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Applied Surface Science (v.262, #)

Editorial Board (pp. ii).

Surface and interface of biomaterials – Structure and effect from macro to nano by Fumio Watari (pp. 1-1).

Characterization and fatigue damage of TiO₂ layer on spark-anodized titanium before and after hot water treatment by Z.X. Chen; W.X. Wang; Y. Takao; T. Matsubara; L.M. Ren (pp. 2-7).

► Water treatment of anodic TiO₂ results in a nanostructured surface layer. ► Spark anodizing at 200V has no significant influence on the fatigue life of titanium. ► Water-treated TiO₂ layer shows severer fatigue damage than untreated one.Spark anodizing of titanium in phosphoric acid solution produced porous TiO₂ layer on its surface, and subsequent hot water treatment was performed to modify the surface characteristics of resultant oxide layer. Results show that water treatment not only transformed the original amorphous TiO₂ into crystalline anatase, but also resulted in a nanostructured surface. Also, the influence of spark anodizing treatment on fatigue behavior of titanium and the fatigue damage of TiO₂ layer before and after water treatment was investigated. Results show that the present spark anodizing at 200V had no significant influence on the fatigue life of titanium. However, significant fatigue damage was induced in the TiO₂ layer. Before water treatment, tensile fatigue loading first caused transverse cracking to occur in the TiO₂ layer, and then inclined spallation of oxide layer driven by slip bands in the titanium substrate was observed at a maximum cyclic stress of 140MPa. In comparison, after water treatment, TiO₂ layer exhibited severer fatigue damage, and extensive cracking and delamination of TiO₂ layer occurred at a lower maximum cyclic stress of 120MPa.

Keywords: Titanium; Spark anodizing; Hot water treatment; Fatigue; Damage

Arrangement techniques of proteins and cells using amorphous calcium phosphate nanofiber scaffolds by Takayuki Nonoyama; Takatoshi Kinoshita; Masahiro Higuchi; Kenji Nagata; Masayoshi Tanaka; Mari Kamada; Kimiyasu Sato; Katsuya Kato (pp. 8-12).

Display Omitted► An immunoglobulin G is absorbed and confined on an ACP nanofiber scaffold. ► A mouse osteoblast-like MC3T3-E1 cell is elongated unidirectionaly. ► A fibronectin controls degree of cellular extension.We demonstrate arrangement techniques of proteins and cells using an amorphous calcium phosphate (ACP) nanofiber scaffold. It is well known that protein andosteoblastic cell are preferably adsorbed onto ACP surface. The ACP nanofiber scaffold was prepared by calcium phosphate mineralization on a polypeptide monolayer-coated mica substrate, and ACP nanofibers were oriented unidirectionaly. In a protein system, the ACP nanofiber scaffold was soaked in a fluorescein isothiocyanate conjugated immunoglobulin G (IgG-FITC) aqueous solution. From fluorescence microscopic measurement, the adsorbed IgG-FITC was highly confined and arranged on the ACP nanofiber. In a cell system, a mouse osteoblast-like cell (MC3T3-E1) behavior on the ACP nanofiber scaffold was observed. The cell was elongated unidirectionaly, and its cytoskeletal shape showed high aspect ratio. These results are clearly different from an ACP bulk template or bare mica substrate, and the arrangement technique enable to fabricate a fine-tuned biomaterial template.

Keywords: Arrangement technique; Osteoblast-like cell; Antibody protein; Mineralization; β-Sheet peptide template; Amorphous calcium phosphate

Rapid and non-destructive analysis of metallic dental restorations using X-ray fluorescence spectra and light-element sampling tools by K. Furuhashi; M. Uo; Y. Kitagawa; F. Watari (pp. 13-18).

► A non-destructive extraction method for dental prostheses was developed. ► A rapid and precise elemental analysis was used after extraction. ► Sample dental prostheses were polished using pastes and disposable buffs. ► We investigated sampling/analysis efficiencies and optimal buff-paste mixtures. ► The method was effective for all three dental alloys and clinical use.Recently, allergic diseases caused by dental metals have been increasing. Therefore, rapid and accurate analytical methods for the metal restorations in the oral cavities of patients are required. The purpose of this study was to develop a non-destructive extraction method for dental alloys, along with a subsequent, rapid and accurate elemental analysis.Samples were obtained by polishing the surfaces of metal restorations using a dental rotating tool with disposable buffs and polishing pastes. As materials for the analysis, three dental alloys were used. To compare the sampling and analysis efficiencies, two buffs and seven pastes were used. After polishing the surface of a metal restoration, the buff was analyzed using X-ray scanning analytical microscopy (XSAM).The efficiency of the analysis was judged based on the sampling rate achieved and the absence of disturbing elements in the background in fluorescence X-ray spectra. The best results were obtained for the combination of TexMet as a buff with diamond as a paste. This combination produced a good collection efficiency and a plain background in the fluorescence X-ray spectra, resulting in a high precision of the analysis.

Keywords: X-ray scanning analytical microscope; Elemental analysis; Light-element sampling tools; Fluorescent X-ray analysis; Metal restoration; Allergic diseases

Protein adsorption kinetics from single- and binary-solution by Naris Barnthip; Erwin A. Vogler (pp. 19-23).

► Protein mass-adsorption rates are shown to be fast compared to rates-of-change of interfacial tensions. ► Decoupling of mass and interfacial energetics is inconsistent with adsorption-kinetic theories based on energy-barriers to mass adsorption. ► Motivates revision of the basic mechanism of protein adsorption. ► Revised protein-adsorption mechanism explains unusual adsorption competition effects between two proteins.Comparison of protein mass-adsorption-rates to rates-of-change in interfacial tensions reveals that mass adsorption is decoupled from interfacial energetics. This implies that energy-barrier theories describing protein-adsorption kinetics do not accurately capture the physics of the process. An alternative paradigm in which protein molecules rapidly diffuse into an inflating interphase which subsequently slowly shrinks in volume, concentrating adsorbed protein and causing slow concomitant decrease in interfacial tensions, is shown to be consistent with adsorption kinetics measured by solution depletion and tensiometry. Mass adsorption kinetics observed from binary-protein solution is compared to adsorption kinetics from single-protein solution, revealing that organization of two different-sized proteins within the interphase can require significantly longer than that adsorbed from single-protein solution and may require expulsion of initially adsorbed protein which is not observed in the single-protein case.

Keywords: Protein adsorption; Kinetics; Interphase; Surface; Adsorption competition

Carbon nanotube-coating accelerated cell adhesion and proliferation on poly (L-lactide) by Eri Hirata; Tsukasa Akasaka; Motohiro Uo; Hiroko Takita; Fumio Watari; Atsuro Yokoyama (pp. 24-27).

► The surface of a polylactic acid (PLLA) was coated multiwalled carbon nanotubes (MWCNTs). ► MWCNT-coated PLLA showed remarkable higher wettability than uncoated PLLA. ► More Human osteosarcoma cell line (Saos2) adhered on the CNT-coated than those on uncoated PLLA at 2h after seeding. ► MWCNT-coating on PLLA improved the surface wettability and initial cell attachment at early stage.The surface of a polylactic acid (PLLA) was coated multiwalled carbon nanotubes (MWCNTs) in order to improve the surface properties. In addition, its surface characteristics and cell culturing properties were examined. Whole surface of PLLA was homogeneously covered by MWCNTs maintained a unique tubular structure. MWCNT-coated PLLA showed remarkable higher wettability than uncoated PLLA. Human osteosarcoma cell line (Saos2) adhered well on the CNT-coated PLLA whereas there are few cells attached on the uncoated PLLA at 2h after seeding. The number of the cells on uncoated PLLA was still smaller than on the MWCNT-coated PLLA at 1 and 3 days. Moreover, The DNA content in the cells attached to the MWCNT-coated PLLA was significantly higher than that on the uncoated PLLA ( p<0.05) at 1 and 3 days. There was no significant difference between the scaffolds for ALP activity normalized by DNA content at both term ( p>0.1). Therefore MWCNT-coating on PLLA improved the surface wettability and initial cell attachment at early stage.

Keywords: Carbon nanotubes; Poly(; l; -lactide); Cell adhesion; Osteoblast like cell

Ionically crosslinked alginate–carboxymethyl cellulose beads for the delivery of protein therapeutics by Min Sup Kim; Sang Jun Park; Bon Kang Gu; Chun-Ho Kim (pp. 28-33).

