reducing the polymerization stress.

Effects of systematically varied thiourethane-functionalized filler concentration on polymerization behavior and relevant clinical properties of dental composites.

Introduction of thiourethane (TU) oligomer to resin-based dental restorative materials reduces stress and improves fracture toughness without compromising conversion. Localization of TU at the resin-filler interface via silanization procedures may lead to more substantial stress reduction and clinical property enhancements. The objective of this study was to evaluate composite properties as a function of TU-functionalized filler concentration. TU oligomers were synthesized using click-chemistry techniques and subsequently silanized to barium glass filler. Resin-based composites were formulated using varying ratios of TU-functionalized filler and conventional methacrylate-silanized barium filler. Material property testing included thermogravimetric analysis, real-time polymerization kinetics and depth of cure, polymerization stress, stress relaxation and fracture toughness. Clinical property testing included water sorption/solubility, composite paste viscosity, and gloss and surface roughness measured before and after subjecting the samples to 6 h of continuous tooth brushing in a custom-built apparatus using a toothpaste/water mixture. Increasing TU-filler in the composite resulted in as much as a 78% reduction in stress, coupled with an increase in fracture toughness. Conversion was similar for all groups. After simulated tooth brushing, gloss reduction was lower for TU-containing composites and surface roughness was less than or equal to the control.

Effect of the addition of thiourethane oligomers on the solâ¿¿gel composition of BisGMA/TEGDMA polymer networks.

OBJECTIVES: Thiourethane oligomers have been shown to increase the fracture toughness and reduce the polymerization stress of methacrylate-based materials. However, network formation has not been elucidated in these materials yet. The aim of this study was to evaluate how the addition of a thiourethane oligomer (TU) influences the sol/gel composition and network structure of methacrylate-based materials using dynamic mechanical analysis and extraction methods. MATERIALS AND METHODS: BisGMA/TEGDMA at systematically varied mass ratios (20/80 to 80/20wt%) were mixed with pre-polymerized thiourethane oligomers at 0 (control) or 20wt%, synthesized by combining pentaerythritol tetra-3-mercaptopropionate with dicyclohexylmethane 4,4⿲-Diisocyanate, at 1:2 isocyanate:thiol. 0.1wt% of 2,2-Dimethoxy-2-phenylacetophenone was added as the photoinitiator and 0.3wt% of 2,6-di-tert-butyl-4-methylphenol was added as a free radical inhibitor. Disk specimens (0.8ÿ10mm in diameter, n=3) were photoactivated at 270mW/ (320â¿¿500nm) for 1min. The degree of conversion (DC) was measured in near-IR (Ë¿6165cmâ¿¿1). Specimens were immersed in two different solvents (water for 7 days or dicholoromethane for 48h). Water sorption (WS) and solubility (SL) were obtained according to ISO 4049. The leachates for both solutions were analyzed with 1H-NMR (400MHz, CDCL3). Bar specimens (1ÿ3ÿ25mm, photocured and then post-processed at 180°C for 8h to DC>95%) were subjected to dynamic mechanical analysis (â¿¿30 to 230°C) to obtain glass transition temperature (Tg), tan delta curves and crosslinking density (ν). Data was analyzed with two-way ANOVA/Tukeyâ¿¿s test (95%). RESULTS: In general, the presence of TU increased the overall conversion. The WS was similar for all groups, but the SL decreased by 2-fold with the addition of the TU oligomer for all compositions, except BisGMA/TEGDMA 80/20. The BisGMA concentration of the leachates increased with increasing BisGMA in the initial mixture, and with the presence of thiourethane. This compositional drift of the gel with the presence of TU was attributed to the preferential dissolution of TEGDMA into the TU network. Tg and ν decreased with the addition of TU, as expected. The addition of TU produced more homogeneous networks, as evidenced by narrower breadth of the tan delta curve. CONCLUSION: The addition of TU affected the composition of the sol/gel in crosslinked networks, which were more homogeneous and presented 2-fold less potentially toxic leachates than the methacrylate controls. CLINICAL SIGNIFICANCE: The addition of TU may produce less cytotoxic materials based on the increased conversion and reduced amount of unreacted extractables from its network after water storage.

Construction of monomer-free, highly crosslinked, water-compatible polymers.

