Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation.

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.

Functionalised amyloid fibrils for roles in cell adhesion

We describe experiments designed to explore the possibility of using amyloid fibrils as new nanoscale biomaterials for promoting and exploiting cell adhesion, migration and differentiation in vitro. We created peptides that add the biological cell adhesion sequence (RGD) or a control sequence (RAD) to the C-terminus of an 11-residue peptide corresponding to residues 105-115 of the amyloidogenic protein transthyretin. These peptides readily self-assemble in aqueous solution to form amyloid fibrils, and X-ray fibre diffraction shows that they possess the same strand and sheet spacing in the characteristic cross-beta structure as do fibrils formed by the parent peptide. We report that the fibrils containing the RGD sequence are bioactive and that these fibrils interact specifically with cells via the RGD group displayed on the fibril surface. As the design of such functionalized fibrils can be systematically altered, these findings suggest that it will be possible to generate nanomaterials based on amyloid fibrils that are tailored to promote interactions with a wide variety of cell types. (c) 2007 Elsevier Ltd. All rights reserved.

Hydrogelation of self-assembling RGD-based peptides

The self-assembly of tripeptides based on the RGD cell adhesion motif is investigated. Two tripeptides containing the Fmoc [N-(fluorenyl)-9-methoxycarbonyl] aromatic unit were synthesized, Fmoc-RGD and a control peptide containing a scrambled sequence, Fmoc-GRD. The Fmoc is used to control selfassembly via aromatic stacking interactions. The self-assembly and hydrogelation properties of the two Fmoc-tripeptides are compared. Both form well defined amyloid fibrils (as shown by cryo-TEM and SAXS) with b-sheet features in their circular dichroism and FTIR spectra. Both peptides form selfsupporting hydrogels, the dynamic shear modulus of which was measured. Preliminary cell culture experiments reveal that Fmoc-RGD can be used as a support for bovine fibroblasts, but not Fmoc- GRD, consistent with the incorporation of the cell adhesion motif in the former peptide.

Alanine-rich amphiphilic peptide containing the RGD cell adhesion motif: a coating material for human fibroblast attachment and culture

We studied the self-assembly of peptide A6RGD (A: alanine, R: arginine, G: glycine, D: aspartic acid) in water, and the use of A6RGD substrates as coatings to promote the attachment of human cornea stromal fibroblasts (hCSFs). The self-assembled motif of A6RGD was shown to depend on the peptide concentration in water, where both vesicle and fibril formation were observed. Oligomers were detected for 0.7 wt% A6RGD, which evolved into short peptide fibres at 1.0 wt% A6RGD, while a co-existence of vesicles and long peptide fibres was revealed for 2–15 wt% A6RGD. A6RGD vesicle walls were shown to have a multilayer structure built out of highly interdigitated A6 units, while A6RGD fibres were based on β-sheet assemblies. Changes in the self-assembly motif with concentration were reflected in the cell culture assay results. Films dried from 0.1–1.0 wt% A6RGD solutions allowed hCSFs to attach and significantly enhanced cell proliferation relative to the control. In contrast, films dried from 2.5 wt% A6RGD solutions were toxic to hCSFs.

Interactions between lipid-free apolipoprotein-AI and a lipopeptide incorporating the RGDS cell adhesion motif

The interaction of a designed bioactive lipopeptide C16-GGGRGDS, comprising a hexadecyl lipid chain attached to a functional heptapeptide, with the lipid-free apoliprotein, Apo-AI, is examined. This apolipoprotein is a major component of high density lipoprotein and it is involved in lipid metabolism and may serve as a biomarker for cardiovascular disease and Alzheimers’ disease. We find via isothermal titration calorimetry that binding between the lipopeptide and Apo-AI occurs up to a saturation condition, just above equimolar for a 10.7 μM concentration of Apo-AI. A similar value is obtained from circular dichroism spectroscopy, which probes the reduction in α-helical secondary structure of Apo-AI upon addition of C16-GGGRGDS. Electron microscopy images show a persistence of fibrillar structures due to self-assembly of C16-GGGRGDS in mixtures with Apo-AI above the saturation binding condition. A small fraction of spheroidal or possibly “nanodisc” structures was observed. Small-angle X-ray scattering (SAXS) data for Apo-AI can be fitted using a published crystal structure of the Apo-AI dimer. The SAXS data for the lipopeptide/ Apo-AI mixtures above the saturation binding conditions can be fitted to the contribution from fibrillar structures coexisting with flat discs corresponding to Apo-AI/lipopeptide aggregates.