► We prepared Fe³⁺ crosslinked alginate–carboxymethyl cellulose (AC) beads. ► Different surface and inner morphology of AC beads were observed on volume of CMC. ► AC beads showed minimum swelling degree in acidic condition. ► Protein release from AC beads was to control in gastrointestinal condition.We developed Fe³⁺-crosslinked alginate–carboxymethyl cellulose (AC) beads in various volume ratios by dropping an AC solution into a ferric chloride solution to form protein therapeutic carrier beads. Scanning electron microscopy revealed that the roughness and pore size of the crosslinked beads increased with the volume ratio of the carboxymethyl cellulose. Fourier transform-infrared analysis revealed the formation of a three-dimensional bonding structure between the anionic polymeric chains of AC and the Fe³⁺ ions. The degree of swelling and the release profile of albumin from the beads were investigated under simulated gastrointestinal conditions (pH 1.2, 4.5, and 7.4). The Fe³⁺-crosslinked AC beads displayed different degrees of swelling and albumin release for the various AC volume ratios and under various pH conditions. An in vitro release test was used to monitor the controlled release of albumin from the AC beads under simulated gastrointestinal conditions over 24h. The Fe³⁺-crosslinked AC beads protected and controlled the release of protein, demonstrating that such beads present a promising protein therapeutic carrier for the oral delivery.

Keywords: Alginate; Carboxymethyl cellulose; Ionic crosslinked beads; Controlled release; Oral protein delivery; Tissue engineering

Micro-arc oxidation treatment to improve the hard-tissue compatibility of Ti–29Nb–13Ta–4.6Zr alloy by Yusuke Tsutsumi; Mitsuo Niinomi; Masaaki Nakai; Harumi Tsutsumi; Hisashi Doi; Naoyuki Nomura; Takao Hanawa (pp. 34-38).

► Thick and porous oxide layer was formed on Ti alloy with micro-arc oxidation treatment. ► Ca, P, and Mg from the electrolyte were incorporated in the resultant oxide layer. ► Calcium phosphate formation was much accelerated on the treated sample. ► Micro-arc oxidation enhances the bioactivity of the biocompatible Ti alloy.Micro-arc oxidation (MAO) was performed on a β-type Ti–29Nb–13Ta–4.6Zr alloy (TNTZ) in this study to improve its bioactivity in a body fluid and its hard-tissue compatibility. The surface oxide layer formed on TNTZ by MAO treatment in a mixture of calcium glycerophosphate and magnesium acetate was characterized using various surface analyses. The oxide layer was mainly composed of two types of TiO₂ (rutile and anatase), and it also contained Ca, P, and Mg, which were incorporated from the electrolyte during the treatment. The calcium phosphate formation on the surface of the specimens after immersion in Hanks’ solution was evaluated to determine the bioactivity of TNTZ with and without MAO treatment. As a result, thick calcium phosphate layers formed on the TNTZ specimen that underwent MAO treatment, whereas only a small amount of precipitate was observed on TNTZ without treatment. Thus, the MAO treatment is a promising method to improve the bioactivity and hard-tissue compatibility of TNTZ.

Keywords: Titanium alloy; Micro-arc oxidation; Calcium phosphate; Bioactivity; Hard-tissue compatibility

Solvent-free formation of hydroxyapatite coated biodegradable particles via nanoparticle-stabilized emulsion route by Masahiro Okada; Syuji Fujii; Taiki Nishimura; Yoshinobu Nakamura; Shoji Takeda; Tsutomu Furuzono (pp. 39-44).

► Hydroxyapatite (HAp) nanoparticles stabilized polymer melt-in-water emulsions without any molecular surfactants. ► Interaction between polymer and HAp played a crucial role. ► HAp-coated polymer particles were obtained from the emulsions without any organic solvents.Hydroxyapatite (HAp) nanoparticle-coated biodegradable polymer particles were fabricated from a nanoparticle-stabilized emulsion in the absence of any molecular surfactants or organic solvents. First, a polymer melt-in-water emulsion was prepared by mixing a water phase containing nanosized HAp particles as a particulate emulsifier and an oil phase consisting of poly( ɛ-caprolactone) (PCL) or poly(l-lactide- co- ɛ-caprolactone) (P(LLA-CL)) above its melting point. It was clarified that the interaction between ester/carboxyl groups of the polymers and the HAp nanoparticles at the polymer–water interface played a crucial role to prepare the nanoparticle-stabilized emulsion. The HAp nanoparticle-coated biodegradable polymer particle (a polymer solid-in-water emulsion) was fabricated by cooling the emulsion. The particle morphology and particle size were evaluated using scanning electron microscope.

Keywords: Hydroxyapatite; Nanoparticle; Pickering emulsion; Microsphere; Coating

Surface properties of Al₂O₃-YSZ ceramic composites modified by a combination of biomimetic coatings and electric polarization by Akiko Nagai; Chufan Ma; Shigeki Kishi; Masahiro Inuzuka; Miho Nakamura; Naohiro Horiuchi; Keishi Nishio; Kimihiro Yamashita (pp. 45-50).

