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Kohei Kawamoto,1 Hirofumi Miyaji,1 Erika Nishida,1 Saori Miyata,1 Akihito Kato,1 Akito Tateyama,1 Tomokazu Furihata,1 Kanako Shitomi,1 Toshihiko Iwanaga,2 Tsutomu Sugaya1 1Department of Periodontology and Endodontology, Faculty of Dental Medicine, Hokkaido University, Sapporo, Japan; 2Department of Histology and Cytology, Faculty of Medicine, Hokkaido University, Sapporo, Japan Introduction: The 3-dimensional scaffold plays a key role in volume and quality of repair tissue in periodontal tissue engineering therapy. We fabricated a novel 3D collagen scaffold containing carbon-based 2-dimensional layered material, named graphene oxide (GO). The aim of this study was to characterize and assess GO scaffold for periodontal tissue healing of class II furcation defects in dog.Materials and methods: GO scaffolds were prepared by coating the surface of a 3D collagen sponge scaffold with GO dispersion. Scaffolds were characterized using cytotoxicity and tissue reactivity tests. In addition, GO scaffold was implanted into dog class II furcation defects and periodontal healing was investigated at 4 weeks postsurgery.Results: GO scaffold exhibited low cytotoxicity and enhanced cellular ingrowth behavior and rat bone forming ability. In addition, GO scaffold stimulated healing of dog class II furcation defects. Periodontal attachment formation, including alveolar bone, periodontal ligament-like tissue, and cementum-like tissue, was significantly increased by GO scaffold implantation, compared with untreated scaffold.Conclusion: The results suggest that GO scaffold is biocompatible and possesses excellent bone and periodontal tissue formation ability. Therefore, GO scaffold would be beneficial for periodontal tissue engineering therapy. Keywords: artificial collagen scaffold, cytotoxicity, nanocarbon, periodontal attachment, periodontal tissue engineering, rat cranial bone augmentation

Saori Miyata,1 Hirofumi Miyaji,1 Hideya Kawasaki,2 Masaki Yamamoto,2 Erika Nishida,1 Hiroko Takita,3 Tsukasa Akasaka,4 Natsumi Ushijima,3 Toshihiko Iwanaga,5 Tsutomu Sugaya1 1Department of Periodontology and Endodontology, Hokkaido University Graduate School of Dental Medicine, Kita-ku, Sapporo, 2Department of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita-shi, Osaka, 3Support Section for Education and Research, Hokkaido University Graduate School of Dental Medicine, 4Department of Biomedical, Dental Materials and Engineering, Graduate School of Dental Medicine, Hokkaido University, 5Department of Anatomy, Laboratory of Histology and Cytology, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo, Japan Abstract: 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. Keywords: Aggregatibacter actinomycetemcomitans, antimicrobial photodynamic therapy, photosensitizer, Porphyromonas gingivalis, singlet oxygen, Streptococcus mutans

