Natsumi Ushijima

3D collagen scaffolds coated with multiwalled carbon nanotubes : Initial cell attachment to internal surface

The cell adhesion in a multiwalled carbon nanotube-coated collagen sponge (MWCNT-coated sponge) was investigated. Immediately after seeding, the cells adhered to the inner surface of the MWCNT-coated sponge and a significantly larger number of cells were observed there than for a pure collagen sponge used as control. On the MWCNT-coated sponge, the cells appeared favorable adhesion and spread in the early stages in the center part of the sponge which cells rarely attached without MWCNT-coating. It was suggested that the physical structure of MWCNTs was effective for initial adhesion of cells from the result of serum-free culture. MWCNT-coating makes the material a suitable three-dimensional scaffold for cell culturing, as opposed to other scaffold systems where such an effect is not seen.

Distribution and organization of lymphatic vessels in the mouse gingiva: An immunohistochemical study with LYVE-1

OBJECTIVE This study introduced the usefulness of LYVE-1 immunoreactivity for identification of lymphatic vessels in decalcified tissues, and demonstrated the fine distribution and organization of these vessels in mouse gingiva. DESIGN After confirming the specificity of anti-mouse LYVE-1, frozen sections of mouse decalcified gingiva were immunostained with the antibody. RESULTS The LYVE-1-positive lymphatic vessels were clearly found in the connective tissue under the gingival epithelium; these vessels appeared to pass through the lamina propria of the gingiva toward the alveolar crest and run along the external surface of the alveolar bone. The lymphatic vessels were sparse and apart from the oral gingival and sulcular epithelia, while they were dense adjacent to the junctional epithelium. CONCLUSIONS The dense network of the lymphatic vessels adjacent to the junctional epithelium, which is apparently exposed to foreign antigens, may act as an efficient drainage pathway of the excessive interstitial fluid and immune cells, and play an active role in the immune defense of the gingiva. The present study also revealed the absence of lymphatic connection between gingiva and periodontal ligament.

Antimicrobial photodynamic activity and cytocompatibility of Au25(Capt)18 clusters photoexcited by blue LED light irradiation

Antimicrobial photodynamic therapy (aPDT) has beneficial effects in dental treatment. We applied captopril-protected gold (Au25(Capt)18) clusters as a novel photosensitizer for aPDT. Photoexcited Au clusters under light irradiation generated singlet oxygen (1O2). Accordingly, the antimicrobial and cytotoxic effects of Au25(Capt)18 clusters under dental blue light-emitting diode (LED) irradiation were evaluated. 1O2 generation of Au25(Capt)18 clusters under blue LED irradiation (420–460 nm) was detected by a methotrexate (MTX) probe. The antimicrobial effects of photoexcited Au clusters (0, 5, 50, and 500 μg/mL) on oral bacterial cells, such as Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis, were assessed by morphological observations and bacterial growth experiments. Cytotoxicity testing of Au clusters and blue LED irradiation was then performed against NIH3T3 and MC3T3-E1 cells. In addition, the biological performance of Au clusters (500 μg/mL) was compared to an organic dye photosensitizer, methylene blue (MB; 10 and 100 μg/mL). We confirmed the 1O2 generation ability of Au25(Capt)18 clusters through the fluorescence spectra of oxidized MTX. Successful application of photoexcited Au clusters to aPDT was demonstrated by dose-dependent decreases in the turbidity of oral bacterial cells. Morphological observation revealed that application of Au clusters stimulated destruction of bacterial cell walls and inhibited biofilm formation. Aggregation of Au clusters around bacterial cells was fluorescently observed. However, photoexcited Au clusters did not negatively affect the adhesion, spreading, and proliferation of mammalian cells, particularly at lower doses. In addition, application of Au clusters demonstrated significantly better cytocompatibility compared to MB. We found that a combination of Au25(Capt)18 clusters and blue LED irradiation exhibited good antimicrobial effects through 1O2 generation and biosafe characteristics, which is desirable for aPDT in dentistry.

Apoptosis of odontoclasts under physiological root resorption of human deciduous teeth

This study was designed to establish the apoptosis of odontoclasts during physiological root resorption of human deciduous teeth. Deciduous teeth were fixed, decalcified, and embedded in paraffin for immunohistochemical (IHC) observations and in Epon for transmission electron microscopy (TEM). Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and then tartrate-resistant acid phosphatase (TRAP) activity was determined on the same sections. Epon-embedded specimens were sectioned serially into 0.5-μm semithin sections; some of these sections were re-embedded in Epon, sectioned into 0.1-μm ultrathin sections, and observed by TEM. IHC revealed that the nuclei of TRAP-positive odontoclasts on the dentine were generally TUNEL-negative. Around these odontoclasts, a few TRAP-positive structures were present together with TUNEL-positive structures, e.g., a TRAP-positive structure with one TUNEL-positive nucleus, a TRAP-positive structure with one TUNEL-positive nucleus plus one or two TUNEL-negative nuclei, or a TRAP-positive structure with no nucleus. By TEM, some odontoclasts showed nuclear fragments including compacted chromatin. The results suggest that, during apoptosis, odontoclasts fragment into variously sized cellular parts including three or fewer nuclei.

Carbon nanohorns allow acceleration of osteoblast differentiation via macrophage activation

Carbon nanohorns (CNHs), formed by a rolled graphene structure and terminating in a cone, are promising nanomaterials for the development of a variety of biological applications. Here we demonstrate that alkaline phosphatase activity is dramatically increased by coculture of human monocyte derived macrophages (hMDMs) and human mesenchymal stem cells (hMSCs) in the presence of CNHs. CNHs were mainly localized in the lysosome of macrophages more than in hMSCs during coculturing. At the same time, the amount of Oncostatin M (OSM) in the supernatant was also increased during incubation with CNHs. Oncostatin M (OSM) from activated macrophage has been reported to induce osteoblast differentiation and matrix mineralization through STAT3. These results suggest that the macrophages engulfed CNHs and accelerated the differentiation of mesenchymal stem cells into the osteoblast via OSM release. We expect that the proof-of-concept on the osteoblast differentiation capacity by CNHs will allow future studies focused on CNHs as ideal therapeutic materials for bone regeneration.

Transmission electron microscopic observation of cells cultured on multiwalled carbon nanotube-coated sponges

The cell structure and interface between cultured cells and a multiwalled carbon nanotube (MWCNT)-coated sponge (MWCNT-coated sponge) were observed by transmission electron microscopy (TEM). Moreover, the atomic structure of MWCNTs that entered the cells was also examined by means of high-resolution TEM (HRTEM). MWCNTs were observed in the cytoplasm, and a few MWCNTs were recognized in the cell nuclei. Those MWCNTs maintained their structure there. Subcellular organelles did not appear to be different from those on the collagen sponge despite the cellular uptake of MWCNTs.