► New surface modifications of Al-YSZ. ► It is the combination of a biomimetic coatings and electric polarization treatment. ► Al-YSZ was polarizable material. ► After polarization, the calcium phosphate deposited by the coatings became thick.Alumina-containing yttria-stabilized zirconia composite (Al-YSZ) has a higher flexural strength than YSZ, although Al-YSZ cannot directly bond with bone tissues. To expand the biomedical applications of Al-YSZ, new surface modifications were investigated to improve the bioactivity in the present study. The combination of a biomimetic coatings method using an alternate soaking technique and electric polarization treatment was performed on Al-YSZ. The crystalline phases of the starting Al-YSZ sintered disks were assigned to tetragonal ZrO₂ and α-Al₂O₃ phases. The analyses of the electrical properties were carried out using complex impedance and thermally stimulated depolarized current measurements for polarization treatments. Our results indicate that the stored charges in Al-YSZ via polarization decreased in YSZ, because of the added alumina, while the calcium phosphate film deposited by the biomimetic coatings method on polarized Al-YSZ was observed to be thicker than the non-polarized film. The coating method would be suitable for the complex geometry of biomaterials. As a result, polarized Al-YSZ fabricated using an alternate soaking process is expected to be useful as a biomaterial under load-bearing conditions.

Keywords: Al-YSZ; Electric polarization; Biomimetic coating; a.c. complex impedance

Surface structure and biocompatibility of demineralized dentin matrix granules soaked in a simulated body fluid by Toshiyuki Akazawa; Masaru Murata; Jun Hino; Futami Nagano; Tatsuhiro Shigyo; Takafumi Nomura; Hiroyuki Inano; Kohji Itabashi; Tohru Yamagishi; Katsuo Nakamura; Touru Takahashi; Shunji Iida; Haruhiko Kashiwazaki (pp. 51-55).

► Demineralized dentin matrix (DDM) is prepared by a pulverizing-dissolution method. ► Human tooth is rapidly pulverized with saline ice and dissolved in acidic solutions. ► DDM granules are soaked in a simulated body fluid to precipitate apatite crystals. ► Microstructure of precipitates depends on the demineralization situation of granules. ► In in-vivo, bio-absorption is recognized around the surface layers of DDM granules.Demineralized dentin matrix (DDM) granules with excellent biocompatibility were easily prepared using unnecessary human teeth by a new cooling-pulverizing and demineralizing technique. Extracted human teeth were pulverized together with saline ice at 12,000rpm-rotation number of a ZrO₂ blade for 30s in a ZrO₂ vessel. The pulverized granules exhibited the particle size distribution of 0.5–2mm that was efficient for regeneration of alveolar bone. The (Ca/P) ratios of the granules were 1.60–1.66, which were close to the stoichiometric value of 1.67 for standard hydroxyapatite (HAp). Small amounts of Na⁺ and Mg²⁺ ions present at less than 1% were detected. The pulverized granules were dissolved with stirring under 500rpm for 10–60min in 2.0%-HNO₃ solutions to obtain partial or complete DDM granules. As the dissolution time increased, crystallinity of HAp phase lowered and asperity on surfaces of the granules became outstanding due to elution of mineral components. At the dissolution of 60min, the pulverizing granules were completely demineralized and the weight decreased to about one-fifth. To improve surface activity of the DDM granules without denaturation of bone growth factors, the DDM granules were soaked at 309.5K and pH 7.40 in a simulated body fluid (SBF). HAp microcrystals were gradually precipitated on surfaces of the DDM granules with increasing the soaking time. Different morphology of the precipitates was observed, depending on the demineralization situation of the pulverized granules. For the DDM with low dissolution efficiency of 42%, porous bone-like apatites at 24h after the soaking and fiber-oriented aggregates at 144h were recognized. The bioactive DDM granules were implanted into the subcutaneous tissues of the back region of rats. At 4 weeks after the implantation, bio-absorption by comparatively small amounts of multi-giant cells was recognized around the surface layers of DDM granules.

Keywords: Surface structure; Biocompatibility; Demineralized dentin matrix; Simulated body fluid; Biomimetic environment; Extracted human teeth

The effect of enhancing the hydrophobicity of OMMT on the characteristics of PMMA/OMMT nanocomposites by Shuichi Yamagata; Yusuke Hamba; Tsukasa Akasaka; Natsumi Ushijima; Motohiro Uo; Junichiro Iida; Fumio Watari (pp. 56-59).