Erika Nishida,1 Hirofumi Miyaji,1 Akihito Kato,1 Hiroko Takita,2 Toshihiko Iwanaga,3 Takehito Momose,1 Kosuke Ogawa,1 Shusuke Murakami,1 Tsutomu Sugaya,1 Masamitsu Kawanami11Department of Periodontology and Endodontology, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan; 2Support Section for Education and Research, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan; 3Laboratory of Histology and Cytology, Hokkaido University Graduate School of Medicine, Sapporo, JapanAbstract: Graphene oxide (GO) consisting of a carbon monolayer has been widely investigated for tissue engineering platforms because of its unique properties. For this study, we fabricated a GO-applied scaffold and assessed the cellular and tissue behaviors in the scaffold. A preclinical test was conducted to ascertain whether the GO scaffold promoted bone induction in dog tooth extraction sockets. For this study, GO scaffolds were prepared by coating the surface of a collagen sponge scaffold with 0.1 and 1 µg/mL GO dispersion. Scaffolds were characterized using scanning electron microscopy (SEM), physical testing, cell seeding, and rat subcutaneous implant testing. Then a GO scaffold was implanted into a dog tooth extraction socket. Histological observations were made at 2 weeks postsurgery. SEM observations show that GO attached to the surface of collagen scaffold struts. The GO scaffold exhibited an interconnected structure resembling that of control subjects. GO application improved the physical strength, enzyme resistance, and adsorption of calcium and proteins. Cytocompatibility tests showed that GO application significantly increased osteoblastic MC3T3-E1 cell proliferation. In addition, an assessment of rat subcutaneous tissue response revealed that implantation of 1 µg/mL GO scaffold stimulated cellular ingrowth behavior, suggesting that the GO scaffold exhibited good biocompatibility. The tissue ingrowth area and DNA contents of 1 µg/mL GO scaffold were, respectively, approximately 2.5-fold and 1.4-fold greater than those of the control. Particularly, the infiltration of ED2-positive (M2) macrophages and blood vessels were prominent in the GO scaffold. Dog bone-formation tests showed that 1 µg/mL GO scaffold implantation enhanced bone formation. New bone formation following GO scaffold implantation was enhanced fivefold compared to that in control subjects. These results suggest that GO was biocompatible and had high bone-formation capability for the scaffold. The GO scaffold is expected to be beneficial for bone tissue engineering therapy.Keywords: biocompatibility, biomaterial, bone tissue engineering, cell ingrowth, collagen sponge, macrophage, nanocarbon

Izumi Kanayama,1 Hirofumi Miyaji,1 Hiroko Takita,2 Erika Nishida,1 Maiko Tsuji,3 Bunshi Fugetsu,4,5 Ling Sun,4,5 Kana Inoue,1 Asako Ibara,1 Tsukasa Akasaka,6 Tsutomu Sugaya,1 Masamitsu Kawanami1 1Department of Periodontology and Endodontology, 2Support Section for Education and Research, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan; 3Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan; 4Division of Frontier Research, Research Department, Creative Research Institution Sousei, 5Graduate School of Environmental Science, 6Department of Biomedical, Dental Materials and Engineering, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan Background: Graphene oxide (GO) is a single layer carbon sheet with a thickness of less than 1 nm. GO has good dispersibility due to surface modifications with numerous functional groups. Reduced graphene oxide (RGO) is produced via the reduction of GO, and has lower dispersibility. We examined the bioactivity of GO and RGO films, and collagen scaffolds coated with GO and RGO. Methods: GO and RGO films were fabricated on a culture dish. Some GO films were chemically reduced using either ascorbic acid or sodium hydrosulfite solution, resulting in preparation of RGO films. The biological properties of each film were evaluated by scanning electron microscopy (SEM), atomic force microscopy, calcium adsorption tests, and MC3T3-E1 cell seeding. Subsequently, GO- and RGO-coated collagen scaffolds were prepared and characterized by SEM and compression tests. Each scaffold was implanted into subcutaneous tissue on the backs of rats. Measurements of DNA content and cell ingrowth areas of implanted scaffolds were performed 10 days post-surgery.Results: The results show that GO and RGO possess different biological properties. Calcium adsorption and alkaline phosphatase activity were strongly enhanced by RGO, suggesting that RGO is effective for osteogenic differentiation. SEM showed that RGO-modified collagen scaffolds have rough, irregular surfaces. The compressive strengths of GO- and RGO-coated scaffolds were approximately 1.7-fold and 2.7-fold greater, respectively, when compared with the non-coated scaffold. Tissue ingrowth rate was 39% in RGO-coated scaffolds, as compared to 20% in the GO-coated scaffold and 16% in the non-coated scaffold.Conclusion: In summary, these results suggest that GO and RGO coatings provide different biological properties to collagen scaffolds, and that RGO-coated scaffolds are more bioactive than GO-coated scaffolds. Keywords: GO, RGO, tissue engineering, regenerative scaffold, cell ingrowth, biomaterials