Polymeric dental adhesives require the formation of densely crosslinked network structures to best ensure mechanical strength and durability in clinical service. Monomeric precursors to these materials typically consist of mixtures of hydrophilic and hydrophobic components that potentially undergo phase separation in the presence of low concentrations of water, which is detrimental to material performance and has motivated significant investigation into formulations that reduce this effect. We have investigated an approach to network formation based on nanogels that are dispersed in inert solvent and directly polymerized into crosslinked polymers. Monomers of various hydrophilic or hydrophobic characteristics were copolymerized into particulate nanogels bearing internal and external polymerizable functionality. Nanogel dispersions were stable at high concentrations in acetone or, with some exceptions, in water and produced networks with a wide range of mechanical properties. Networks formed rapidly upon light activation and reached high conversion with extremely low volumetric shrinkage. Prepolymerizing monomers into reactive nanostructures significantly changes how hydrophobic materials respond to water compared with networks obtained from polymerizations involving free monomer. The modulus of fully hydrated networks formed solely from nanogels was shown to equal or exceed the modulus in the dry state for networks based on nanogels containing a hydrophobic dimethacrylate and hydrophilic monomethacrylate, a result that was not observed in a hydroxyethyl methacrylate (HEMA) homopolymer or in networks formed from nanogels copolymerized with HEMA. These results highlight the unique approach to network development from nanoscale precursors and properties that have direct implications in functional dental materials.

Improved dental adhesive formulations based on reactive nanogel additives.

Current challenges in adhesive dentistry include over-hydrophilic bonding formulations, which facilitate water percolation through the hybrid layer and result in unreliable bonded interfaces. This study introduces nanogel-modified adhesives as a way to control the material's hydrophobic character without changing the basic monomer formulation (keeping water-chasing capacity and operatory techniques unaltered). Nanogel additives of varied hydrophobicity were synthesized in solution, rendering 10- to 100-nm-sized particles. A model BisGMA/HEMA solvated adhesive was prepared (control), to which reactive nanogels were added. The increase in adhesive viscosity did not impair solvent removal by air-thinning. The degree of conversion in the adhesive was similar between control and nanogel-modified materials, while the bulk dry and, particularly, the wet mechanical properties were significantly improved through nanogel-based network reinforcement and reduced water solubility. As preliminary validation of this approach, short-term micro-tensile bond strengths to acid-etched and primed dentin were significantly enhanced by nanogel inclusion in the adhesive resins.

Transmural variation of myocardial attenuation measured with a clinical imager.

The objective of this study was to quantify the transmural variation in attenuation for the septal and lateral walls of the heart. Our approach was to utilize a commercially available ultrasonic imaging system to acquire images of excised sections of eight sheep hearts with an orientation similar to that encountered in the apical four-chamber view. The measured values (mean +/-SE) of the slope of attenuation for the transmural regions of the septum are: 1.40 +/-0.11, 0.99 +/-0.09, and 1.85 +/-0.16 (dB/cm/MHz) for the left subendocardial, midmyocardial, and right subendocardial zones, respectively. The analogous data from the lateral wall are: 1.42 +/-0.11, 0.83 +/-0.07, and 1.20 +/- 0.16 (dB/cm/MHz) for the subendocardial, midmyocardial, and subepicardial zones, respectively. These data demonstrate that ultrasonic attenuation associated with the septum and the lateral wall, when imaged in a manner similar to that of the apical four-chamber view, is anisotropic.

The placenta and its significance in neonatal outcome.

The placenta should not be overlooked as a source of much valuable diagnostic information. Close evaluation of the placenta and its attached membranes may reveal further information. Essential data may be obtained from pathologic examination, and if questions exist, specimens should be retained with proper care.

Effects of tissue anisotropy on the spectral characteristics of ultrasonic backscatter measured with a clinical imaging system.

In this paper, we report the effects of inherent tissue anisotropy on the spectral properties of backscattered ultrasound when measured with a commercially-available imaging system. We insonified five specimens of bovine tendon immersed in a water tank and rotated in 10 degrees increments while being imaged with a Hewlett-Packard Sonos 1500 system. The backscattered RF signals corresponding to each angle of insonification were digitized and the spectral characteristics of the backscattered ultrasound were determined. The mean anisotropy, defined as the average difference between values at perpendicular and parallel insonification, for band-limited estimates of backscattered power, centroid frequency, upper-band to lower-band power ratio, and upper-band to total-band power ratio were found to be 24.6 +/- 1.1 dB, 142 +/- 27 kHz, 32 +/- 13%, and 22 +/- 5%, respectively (mean +/- SE). The magnitude of each of these backscatter spectral parameters was larger at perpendicular insonification compared with the corresponding values at parallel insonification, consistent with previous measurements of the inherent anisotropy of ultrasonic attenuation and backscatter in tissue.

