Youssef Haikel

Dynamic and cyclic fatigue of engine-driven rotary nickel-titanium endodontic instruments.

The absence of adequate testing standards for engine-driven nickel-titanium (NiTi) instruments necessitates further study of these instruments in all areas. This study examined three groups of engine-driven rotary NiTi endodontic instruments (Profile, Hero, and Quantec) and assessed the times for dynamic fracture in relation to the radius of curvature to which the instruments were subjected during preparation, with the instrument diameter determined by size and taper and the mode by which the fracture occurred. Ten instruments were randomly selected representing each size and taper for each group and for each radius of curvature: 600 in total. The instruments were rotated at 350 rpm and introduced into a tempered steel curve that simulated a canal. Two radii of curvature of canals were used: 5 and 10 mm. Time at fracture was noted for all files, and the fracture faces of each file were analyzed with scanning electron microscopy. Radius of curvature was found to be the most significant factor in determining the fatigue resistance of the files. As radius of curvature decreased, fracture time decreased. Taper of files was found to be significant in determining fracture time. As diameter increased, fracture time decreased. In all cases, fracture was found to be of a ductile nature, thus implicating cyclic fatigue as a major cause of failure and necessitating further analyses and setting of standards in this area.

Expression of mRNAs Encoding for α and β Integrin Subunits, MMPs, and TIMPs in Stretched Human Periodontal Ligament and Gingival Fibroblasts

The biological mechanisms of tooth movement result from the cellular responses of connective tissues to exogenous mechanical forces. Among these responses, the degradation of the extracellular matrix takes place, but the identification of the molecular basis as well as the components implicated in this degradation are poorly understood. To contribute to this identification, we subjected human fibroblasts obtained from the periodontal ligament (PDLs) and from the gingiva (HGFs) to a continuous stretch to quantify the mRNAs encoding for various metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and a and β integrin subunits. Both cell lines reacted by inducing the expression of the mRNAs encoding for MMP-1, MMP-2, TIMP-1, and TIMP-2, while other mRNAs did not vary (MTI-MMP, TIMP-3) or were not expressed (MMP-9). PDLs expressed selectively the mRNAs encoding for a4 and av, with no difference measurable under stretching, while the mRNAs encoding for a6 and β1 were increased and the one encoding for a5 was decreased. HGFs increased the mRNAs encoding for α2, α6, β1, and β3 and decreased the one encoding for a3. Analysis of our data indicated that stretched HGFs and PDLs induced the same pattern of mRNAs encoding for MMPs and TIMPs but differed for those encoding various integrin subunits, known to act as protein receptors in mechanotransduction.

A new method for the quantitative analysis of endodontic microleakage

The aim of this in vitro study was to evaluate the apical seal obtained with three commonly used root canal sealing cements: Sealapex, AH Plus or Topseal, and Sealite, using a new method based on the quantitative analysis of 125I-radiolabeled lysozyme penetration. One hundred thirteen teeth with straight single root canals were instrumented to master apical point #25/30. These were divided into three groups: (i) negative control (4 roots) covered with two layers of nail polish, (ii) test group (105 roots) obturated by laterally condensed guttapercha with the three cements; and (iii) positive control (4 roots) obturated without cement. The groups were then immersed in 125I lysozyme solution for a period of 1, 7, 14, or 28 days. After removal, six sections of 0.8 mm length each were made of each root with a fine diamond wire. Each section was analyzed for activity by a gamma counter, corrected for decay, and used to quantify protein penetration. Leakage was high in the positive control and almost negligible in the negative control. AH Plus (Topseal) and Sealapex showed similar leakage behavior over time, with AH Plus (Topseal) performing better. Sealite showed acceptable leakage up until day 14, after which a large increase occurred, presumably due to three-dimensional instability.

Bortezomib/proteasome inhibitor triggers both apoptosis and autophagy-dependent pathways in melanoma cells.