► PMMA/OMMT nanocomposites were fabricated using a solution intercalation method. ► The silicate platelets were largely well dispersed independent of the hydrophobicity. ► The partially exfoliation of the silicate layers were observed. ► The flexural modulus of the nanocomposites increased favorably. ► The strain at breakage of the nanocomposites remained suitable for our use.Transparent poly(methyl methacrylate)/organically modified montmorillonite (PMMA/OMMT) nanocomposites were fabricated using a solution intercalation method. Two grades of OMMT modified with quaternary alkylammonium ions and containing different amounts of organic matter, NZ70 and NX, were used. X-ray diffraction patterns showed that the peaks regarded as a (001) d-spacing and a second peak were shifted toward lower 2 θ values, implying the expansion or the partial exfoliation of the silicate layers, respectively. TEM images showed that the silicate platelets were largely well dispersed independent of the hydrophobicity, although some aggregates were observed. The flexural modulus of the PMMA/OMMT nanocomposites increased favorably with an increasing amount of OMMT of either grade. However, only the NX displayed comparable strain at breakage to that of the PMMA. These results suggest that the highly hydrophobic grade NX shows great promise for use in nanocomposites made via solution intercalation.

Keywords: Nanocomposite; PMMA; Montmorillonite; Orthodontic wire; Esthetics

Effects of implantation of three-dimensional engineered bone tissue with a vascular-like structure on repair of bone defects by Masanori Nishi; Rena Matsumoto; Jian Dong; Toshimasa Uemura (pp. 60-63).

► Bone marrow MSCs and MSC-derived ECs were co-cultured by an RWV bioreactor. ► The engineered tissue was implanted into defects in femurs of immunodeficient rats. ► The bone defects were better repaired in the implanted group after 3 weeks.Previously, to create an implantable bone tissue associated with blood vessels, we co-cultured rabbit bone marrow mesenchymal stem cells (MSCs) with MSC-derived endothelial cells (ECs) within a porous polylactic acid-based scaffold utilizing a rotating wall vessel (RWV) bioreactor. Here, this engineered tissue was orthotopically implanted into defects made in femurs of immunodeficient rats, and histological analysis were carried out to examine the repair of the damage and the formation of bone around the implant. The bone defects were better repaired in the implanted group than control group after 3 weeks. The results indicate that the engineered bone could repair bone defects.

Keywords: Bone regeneartion; Bone tissue engineering; Implantation; Mesenchymal stem cell; Three-dimentional culture; Rotating wall vessel bioreactor

Confocal laser scanning microscopy in study of bone calcification by Tetsunari Nishikawa; Mayu Kokubu; Hirohito Kato; Koichi Imai; Akio Tanaka (pp. 64-68).

► High-magnification images with depth selection, and thin sections were observed using CLSM. ► The direction and velocity of calcification of the bone was observed by administration of 2 fluorescent dyes. ► In dog femora grafted with coral blocks, newly-formed bone was observed in the coral block space with a rough surface. ► Twelve weeks after dental implant was grafted in dog femora, the space between screws was filled with newly-formed bones.Bone regeneration in mandible and maxillae after extraction of teeth or tumor resection and the use of rough surface implants in bone induction must be investigated to elucidate the mechanism of calcification. The calcified tissues are subjected to chemical decalcification or physical grinding to observe their microscopic features with light microscopy and transmission electron microscopy where the microscopic tissue morphology is significantly altered. We investigated the usefulness of confocal laser scanning microscopy (CLSM) for this purpose. After staggering the time of administration of calcein and alizarin red to experimental rats and dogs, rat alveolar bone and dog femur grafted with coral as scaffold or dental implants were observed with CLSM. In rat alveolar bone, the calcification of newly-formed bone and net-like canaliculi was observed at the mesial bone from the roots progressed at the rate of 15μm/day. In dog femur grafted with coral, newly-formed bones along the space of coral were observed in an orderly manner. In dog femur with dental implants, after 8 weeks, newly-formed bone proceeded along the rough surface of the implants. CLSM produced high-magnification images of newly-formed bone and thin sections were not needed.

Keywords: CLSM; Bone formation; Implant surface

Characterization of silica particles prepared via urease-catalyzed urea hydrolysis and activity of urease in sol–gel silica matrix by Katsuya Kato; Masakazu Nishida; Kimiyasu Ito; Masahiro Tomita (pp. 69-75).