Etch and rinse versus self-etching adhesives systems: Tridimensional micromechanical analysis of dentin/adhesive interface

The purpose of this study was to evaluate stress distribution in the hybrid layer produced by two adhesive systems using three-dimensional finite element analysis (FEA). Four FEA models (M) were developed: Mc, a representation of a dentin specimen (41 x 41 x 82 mu m) restored with composite resin, exhibiting the adhesive layer, hybrid layer (HL), resin tags, peritubular dentin, and intertubular dentin to simulate the etch-and-rinse adhesive system; Mr, similar to Mc, with lateral branches of the adhesive; Ma, similar to Mc, however without resin tags and obliterated tubule orifice, to simulate the environment for the self-etching adhesive system; Mat, similar to Ma, with tags. A numerical simulation was performed to obtain the maximum principal stress (sigma(max)). The highest sigma(max) in the HL was observed for the etch-and-rinse adhesive system. The lateral branches increased the sigma(max) in the HL. The resin tags had a little influence on stress distribution with the self-etching system. (C) 2012 Elsevier Ltd. All rights reserved.

Bonding Efficacy of New Self-etching, Self-adhesive Dual-curing Resin Cements to Dental Enamel

Purpose: This study evaluated the efficacy of the union between two new self-etching self-adhesive resin cements and enamel using the microtensile bond strength test.Materials and Methods: Buccal enamel of 80 bovine teeth was submitted to finishing and polishing with metallographic paper to a refinement of #600, in order to obtain a 5-mm(2) flat area. Blocks (2 x 4 x 4 mm) of laboratory composite resin were cemented to enamel according to different protocols: (1) untreated enamel + RelyX Unicem cement (RX group); (2) untreated enamel + Bifix SE cement (BF group); (3) enamel acid etching and application of resin adhesive Single Bond + RelyX Unicem (RXA group); (4) enamel acid etching and application of resin adhesive Solobond M + Bifix SE (BFA group). After 7 days of storage in distillated water at 37 degrees C, the blocks were sectioned for obtaining microbar specimens with an adhesive area of 1 mm(2) (n = 120). Specimens were submitted to the microtensile bond strength test at a crosshead speed of 0.5 mm/min. The results (in MPa) were analyzed statistically by ANOVA and Tu key's test.Results: Enamel pre-treatment with phosphoric acid and resin adhesive (27.9 and 30.3 for RXA and BFA groups) significantly improved (p <= 0.05) the adhesion of both cements to enamel compared to the union achieved with as-polished enamel (9.9 and 6.0 for RX and BF).Conclusion: Enamel pre-treatment with acid etching and the application of resin adhesive significantly improved the bond efficacy of both luting agents compared to the union achieved with as-polished enamel.

Effects of Surface Hydration and Application Method on the Bond Strength of Self-Etching Adhesives to Dentin

The effect of application methods and dentin hydration on the bond strength of three self-etching adhesives (SEA) were evaluated; 195 extracted bovine incisors were used. The buccal surface was ground in order to expose the dentin, which remained 2-mm minimum thickness, measured by a thickness meter through an opening on the lingual surface. Adper Single Bond 2 (TM) was used for the control group. The SEA were applied following two modes of application: passive or active and two hydration states of the dentin surface-dry and wet. After light-curing, composite buildups were made using Grandio (TM) composite. The specimens were sectioned and tested with a microtensile bond strength test. The application method and the hydration state resulted in statistical differences (p = 0.000) making the values of active application for mu TBS to dentin higher than passive application. The wet surfaces showed higher mu TBS to dentin ratios than dry surfaces. There were no statistical differences in mu TBS among the SEA tested but there were differences regarding to control group.

Effects of Self-Etching Adhesive Systems Used in the Dental Modelling Technique on the Cohesive Strength of Composite Resin

This study evaluated the cohesive strength of composite using self-etching adhesive systems (SE) in the lubrication of instruments between layers of composite. The specimens were made by using a Teflon (R) device. SE were used at the interface to lubricate the instruments: Group 1(G1) - control group, no lubricant was used; Group 2(G2) -Futurabond (R) M; Group 3(G3) - Optibond (R) All-In-One; Group 4(G4) - Clearfil (R) SE Bond; Group 5(G5) - Futurabond (R) NR; Group 6(G6) - Adper (R) SE Plus; Group 7(G7) - One Up Bond (R) F. Specimens were submitted to the tensile test to evaluate the cohesive strength. Data were submitted to the ANOVA and Tukey tests. ANOVA showed a value of p = 0.00. The average means (SD): G2 = 11.33(+/-3.44) a, G3 = 15.36(+/-4.06) ab, G4 = 18.9(+/-4.72) bc, G7 = 19.62(+/-4.46) bc, G5 = 21.02(+/-5.09) bc, G6 = 23.39(+/-4.17) cd, and G1 = 28.49(+/-2.89) d. All SE decreased the cohesive strength of the composite, except for Adper (R) SE Plus.

Evaluation of the shear bond strength of a composite resin in sound, demineralized and hypermineralized bovine dentin after irradiation with Er:YAG and Nd:YAG lasers, employing a self-etching adhesive system

Background and Objectives. The adhesion of dental materials is important for the success of treatment. The aim of this study is to evaluate the bond strength of a composite resin applied with a self-etching adhesive system in different dentins after irradiation with Er:YAG and Nd:YAG lasers, observing their morphologic pattern using Scanning Electronic Microscopy (SEM). Materials and Methods. The buccal surface of 72 bovine incisors was worn until exposure of medium depth dentin. The specimens were divided into three groups; GI: normal, GII: demineralized and GIII: hypermineralized dentin. These were also divided into two subgroups; A-irradiated for 30 s with Er:YAG laser in noncontact mode at 40 mJ and 6 Hz and B- irradiated for 30 s with Nd:YAG laser in contact mode at 60 mJ and 10 Hz. The adhesive system Clearfil SE. Bond (Kuraray) and composite resin Tetric Ceram (Vivadent) were applied on the irradiated area by the incremental technique. After storage for 24 h in distilled water at 37 degrees C, the specimens were submitted to the shear strength test in a universal testing machine (EMIC) at a crosshead speed of 1.0 mm/min. Other specimens were made to be analyzed by SEM. Results. The results were statistically analyzed by Analysis of Variance and the Tukey test. Regardless of the type of dentin, the bond strength of specimens irradiated with the Nd:YAG laser (8,94 +/- 2,07) was higher compared to specimens irradiated with the Er:YAG laser (7,03 +/- 2,47); the highest bond strength was obtained for the group of hypermineralized dentin irradiated with the Nd:YAG laser. The SEM analysis showed that the Er:YAG laser caused opening of tubules and the Nd:YAG laser produced areas of fusion as well as regions of opening of dentinal tubules. Conclusions. The dentin showed different morphological patterns and the laser promote alterations on their surfaces, influencing the bond strength of the composite resin. (C) 2010 Laser Institute of America.

Adhesive Quality of Self-adhesive and Conventional Adhesive Resin Cement to Y-TZP Ceramic Before and After Aging Conditions

Purpose: This study evaluated the adhesive quality of simplified self-adhesive and conventional resin cements to Y-TZP in dry and aged conditions. Methods: Y-TZP ceramic blocks (N=192) (5 x 5 x 2 mm) were embedded in acrylic resin and randomly divided into two groups, based on surface conditioning: 96% isopropanol or chairside tribochemical silica coating and silanization. Conditioned ceramics were divided into four groups to receive the resin cements (Panavia F 2.0, Variolink II, RelyX U100 and Maxcem). After 24 hours, half of the specimens (n=12) from each group were submitted to shear bond strength testing (0.5 nun/minute). The remaining specimens were tested after 90 days of water storage at 37 degrees C and thermocycling (12,000x, 5 degrees C-55 degrees C). Failure types were then assessed. The data were analyzed using three-way ANOVA and the Tukey's test (alpha=0.05). Results: Significant effects of ceramic conditioning, cement type and storage conditions were observed (p<0.0001). The groups cleaned using alcohol only showed low bond strength values in dry conditions and the bond strength was reduced dramatically after aging. Groups conditioned using silica coating and silanization showed higher bond strengths both in dry and aged conditions. A high number of specimens failed prematurely prior to testing when they were cleaned using 96% isopropanol. Conclusion: Overall, silica coating and silanization showed higher, stable bond strengths with and without aging. The durability of resin-ceramic adhesion varied, depending on the adhesive cement type.

