Hiroshi Egusa

Three-dimensional cell and tissue patterning in a strained fibrin gel system.

Techniques developed for the in vitro reproduction of three-dimensional (3D) biomimetic tissue will be valuable for investigating changes in cell function in tissues and for fabricating cell/matrix composites for applications in tissue engineering techniques. In this study, we show that the simple application of a continuous strain to a fibrin gel facilitates the development of fibril alignment and bundle-like structures in the fibrin gel in the direction of the applied strain. Myoblasts cultured in this gel also exhibited well-aligned cell patterning in a direction parallel to the direction of the strain. Interestingly, the direction of cell proliferation was identical to that of cell alignment. Finally, the oriented cells formed linear groups that were aligned parallel to the direction of the strain and replicated the native skeletal muscle cell patterning. In addition, vein endothelial cells formed a linear, aligned vessel-like structure in this system. Thus, the system enables the in vitro reproduction of 3D aligned cell sets replicating biological tissue patterns.

Suspected association of an allergic reaction with titanium dental implants: A clinical report

Recent reports have questioned whether metal sensitivity may occur after exposure to titanium. This clinical report demonstrates the emergence of facial eczema in association with a titanium dental implant placed for a mandibular overdenture supported by 2 implants. Complete remission was achieved by the removal of the titanium material. This clinical report raises the possibility that in rare circumstances, for some patients, the use of titanium dental implants may induce an allergic reaction.

NEURONAL DIFFERENTIATION OF BONE MARROW-DERIVED STROMAL STEM CELLS INVOLVES SUPPRESSION OF DISCORDANT PHENOTYPES THROUGH GENE SILENCING

Tissue engineering involves the construction of transplantable tissues in which bone marrow aspirates may serve as an accessible source of autogenous multipotential mesenchymal stem cells. Increasing reports indicate that the lineage restriction of adult mesenchymal stem cells may be less established than previously believed, and stem cell-based therapeutics await the establishment of an efficient protocol capable of achieving a prescribed phenotype differentiation. We have investigated how adult mouse bone marrow-derived stromal cells (BMSCs) are guided to neurogenic and osteogenic phenotypes. Naïve BMSCs were found surprisingly active in expression of a wide range of mRNAs and proteins, including those normally reported in terminally differentiated neuronal cells and osteoblasts. The naïve BMSCs were found to exhibit voltage-dependent membrane currents similar to the neuronally guided BMSCs, although with smaller amplitudes. Once BMSCs were exposed to the osteogenic culture condition, the neuronal characteristics quickly disappeared. Our data suggest that the loss of discordant phenotypes during BMSC differentiation cannot be explained by the selection and elimination of unfit cells from the whole BMSC population. The percent ratio of live to dead BMSCs examined did not change during the first 8–10 days in either neurogenic or osteogenic differentiation media, and cell detachment was estimated at <1%. However, during this period, bone-associated extracellular matrix genes were selectively down-regulated in neuronally guided BMSCs. These data indicate that the suppression of discordant phenotypes of differentiating adult stem cells is achieved, at least in part, by silencing of superfluous gene clusters.

Modification of Apatite Materials for Bone Tissue Engineering and Drug Delivery Carriers

Apatite-related calcium phosphate, the main component of biological hard tissue, has good biocompatibility and is an economical material. Methods for the synthesis of apatite materials including hydroxyapatite (HAp) have previously been established. Therefore, for many years, apatite materials have been utilized as substitute materials for bone in orthopedic and dental fields. Such types of conventional substitute materials, which are implanted in the human body, should ostensibly be chemically stable to maintain their quality over time. However, recent advances in tissue engineering have altered this concept. Physicians and researchers now seek to identify materials that alter their properties temporally and spatially to achieve ideal tissue regeneration. In order to use apatite materials for tissue engineering and as drug delivery systems, the materials require both a high affinity for cells, tissues and/or functional molecules (e.g. growth factors and genes) and controllable bioabsorbability. To achieve these properties, various physicochemical modifications of apatite materials have been attempted. In addition, fabrication desiring three-dimensional structures (e.g. size, morphology and porosity) of apatite materials for implant sites could be one of the crucial techniques used to obtain ideal prognoses. In this review, the latest research trends relating to the techniques for the fabrication and modification of apatite materials are introduced.

Enhanced bone regeneration via multimodal actions of synthetic peptide SVVYGLR on osteoprogenitors and osteoclasts

Recently, the binding sequence Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) was found adjacent to the RGD sequence in osteopontin, suggesting involvement in osteo-immune cross-talk. The aim of this study was to investigate bioactive functions of a synthetic SVVYGLR peptide in osteoprogenitor cells and osteoclasts, and to examine potential applications in bone regeneration. The SVVYGLR peptide significantly enhanced the adhesion and proliferation of several human mesenchymal cells including bone marrow-derived mesenchymal stem cells, and alphavbeta3 integrin was involved in cell attachment to the peptide. Additionally, the peptide reduced the number of TRAP-positive multinucleated cells during osteoclastogenesis of RAW264.7 cells and normal murine pre-osteoclasts, and also suppressed NFAT activity and expression of osteoclastogenesis-related mRNAs. When standardized bone defects in rat calvariae were filled with a collagen sponge containing the peptide or PBS (control), the number of TRAP-positive osteoclasts in the grafted sites after 3 weeks was significantly lower in the peptide group. By the 5th week, significantly enhanced resorption of the grafted collagen sponge and new bone formation was observed within and surrounding the sponge in the peptide group. These data suggest that SVVYGLR is an effective bioactive peptide for bone tissue regeneration that promotes attachment and proliferation of osteogenic cells while also suppressing osteoclastogenesis.