► Silica precipitation occurred via urease-catalytic reactions. ► Higher urease activity for silica synthesis enables mesostructure of silica–urease composites. ► Urease encapsulating in silica matrix retained high activity.Urease templated precipitation of silica synthesized by sol–gel chemistry produces a composite material allowing high urease activity. This study investigates the structural properties of the composite material that allow for the retention of the urease hydrolysis activity. Scanning (SEM) and transmission (TEM) electron microscopy reveal that the composite has a mesoporous structure composed of closely packed spherical structures ∼20–50nm in diameter. Brunauer–Emmett–Teller (BET) analysis revealed that the surface area and pore volume of the composite prepared under the conditions of 50mM urea and 25°C is relatively high (324m²/g and 1.0cm³/g). These values are equivalent to those of usual mesoporous silica materials synthesized from the self-assembly of triblock copolymers as organic templates. In addition, after encapsulating in a sol–gel silica matrix, urease retained high activity (∼90% of the activity compared with native urease). Our results suggest a new method for synthesizing mesoporous silica materials with highly tunable pore sizes and shapes under mild conditions.

Keywords: Encapsulation; Enzyme; Silica particle; Stability; Urease

Synthesis and antibacterial properties of water-dispersible silver nanoparticles stabilized by metal–carbon σ-bonds by Koji Kawai; Takashi Narushima; Kotaro Kaneko; Hayato Kawakami; Miyuki Matsumoto; Atsushi Hyono; Hiroshi Nishihara; Tetsu Yonezawa (pp. 76-80).

► A new water-soluble stabilizer for metal nanoparticles (NPs), 4-diazoniumcarboxylbenzene tetrafluoroborate, was synthesized. ► A stable aqueous dispersion of Ag NPs was successfully produced by a simultaneous aqueous reduction of this diazonium salt and silver nitrate by NaBH₄. ► Characterization of the obtained Ag NPs by UV-Vis spectra, TEM images, XRD patterns, and XPS spectra revealed that they were stabilized by Ag–C σ-bonds. ► These NPs showed excellent antimicrobial properties against Staphylococcus aureus.The synthesis of 4-diazoniumcarboxylbenzene fluoroborate, a new water-soluble stabilizer for metal nanoparticles (NPs), is described. A stable dispersion of Ag NPs in water was successfully produced by a simultaneous aqueous reduction of this diazonium salt and silver nitrate by NaBH₄. UV–vis spectra, TEM images, XRD patterns, and XPS spectra of the obtained Ag NPs revealed that they were stabilized by Ag–C σ-bonds. These NPs showed excellent antimicrobial properties against Staphylococcus aureus.

Keywords: Silver; Nanoparticles; Diazonium; Sigma-bonds; Antibacterial

A comparative biomechanical study of bone ingrowth in two porous hydroxyapatite bioceramics by Li-Mei Ren; Mitsugu Todo; Takaaki Arahira; Hideki Yoshikawa; Akira Myoui (pp. 81-88).

► Apaceram-AX^® shows enhanced initial bone regeneration and a faster increase in stiffness compared with NEOBONE^®. ► Micropores in Apaceram-AX^® results in favorable rough surface and leads to progressive degradation. ► Strain energy density exhibits severe concentration in the early postoperative period of NEOBONE^® and afterwards such concentration becomes less.Calcium phosphate-based bioceramics have been widely used as artificial bone substitute materials because of their superior biocompatibility and osteoconductivity. In the present study, mechanical properties changes of two hydroxyapatite (HA) ceramics induced by bone ingrowth were tested and evaluated in a rabbit model. Both materials (NEOBONE^®, Apaceram-AX^®) have highly interconnected pores with a porosity of 75–85%. The major structural difference between them lies in that Apaceram-AX^® has micropores smaller than 10 micrometers in diameter, whereas NEOBONE^® does not contain such micropores. Both materials were implanted into the femoral condyles of rabbits for the specified observation period (1, 5, 12, 24, and 48 weeks) and then evaluated by experimental approach in combination with finite element method (FEM). Results indicate that two porous bioceramics exhibit different degradability in vivo, and remarkably different variation of total stiffness, elastic modulus distribution, as well as strain energy density distribution calculated by FE simulation. These results demonstrate how the internal microstructures affect the progress of bone regeneration and mechanical properties with the duration of implantation, emphasizing the importance of biomaterial design tailored to various clinic applications. Additionally, this study showed a potential for applying the computational method to monitor the time-dependent biomechanical changes of implanted porous bioceramics.