We investigated patterning with curable dental materials via micro/nano-molding as a screening trial. We used typical curable dental materials such as a composite resin (CR), acrylic resin, varnish, adhesive resin, restoration, sealer, cement, tooth desensitizer, and gypsum with either a cyclo-olefin polymer (COP) mold or polydimethylsiloxane (PDMS) mold that had holes, pillars, and grooves with 0.5 µm diameters and 1 µm heights. The COP mold was suitable for patterning of holes and grooves with Bondfill SB after 2 h curing, while PDMS mold was suitable for patterning of pillars with Bondfill SB at the 0.5 µm scale after 2 h curing. Most of the curable dental materials could be patterned using the COP or the PDMS mold. However, dental impression materials could not be patterned as required. In addition, the COP mold was suitable for many organic pillars, while the PDMS mold was suitable for inorganic pillars. Further, we achieved patterning on the curved surface of an artificial tooth with flowable CR using the COP thin film mold. We conclude that for the formation of uniform shaped patterns, it is important to cure within a suitable time and to select mold types depending on the type of curable dental material. [ABSTRACT FROM AUTHOR]

Background and Objective Nanoparticle bioceramics are being investigated for biomedical applications. We fabricated a regenerative scaffold comprising type I collagen and beta-tricalcium phosphate (β- TCP) nanoparticles. Fibroblast growth factor-2 ( FGF-2) is a bioeffective signaling molecule that stimulates cell proliferation and wound healing. This study examined the effects, on bioactivity, of a nano-β- TCP/collagen scaffold loaded with FGF-2, particularly on periodontal tissue wound healing. Material and Methods Beta-tricalcium phosphate was pulverized into nanosize particles (84 nm) and was then dispersed. A nano-β- TCP scaffold was prepared by coating the surface of a collagen scaffold with a nanosize β- TCP dispersion. Scaffolds were characterized using scanning electron microscopy, compressive testing, cell seeding and rat subcutaneous implant testing. Then, nano-β- TCP scaffold, nano-β- TCP scaffold loaded with FGF-2 and noncoated collagen scaffold were implanted into a dog one-wall infrabony defect model. Histological observations were made at 10 d and 4 wk postsurgery. Results Scanning electron microscopy images show that TCP nanoparticles were attached to collagen fibers. The nano-β- TCP scaffold showed higher compressive strength and cytocompatibility compared with the noncoated collagen scaffold. Rat subcutaneous implant tests showed that the DNA contents of infiltrating cells in the nano-β- TCP scaffold and the FGF-2-loaded scaffold were approximately 2.8-fold and 3.7-fold greater, respectively, than in the collagen scaffold. Histological samples from the periodontal defect model showed about five-fold greater periodontal tissue repair following implantation of the nano-β- TCP scaffold loaded with FGF-2 compared with the collagen scaffold. Conclusion The β- TCP nanoparticle coating strongly improved the collagen scaffold bioactivity. Nano-β- TCP scaffolds containing FGF-2 are anticipated for use in periodontal tissue engineering. [ABSTRACT FROM AUTHOR]

In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased β-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/β-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study. [ABSTRACT FROM AUTHOR]

Proton pump inhibitor triple therapy with clarithromycin and metronidazole has been widely used for Helicobacter pylori eradication. However, the efficacy and the safety of this therapy in patients with liver cirrhosis have not been established. To evaluate the effect of hepatic dysfunction on metabolism of clarithromycin as it is used for H. pylori eradication therapy in patients with liver cirrhosis, and the efficacy of eradication therapy in those patients. Values for the area under the 0–6h concentration-time curve (AUC) for clarithromycin were not significantly different among the groups. However, the AUC (0–6h) values of 14-(R)-hydroxyclarithromycin were significantly lower in the Child­Pugh C group than in either the normal controls or the Child­Pugh A/B group. The cure rate for the peptic ulcer patients was 84% on a per protocol analysis (95% CI: 80%–88%) and 81% on an intention-to-treat analysis (95% CI: 77%–85%), while in the liver cirrhosis patients it was 89% in a per protocol analysis (95% CI: 78%–99%) and 83% in an intention-to-treat analysis (95% CI: 70%–97%). Mild adverse effects were observed in 10% of the peptic ulcer patients and 13% of the liver cirrhosis patients, with none leading to premature withdrawal from the study. The 2-week low-dose lansoprazole-based triple therapy tested is a simple, effective and well-tolerated regimen for H. pylori eradication in patients with liver cirrhosis. [ABSTRACT FROM AUTHOR]