Tensile bond strength of self-etching versus total-etching adhesive systems under different dentinal substrate conditions

The use of acid etchants to produce surface demineralization and collagen network exposure, allowing adhesive monomers interdiffusion and consequently the formation of a hybrid layer, has been considered the most efficient mechanism of dentin bonding. The aim of this study was to compare the tensile bond strength to dentin of three adhesive systems, two self-etching ones (Clearfil SE Bond - CSEB and One Up Bond F - OUBF) and one total-etching one (Single Bond - SB), under three dentinal substrate conditions (wet, dry and re-wet). Ninety human, freshly extracted third molars were sectioned at the occlusal surface to remove enamel and to form a flat dentin wall. The specimens were restored with composite resin (Filtek Z250) and submitted to tensile bond strength testing (TBS) in an MTS 810. The data were submitted to two-way ANOVA and Tukey's test (p = 0.05). Wet dentin presented the highest TBS values for SB and CSEB. Dry dentin and re-wet produced significantly lower TBS values when using SB. OUBF was not affected by the different conditions of the dentin substrate, producing similar TBS values regardless of the surface pretreatments.

Adhesion between fiber post and root dentin: evaluation of post surface conditioning for bond strength improvement.

The aim of this paper was to evaluate two surface conditioning methods associated with the application of adhesive on the post surface for improving the bond to resin cement. Sixty single-rooted bovine teeth were sectioned at 16 mm in length, prepared (9 mm depth), embedded in a PVC cylinder using acrylic resin, and allocated into 3 groups (N.=20) according to post surface treatment: cleaning with ethanol (control group); etching with hydrogen peroxide; etching with hydrofluoric acid. Ten posts for each group were silanized and other 10 posts were silanized and received an adhesive agent. The posts were cemented with self-adhesive resin cement (RelyX U100 resin cement). All teeth were sectioned perpendicularly to the long axis (2 mm thickness per slice), submitted to push out bond strength testing and the type of failure was recorded. The obtained data were submitted to two-way ANOVA and Turkey's test, with the level of significance set at 5%. Neither the hydrofluoric acid or hydrogen peroxide post surface treatment, nor the adhesive application, had an influence on bond strength values. The main type of failure was adhesive between cement and dentin. Etching and the application of an adhesive on the post surface did not presented a significant influence on the bond strength results for the fiber post resin cement-root dentin assembly. The cement appears to adhere very well to the fiber post surface rather than the dentin surface.

Effect of Er:YAG laser and diamond drill on hybrid layer morphology obtained with self-etch adhesive: analysis by SEM and confocal laser scanning microscopy (CLSM)

This study aimed to evaluate the effect of Er:YAG (L) and diamond drills (DD) on: 1) the microshear bond strength (MPa); 2) the adhesive interface of two-step (TS) – Adper Scotchbond Multipurpose and one-step (OS) adhesives – Adper EasyOne, both from 3M ESPE. Material and methods: According to the preparation condition and adhesives, the samples were divided into four groups: DD_TS (control); DD_OS; L_TS and L_OS. 60 bovine incisors were randomly divided into experimental and groups: 40 for microshear bond strength (n = 10) and 20 for the adhesive interface morphology [6 to measure the thickness of the hybrid layer (HL) and length of tags (t) by CLSM (n = 3); 12 to the adhesive interface morphology by SEM (n = 3) and 2 to illustrate the effect of the instruments on dentine by SEM (n = 1)]. To conduct the microshear bond strength test, four cylinders (0.7 mm in diameter and 1 mm in height with area of adhesion of 0.38 mm) were constructed with resin composite (Filtek Z350 XT – 3M ESPE) on each dentin surface treated by either L or DD and after adhesives application. Microshear bond strength was performed in universal testing machine (EMIC 2000) with load cell of 500 kgf and a crosshead speed of 0.5 mm / min. Adhesive interface was characterized by thickness of hybrid layer (HL) and length of tags (t) in nm, with the aid of UTHSCSA ImageTool software. Results: Microshear bond strength values were: L_TS 34.10 ± 19.07, DD_TS 24.26 ± 9.35, L_OS 33.18 ± 12.46, DD_OS 21.24 ± 13.96. Two-way ANOVA resulted in statistically significant differences only for instruments (p = 0.047). Mann-Whitney identified the instruments which determined significant differences for HL thickness and tag length (t). Concerning to the adhesive types, these differences were only observed for (t). Conclusion: It can be concluded that 1) laser Er:YAG results in higher microshear bond strength values regardless of the adhesive system (TS and OS); 2) the tags did not significant affect the microshear bond strength; 3) the adhesive interface was affected by both the instruments for cavity preparation and the type of adhesive system used.