Comparison of Fit Between Zirconia and Metal Copings Fabricated Conventionally or Using Different CAD/CAM Technique

This study was designed to assess the fit of cobalt chromium copings fabricated using direct metal laser sintering and to compare with copings fabricated from nickel chromium (traditional casting) and zirconia (milled from partially sintered blanks). For both cobalt chromium and zirconia groups, impressions were generated using direct or indirect digitization. There were 5 experimental groups of 12 copings fabricated for a prepared maxillary right molar. The intimacy of fit (measured at the margin, axial wall and occlusal) was assessed using a replica method technique. The results showed that zirconia copings produced using direct digitization had significantly smaller marginal, axial and occlusal measurements compared to other groups (p⟨0.05). Nevertheless, all groups had a mean marginal gap within accepted clinical values. Though there was a decrease in the intimacy of fit from the margin to axial wall to the occlusal surface. It can be concluded that the fit of zirconia copings fabricated using direct digitization was significantly better than the other groups. However, it can be expected that further enhancement and refinement of additive technology such as direct metal laser sintering will offer clinicians a viable alternative to nickel chromium analogue production methods in the future

Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages

Novel cell lines isolated from mouse embryonic stem cells exhibiting de novo methylation of the E-cadherin promoter.

Mouse embryonic stem cells (mESCs) and epiblast stem cells represent the naïve and primed pluripotent states, respectively. These cells self‐renew via distinct signaling pathways and can transition between the two states in the presence of appropriate growth factors. Manipulation of signaling pathways has therefore allowed the isolation of novel pluripotent cell types such as Fibroblast growth factor, Activin and BIO‐derived stem cells and IESCs. However, the effect of cell seeding density on pluripotency remains unexplored. In this study, we have examined whether mESCs can epigenetically regulate E‐cadherin to enter a primed‐like state in response to low cell seeding density. We show that low density seeding in the absence of leukaemia inhibitory factor (LIF) induces decreased apoptosis and maintenance of pluripotency via Activin/Nodal, concomitant with loss of E‐cadherin, Signal transducer and activator of transcription phosphorylation, and chimera‐forming ability. These cells, E‐cadherin negative proliferating stem cells (ENPSCs) can be reverted to a naïve phenotype by addition of LIF or forced E‐cadherin expression. However, prolonged culture of ENPSCs without LIF leads to methylation of the E‐cadherin promoter (ENPSCM), which cannot be reversed by LIF supplementation, and increased histone H3K27 and decreased H3K4 trimethylation. Transcript analysis of ENPSCM revealed a primed‐like phenotype and their differentiation leads to enrichment of neuroectoderm cells. The generation of ENPSCs is similar to tumorigenesis as ENPSCs exhibit transcript alterations associated with neoplasia, hyperplasia, carcinoma, and metastasis. We therefore describe a novel cell model to elucidate the role of E‐cadherin in pluripotency and to investigate epigenetic regulation of this gene during mESC differentiation and tumor metastasis

Maternal-zygotic knockout reveals a critical role of Cdx2 in the morula to blastocyst transition

The first lineage segregation in the mouse embryo generates the inner cell mass (ICM), which gives rise to the pluripotent epiblast and therefore the future embryo, and the trophectoderm (TE), which will build the placenta. The TE lineage depends on the transcription factor Cdx2. However, when Cdx2 first starts to act remains unclear. Embryos with zygotic deletion of Cdx2 develop normally until the late blastocyst stage leading to the conclusion that Cdx2 is important for the maintenance but not specification of the TE. In contrast, down-regulation of Cdx2 transcripts from the early embryo stage results in defects in TE specification before the blastocyst stage. Here, to unambiguously address at which developmental stage Cdx2 becomes first required, we genetically deleted Cdx2 from the oocyte stage using a Zp3-Cre/loxP strategy. Careful assessment of a large cohort of Cdx2 maternal-zygotic null embryos, all individually filmed, examined and genotyped, reveals an earlier lethal phenotype than observed in Cdx2 zygotic null embryos that develop until the late blastocyst stage. The developmental failure of Cdx2 maternal-zygotic null embryos is associated with cell death and failure of TE specification, starting at the morula stage. These results indicate that Cdx2 is important for the correct specification of TE from the morula stage onwards and that both maternal and zygotic pools of Cdx2 are required for correct pre-implantation embryogenesis.We thank the Wellcome Trust (Grant no. 098287/Z/12/Z) for supporting this work.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0012160614006307#

Altered subcellular localization of transcription factor TEAD4 regulates first mammalian cell lineage commitment

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification