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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]