Keywords: Porous bioceramics; Microstructure; Finite element analysis; Mechanical properties; Bone ingrowth

Characterization and morphology analysis of degradable poly(l-lactide) film in in-vitro gastric juice incubation by Hao-Ming Chang; Chun-Chiang Huang; Hsieh-Chih Tsai; Toyoko Imae; Po-Da Hong (pp. 89-94).

► PLLA film was fabricated using a hot melting process to a thickness of around 40–50μm. ► Fabricated PLLA film contained amorphous and crystalline regions. ► Decomposition of the PLLA film began with surface erosion in human gastric juice. ► Gastric solution diffused into the swelling PLLA film for the next step of degradation.The purpose of this study was to evaluate the use of the biodegradable poly(l-lactide) (PLLA) as a gastro-jejunal tube anchored in the duodenum for duodenal exclusion. PLLA film was fabricated using a hot melting process to a thickness of around 40–50μm and was then immersed in human gastric juice to estimate the in vitro biodegradability behavior. PLLA film was more biodegradable in human gastric juice than in HCl and PBS. Measurements of weight loss indicated that 60% of original the PLLA was lost after 42 days of incubation. Surface functional group characterization, thermal stability, and surface morphology of the degraded PLLA film in human gastric juice showed that the decomposed sections of the PLLA film were primarily from the amorphous region. The degradation of the PLLA film in human gastric juice began with the erosion of continuous nanocavities in the range of 100–200nm on the PLLA surface over the course of 21 days. The PLLA film collapsed and spiral PLLA fiber was obtained after 42 days of decomposing in human gastric juice.

Keywords: Degradation; Poly(; l; -lactide); Film; Hot melting; Gastric juice

Thermo-responsive wound dressings by grafting chitosan and poly( N-isopropylacrylamide) to plasma-induced graft polymerization modified non-woven fabrics by Jyh-Ping Chen; Chang-Yi Kuo; Wen-Li Lee (pp. 95-101).

► Poly( N-isopropylacrylamide) and chitosan were grafted to polypropylene non-wovens. ► An easily stripped off thermo-responsive wound dressing was developed. ► The wound dressing is biocompatible, has antibacterial and wound healing abilities. ► The bigraft non-woven will be a potential wound dressing for biomedical use.To obtain a chitosan wound dressings with temperature-responsive characteristics, polypropylene (PP) non-woven fabric (NWF) was modified by direct current pulsed oxygen plasma-induced grafting polymerization of acrylic acid (AAc) to improve hydrophilicity and to introduce carboxylic acid groups. Conjugation of chitosan and poly( N-isopropylacrylamide) (PNIPAAm) followed by using water-soluble carbodiimide as a coupling agent to form a novel bigraft PP- g-chitosan- g-PNIPAAm wound dressing. The amount of chitosan and PNIPAAm grafted to PP- g-chitosan- g-PNIPAAm were 83.0±4.6μg/cm² and 189.5±8.2μg/cm², respectively. The surface chemical composition and microstructure of the NWF were studied by electron spectroscopy for chemical analysis (ESCA) and scanning electron microscopy (SEM). The linkages between AAc, chitosan, and PNIPAAm were confirmed with the formation of amide bonds. Physical properties of the NWF were characterized and potentials of these NWFs as wound dressings were evaluated using SD rat as the animal model. NWFs contained PNIPAAm were better than those contained only chitosan in wound healing rates and the wound areas covered by PP- g-chitosan- g-PNIPAAm wound dressings healed completely in 17 days.

Keywords: Plasma-induced polymerization; Biomolecules grafting; Biomedical application; Chitosan; Thermo-responsive; Wound dressing

The effects of general anesthetics on ESR spectra of spin labels in phosphatidylcholine vesicles containing purified Na,K-ATPase or microsomal protein by Makiko Shibuya; Toshifumi Hiraoki; Kunie Kimura; Kazuaki Fukushima; Kuniaki Suzuki (pp. 102-106).