Nuclear Import Mechanism for Myocardin Family Members and Their Correlation with Vascular Smooth Muscle Cell Phenotype

Myocardin (Mycd), which is essential for the differentiation of the smooth muscle cell lineage, is constitutively located in the nucleus, although its family members, myocardin-related transcription factors A and B (MRTF-A/B), mostly reside in the cytoplasm and translocate to the nucleus in response to Rho signaling. The mechanism for their nuclear import is unclear. Here we investigated the mechanism for the nuclear import of Mycd family members and demonstrated any correlation between such mechanism and the phenotype of vascular smooth muscle cells (VSMCs). In cultured VSMCs, the knockdown of importin β1 inhibited the nuclear import of Mycd and MRTF-A/B. Their NH2-terminal basic domain was identified as a binding site for importin α/β1 by in vitro analyses. However, Mycd had a higher affinity for importin α/β1 than did MRTF-A/B, even in the absence of G-actin, and Mycd affinity for importin α1/β1 was stronger than for any other importin α/β1 heterodimers. The binding of Mycd to importin α/β1 was insensitive to G-actin, whereas that of MRTF-A/B was differently inhibited by G-actin. In dedifferentiated VSMCs, the levels of importins α1 and β1 were reduced concomitant with down-regulation of Mycd, serum response factor, and smooth muscle cell markers. By contrast, in differentiated VSMCs, their expressions were up-regulated. Thus, the nuclear import of Mycd family members in VSMCs depends on importin α/β1, and their relative affinities for importin α/β1 heterodimers determine Mycd nuclear import. The expression of Mycd nuclear import machineries is related to the expression levels of VSMC phenotype-dependent smooth muscle cell markers.

Effect of filamentation and mode of growth on antifungal susceptibility of Candida albicans

Biofilm formation involving profuse hyphal growth is a major characteristic of Candida spp. and confers higher antifungal resistance than its planktonic mode of growth. We investigated the antifungal susceptibility of Candida albicans and its hyphal mutants (Delta efg1/efg1, Delta cph1/cph1 and DeltaDelta cph1/cph1 efg1/efg1) to commonly used antifungals during planktonic, adhesion and biofilm modes of growth. The minimum inhibitory concentration (MIC) of each antifungal agent was determined for a lower inoculum (1x10(3) cells/mL) and higher inoculum (1x10(7) cells/mL) of planktonic Candida. Furthermore, MICs of C. albicans biofilms and adhesion modes of growth were determined with a standard XTT assay. Candida albicans in adhesion and biofilm modes of growth, but not in planktonic mode, were resistant to all five antifungal agents tested. Although Delta efg1/efg1 and DeltaDelta cph1/cph1 efg1/efg1 mutants formed less biofilm than wild-type C. albicans SC5314, they were similarly resistant to caspofungin. However, these mutants were more sensitive to amphotericin B and nystatin than the wild-type. Adhesion per se confers increased resistance to antifungal agents, which is further pronounced in the biofilm mode of Candida. Filamentation does not appear to be a major determinant of the antifungal resistance in Candida biofilms.

Trabecular bone deterioration in col9a1+/- mice associated with enlarged osteoclasts adhered to collagen IX-deficient bone.

Introduction: Short collagen IX, the exclusive isoform expressed by osteoblasts, is synthesized through alternative transcription of the col9a1 gene. The function of short collagen IX in bone was characterized in col9a1‐null mutant mice.

Molecular cloning of novel Monad binding protein containing tetratricopeptide repeat domains

MINT‐6551090: Monad (uniprotkb:Q96MX6) physically interacts (MI:0218) with RPAP3 (uniprotkb:Q9H6T3) by anti tag coimmunoprecipitation (MI:0007) MINT‐6551101, MINT‐6551118: Monad (uniprotkb:Q96MX6) physically interacts (MI:0218) with RPAP3 (uniprotkb:Q9H6T3) by pull down (MI:0096) MINT‐6551132: RPAP3 (uniprotkb:Q9H6T3) physically interacts (MI:0218) with Monad (uniprotkb:Q96MX6) by anti bait coimmunoprecipitation (MI:0006)

An in vitro evaluation of the adhesion of Candida species to oral and lung tissue cells

The analysis of the adherence capacity of fungi to surfaces of both oral tissue and different tissues would be of interest in the fungal dissemination as an oral and systemic pathogen. We developed an in vitro adenosine triphosphate (ATP)‐based assay technique to extract the cellular and fungal ATP separately, which allowed the quantitative evaluation of the adhesion of the yeast to monolayers of human gingival epithelial cells (GEC), gingival fibroblasts (GF) and pulmonary fibroblasts (PF). Seven oral isolates of Candida species (three of Candida albicans, three of Candida tropicalis and one of Candida glabrata) were used in the study. The adherent level of the Candida species varied depending on both the isolates and the cell origins, although all the Candida isolates had a significantly higher level of adherence to GEC than to GF except the single isolate of C. tropicalis. Whereas the adherent level of the five isolates to GEC was significantly higher than that to PF, the adherent level of the remaining two isolates of C. tropicalis to GEC was significantly lower than that to PF. These results suggest that candidal adherence to host tissue cells should be regulated in an isolate‐dependent and cell‐origin‐dependent manner, and that the phenomena may be involved in the colonisation and/or dissemination of the fungi.