Generally, both endoplasmic reticulum (ER) stress and mitochondrial dysregulation are a potential therapeutic target of anticancer agents including bortezomib. The treatment of melanoma cells with bortezomib was found to induce apoptosis together with the upregulation of Noxa, Mcl-1, and HSP70 proteins, and the cleavage of LC3 and autophagic formation. Also, bortezomib induced ER-stress as evidenced by the increase of intracellular Ca(2+) release. In addition, bortezomib enhanced the phosphorylation of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), c-jun-N-terminal kinase (JNK) and p38, and the activation of the transcription factors AP-1, ATF-2, Ets-1, and HSF1. Bortezomib-induced mitochondrial dysregulation was associated with the accumulation of reactive oxygen species (ROS), the release of both apoptosis inducing factor (AIF) and cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of Poly (ADP-ribose) polymerase (PARP). The pretreatment of melanoma cells with the inhibitor of caspase-3 (Ac-DEVD-CHO) was found to block bortezomib-induced apoptosis that subsequently led to the increase of autophagic formation. In contrast, the inhibition of ASK1 abrogated bortezomib-induced autophagic formation and increased apoptosis induction. Furthermore, the inhibition of JNK, of HSP70 also increased apoptosis induction without influence of bortezomib-induced autophagic formation. Based on the inhibitory experiments, the treatment with bortezomib triggers the activation of both ER-stress-associated pathways, namely IRE1α-ASK1-p38-ATF-2/ets-1-Mcl-1, and IRE1α-ASK1-JNK-AP-1/HSF1-HSP70 as well as mitochondrial dysregulation-associated pathways, namely ROS-ASK1-JNK-AP-1/HSF1-HS70, and AIF-caspase-3-PARP and Cyt.c, and caspase-9-caspase-3-PARP. Taken together, our data demonstrates for the first time the molecular mechanisms, whereby bortezomib triggers both apoptosis and autophagic formation in melanoma cells.

Immunolocalization of BMP-2/-4, FGF-4, and WNT10b in the Developing Mouse First Lower Molar

Intercellular signaling controls all steps of odontogenesis. The purpose of this work was to immunolocalize in the developing mouse molar four molecules that play major roles during odontogenesis: BMP-2, −4, FGF-4, and WNT10b. BMP-2 and BMP-4 were detected in the epithelium and mesenchyme at the bud stage. Staining for BMP-2 markedly increased at the cap stage. The relative amount of BMP-4 strongly increased from E14 to E15. At E15, BMP-4 was detected in the internal part of the enamel knot where apoptosis was intense. In contrast to TGFβ1, BMP-2 and −4 did not show accumulation at the epithelial-mesenchymal junction where the odontoblast started differentiation. When odontoblasts became functional, BMP-2 and BMP-4 were detected at the apical and basal poles of preameloblasts. BMP-2, which induces ameloblast differentiation in vitro, may also be involved physiologically. The decrease in FGF-4 from E14 to E15 supports a possible role for the growth factor in the control of mesenchymal cell proliferation. The relative amount of FGF-4 was maximal at E17. The subsequent decrease at E19 showed correlation with the withdrawal of odontoblasts and ameloblasts from the cell cycle. WNT10b might also stimulate cell proliferation. At E14-15, WNT10b was present in the mesenchyme and epithelium except for the enamel knot, where the mitotic activity was very low. At E19 there was a decreasing gradient of staining from the cervical loop where cells divide to the tip of the cusp in the inner dental epithelium where cells become postmitotic. The target cells for FGF-4 and WNT10b appeared different.

Selective and uncoupled role of substrate elasticity in the regulation of replication and transcription in epithelial cells.

Actin cytoskeleton forms a physical connection between the extracellular matrix, adhesion complexes and nuclear architecture. Because tissue stiffness plays key roles in adhesion and cytoskeletal organization, an important open question concerns the influence of substrate elasticity on replication and transcription. To answer this major question, polyelectrolyte multilayer films were used as substrate models with apparent elastic moduli ranging from 0 to 500 kPa. The sequential relationship between Rac1, vinculin adhesion assembly, and replication becomes efficient at above 200 kPa because activation of Rac1 leads to vinculin assembly, actin fiber formation and, subsequently, to initiation of replication. An optimal window of elasticity (200 kPa) is required for activation of focal adhesion kinase through auto-phosphorylation of tyrosine 397. Transcription, including nuclear recruitment of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), occurred above 50 kPa. Actin fiber and focal adhesion signaling are not required for transcription. Above 50 kPa, transcription was correlated with αv-integrin engagement together with histone H3 hyperacetylation and chromatin decondensation, allowing little cell spreading. By contrast, soft substrate (below 50 kPa) promoted morphological changes characteristic of apoptosis, including cell rounding, nucleus condensation, loss of focal adhesions and exposure of phosphatidylserine at the outer cell surface. On the basis of our data, we propose a selective and uncoupled contribution from the substrate elasticity to the regulation of replication and transcription activities for an epithelial cell model.

Poly(l-lysine) nanostructured particles for gene delivery and hormone stimulation

In this work, we designed replica particles based on poly (L-lysine) (PLL) polymers crosslinked via a homobifunctional linker to support coadsorption of a plasmid DNA and a peptide hormone for concurrent transfection and induction of a cellular function. PLL replica particles (PLL(RP)) were prepared by infiltrating polymer into mesoporous silica (MS) particles, crosslinking the adsorbed chains by using a homobifunctional crosslinker and finally removing the template particles. Moreover, we verified their cytotoxicity. Furthermore, based on this PLL(RP) gene delivery system, we simultaneously evaluated the melanin stimulation and gene expression in these cells by fluorescence microscopy. To further understand the bi-functionality, we labeled the SPT7pTL and PGA-alpha-MSH with YOYO-1 and Rhodamine, respectively, to follow its intracellular pathway by confocal microscopy. Our data suggests that the PLL(RP) is a promising vector for gene therapy and hormone stimulation.

Vascularization of Engineered Teeth

The implantation of cultured dental cell-cell re-associations allows for the reproduction of fully formed teeth, crown morphogenesis, epithelial histogenesis, mineralized dentin and enamel deposition, and root-periodontium development. Since vascularization is critical for organogenesis and tissue engineering, this work aimed to study: (a) blood vessel formation during tooth development, (b) the fate of blood vessels in cultured teeth and re-associations, and (c) vascularization after in vivo implantation. Ex vivo, blood vessels developed in the dental mesenchyme from the cap to bell stages and in the enamel organ, shortly before ameloblast differentiation. In cultured teeth and re-associations, blood-vessel-like structures remained in the peridental mesenchyme, but never developed into dental tissues. After implantation, both teeth and re-associations became revascularized, although later in the case of the re-associations. In implanted re-associations, newly formed blood vessels originated from the host, allowing for their survival, and affording conditions organ growth, mineralization, and enamel secretion.

Scanning Electron Microscopy of the Human Enamel Surface Layer of Incipient Carious Lesions

A scanning electron microscopic (SEM) study of the enamel surface layer of human natural white and brown spot lesions has been conducted in deciduous as well as permanent teeth. No ultrastructural dif

The antimicrobial peptides derived from chromogranin/secretogranin family, new actors of innate immunity

Chromogranins/secretogranins are members of the granin family present in secretory vesicles of nervous, endocrine and immune cells. In chromaffin cells, activation of nicotinic cholinergic receptors induces the release, with catecholamines, of bioactive peptides resulting from a natural processing. During the past decade, our laboratory has characterized new antimicrobial chromogranin-derived peptides in the secretions of stimulated bovine chromaffin cells. They act at the micromolar range against bacteria, fungi, yeasts, and are non-toxic for the mammalian cells. They are recovered in several biological fluids involved in defence mechanisms (human serum, neutrophil secretions and saliva). These new antimicrobial peptides demonstrate the major role of the adrenal medulla in innate immunity. In this review we focus on the antimicrobial peptides derived from human and bovine chromogranin A (CGA), chromogranin B (CGB) and secretogranin II (SGII) emphasizing their direct action against pathogens and their effects on immune cells.