Comparison of integrated backscatter values obtained with acoustic densitometry with values derived from spectral analysis of digitized signals from a clinical imaging system.

Time-domain-based integrated backscatter values obtained with the use of acoustic densitometry (AD) were compared with values determined from a spectral-based analysis of the radio-frequency (RF) signals with a modified Hewlett-Packard Sonos 1500 imaging system. Integrated backscatter images of five specimens of bovine tendon were acquired in the AD acquisition mode, and the corresponding signals related to the backscattered RF were digitized for each angle of insonification as the specimens were rotated in 10-degree increments. The integrated backscatter images were analyzed with the AD analysis package, and the corresponding values determined from the RF power spectra were obtained from the digitized ultrasonic signals. Good agreement was found between the two methods over the entire range of measured values. The mean anisotropy in the measured integrated backscatter (mean +/- standard error) was found to be 27 +/- 2 dB for time-domain-based analysis and 25 +/- 2 dB for RF spectral-based analysis.

HIV-1 in trophoblastic and villous Hofbauer cells, and haematological precursors in eight-week fetuses.

AIDS in children is usually caused by vertical transmission of human immunodeficiency virus type 1 (HIV-1). Aborted eight-week fetal and placental tissue from HIV-1 positive and negative (enzyme-linked immunosorbent assay and Western blot) women was analysed by immunocytochemistry and in-situ hybridisation. Maternal decidual leucocytes, villous trophoblastic derivatives, villous mesenchymal cells, and embryonic blood cell precursors in tissues from seropositive patients all stained for HIV-1 antigen and hybridised for nucleic acids. These observations suggest that a cytological pathway for vertical transmission of HIV-1 is established by eight weeks gestational age.

Benign mesenchymoma of the round ligament. A report of two cases with immunohistochemistry.

Benign mesenchymomas are tumors composed of an admixture of two or more mature mesenchymal tissue types. Clinically and histologically they are benign lesions. However, they are seldom encapsulated and therefore may recur locally if incompletely excised. The authors report on two benign mesenchymomas composed of mature fat, thin-walled small and medium size vessels, and spindle cells demonstrated to be smooth muscle by immunohistochemical studies. They were diagnosed in women in their 50s. Both were located in the round ligament of the uterus. One was discovered incidentally during hysterectomy, and the other was unusually large, being diagnosed preoperatively as a retroperitoneal liposarcoma. Most round ligament tumors are leiomyomas or fibromas. To the authors' knowledge, this is the first report of benign mesenchymomas occurring in this location.

Correlation of karyotype and immunophenotype in childhood acute lymphoblastic leukemia.

To correlate leukemic cell karyotype with immunophenotype, we studied 364 children with acute lymphoblastic leukemia (ALL). A prognostically favorable cytogenetic feature, hyperdiploidy greater than 50 chromosomes, was found in 33% of cases classified as common ALL antigen positive (CALLA+) early pre-B (common) ALL, in contrast to 18% of pre-B cases (P = .012), 5% of T cell cases (P less than .001), and none of the B cell cases (P less than .001) or cases of CALLA negative (CALLA-) early pre-B ALL (P = .002). The frequency of translocations, an adverse cytogenetic feature, was significantly lower in CALLA+ early pre-B ALL cases (35%) than in B cell (100%; P less than .0001), pre-B (59%; P less than .001), or CALLA- early pre-B (62%; P = .016) cases. Thus, patterns of chromosomal change differ widely among the major immunophenotypic groups of ALL and may account for reported differences in responsiveness to treatment.

Tumorigenicity of embryonal carcinoma as an assay to study control of malignancy by the murine blastocyst.

A bioassay, based on tumorigenicity, has been developed to determine the mechanism whereby the blastocyst of the mouse controls malignant expression of embryonal carcinoma. The assay is based upon the incidence of tumors obtained when known numbers of cells of the 402AX strain of embryonal carcinoma are injected into strain 129 mice, compared to the incidence obtained when the same number of embryonal carcinoma cells are incorporated into Swiss-Webster blastocysts that are then injected in strain 129 animals. The results indicate that the blastocyst can regulate one embryonal carcinoma cell consistently; it may have a slight effect on three, but it cannot regulate four or five of them. The position of the embryonal carcinoma cell in the blastocyst is important. Regulation occurs if the embryonal carcinoma cell is placed in the blastocoele cavity, but enhancement of tumorigenicity is obtained if it is placed between the zona pellucida and the trophectoderm. By contrast, the blastocyst is unable to regulate a single B-16 melanoma cell placed in the blastocoele cavity, indicating a degree of specificity for the regulatory process.