c-Kit is a receptor tyrosine kinase, and it is encoded by the mouse W locus. Mutant W/Wv mice develop spontaneous gastric antral ulcers. The aim of the present study was to investigate the pathogenesis of these gastric ulcers and to examine the effects of two antiulcer drugs; a proton pump inhibitor (2{[4-(3-methoxypropoxy)-3-methylpyridine-2-yl]methyl-sulfinyl}-1H -benzimidazole sodium salt, rabeprazole) and a mucosal protective drug (geranylgeranylacetone, GGA), on the gastric ulcers. The inhibition of the gastric acid secretion by rabeprazole (30 mg/kg body weight, subcutaneous injection once a day for six weeks) significantly increased the gastric ulcer formation compared to the controls. In contrast, the GGA treatment (100 mg/kg body weight, oral administration for six weeks) significantly inhibited the ulcer formation. Bile reflux was seen in these mutant mice, and they showed no cyclic intense contractions in the gastric antrum. These results suggest that bile reflux due to the disturbance of gastric antral movement is a cause of the spontaneous gastric ulcers in W/Wv mice. [ABSTRACT FROM AUTHOR]

Objectives:The purpose of the present study was to examine the clinical outcomes of using tracheoesophageal diversion for preventing intractable aspiration.Method:We retrospectively reviewed 25 patients who underwent tracheoesophageal diversion from 2003 to 2009 at our hospital (median age, 25 years; range, 0–78 years). End-to-side anastomosis was used in 16 cases and side-to-side anastomosis was used in 9.Results:The average operative time was 141 minutes for end-to-side anastomosis and 191 minutes for side-to-side anastomosis. Peri-operative complications were observed in only two (8 per cent) cases: one with infection and one with haematoma. No fistulas were observed. Aspiration was prevented in all cases, but the nutritional route depended on the swallowing function of the patient. Oral feeding was the main nutritional route after surgery in only four patients (16 per cent).Conclusion:This procedure is well suited to patients who lack speech communication and are at high risk of aspiration. [ABSTRACT FROM PUBLISHER]

Osteoclasts are associated with certain diseases such as osteopetrosis. The development and functions of osteoclasts are controlled by the micro- and nano-surface roughness of biomaterials. However, the effect of fine micro/nano-patterns on osteoclastic differentiation remains poorly understood. In the present study, we investigated the effect of fine shape patterns at the nano-scale on osteoclastic differentiation. A hole pattern of 500 nm in diameter and 500 nm in height was prepared using nanoimprinting of a glycol-modified polyethylene terephthalate (G-PET) film for a basic topological study. RAW264.7 cells were used as an alternative basic model to primary cells. The result of a cell attachment assay showed that the average number of attached cells on the hole patterned-film was 1.5 times higher than that on the planar film. A cell differentiation assay showed that the average number of multinucleated osteoclasts on the hole patterned-film was approximately 1.9 times higher than that on the planar film. However, the patterning effects in this study were quite small (p> 0.05). Although the morphology of the cells was basically similar when observed under an optical microscope, the morphologies of the attached and differentiated cells were slightly different in terms of their cell shape and filopodia shape between the holed and planar films when observed under a scanning electron microscope. Differences in cell attachment and differentiation behaviors between holed and planar films would affect the functions of the osteoclasts. We suggest that fine nano-patterning of the substrate could be one factor to control osteoclastic differentiation and cell function. [ABSTRACT FROM AUTHOR]