► We studied the effects of general anesthetics on liposome using ESR spectra. ► Two spin labels, 5-DSA and 16-DSA, were located in different position in liposome. ► Anesthetics did not change the environment around the spin labels in the liposome. ► Anesthetics remained on the surface of the lipid bilayer of liposome. ► Proteins in the liposome did not change the effects of anesthetics on liposome.We investigated the effects of general anesthetics on liposome containing spin labels, 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA), and purified Na,K-ATPase or membrane protein of microsome using an electron spin resonance (ESR) spectroscopy.The spectra of 16-DSA in liposomes with both proteins showed three sharp signals compared with 5-DSA. The difference in the order parameter S value of 5-DSA and 16-DSA suggested that the nitroxide radical location of 5-DSA and 16-DSA were different in the membrane bilayer. The results were almost the same as those obtained in liposomes without proteins.The addition of sevoflurane, isoflurane, halothane, ether, ethanol and propofol increased the intensity of the signals, but the clinical concentrations of anesthetics did not significantly alter the S and τ values, which are indices of the fluidity of the membrane.These results suggest that anesthetics remain on the surface of the lipid bilayer and do not act on both the inside hydrophobic area and the relatively hydrophilic area near the surface. These results and others also suggest that the existence of Na,K-ATPase and microsomal proteins did not affect the environment around the spin labels in the liposome and the effects of anesthetics on liposome as a model membrane.

Keywords: Electron spin resonance; Liposome; General anesthetics; Membrane proteins; Order parameter; S; Rotational correlation time; τ

Surface characteristics and osteoblastic cell response to titanium–8tantalum–3neobium alloy by De-zhe Cui; Ki-Deog Park; Kyung-Ku Lee; Young-Suk Jung; Bo-Ah Lee; Yang-Jin Lee; Ok-Su Kim; Hyun-Ju Chung; Young-Joon Kim (pp. 107-109).

► The surface properties and osteblastic responses to a titanium–8%tantalum–3%niobium (Ti–8Ta–3Nb) alloy were investigated in vitro. ► The surface of the Ti–8Ta–3Nb alloy was more hydrophilic than those of commercially pure titanium (TiS) and Ti–6Al–4V alloy. ► The present study showed improved surface characteristics and osteoblastic response to the Ti–8Ta–3Nb alloy compared to TiS and Ti–6Al–4V alloy.In the present study, the surface properties and osteblastic responses to a titanium–8%tantalum–3%niobium (Ti–8Ta–3Nb) alloy were investigated in vitro. The surface roughness and morphology of all samples appeared similar. The surface of the Ti–8Ta–3Nb alloy was more hydrophilic than those of commercially pure titanium (TiS) and Ti–6Al–4V alloy. The Ti–8Ta–3Nb alloy had a better cellular response with regard to proliferation and ALP activity. The present study showed improved surface characteristics and osteoblastic response to the Ti–8Ta–3Nb alloy compared to TiS and Ti–6Al–4V alloy.

Keywords: Key words; Titanium–8%tantalum–3%niobium alloy; X-ray photoelectron spectroscopy (XPS); Cell proliferation; Alkaline phosphatase activity

Fabrication of carbon nanotubes/hydroxyapatite nanocomposites via an in situ process by X.Y. Lu; N.Y. Zhang; L. Wei; J.W. Wei; Q.Y. Deng; X. Lu; K. Duan; J. Weng (pp. 110-113).

Carbon nanotubes (CNTs)/hydroxyapatite (HA) nanocomposites with ferromagnetic properties have been fabricated via an in situ process in which a HA-based matrix was used as the catalyst for the growth of CNTs in a chemical vapor deposition system. The results showed that approximately 20wt% of multi-walled CNTs with a mean diameter of 40–60nm have been produced in the nanocomposites. The CNTs in the nanocomposites were observed to have an undamaged structure, with ordered graphitic layers and non-defective lattice structure on their walls. The growth of CNTs are shown to be in situ from the nano-HA crystallites. The compress mechanical properties of these CNTs/HA composites prepared by this method were significantly better than that of the composites prepared by physical mixing CNTs with HA particles. Magnetic measurement showed that the nanocomposites exhibit a ferromagnetic behavior with a saturation magnetization of 0.126emug⁻¹ at a room temperature. These magnetic nanocomposites could have a potential application in the drug delivery system as well as other biomedical fields.

Keywords: Nanocomposite; Hydroxyapatie; Carbon nanotube; In situ preparation

Integrating sol–gel with cold plasmas modified porous polycaprolactone membranes for the drug-release of silver-sulfadiazine and ketoprofen by Dave Mangindaan; Chao-Ting Chen; Meng-Jiy Wang (pp. 114-119).

► Integrated control–release system of sol–gel and biodegradable polycaprolactone membrane. ► Biocompatible and nontoxic chitosan–SiO₂ sol–gel for drug loading. ► Characterizations of the oxygen plasma modified biodegradable PCL porous membrane. ► Achieved controllable in vitro release for ketoprofen and silver sulfadiazine. ► The sol–gel/plasma modified porous membranes showed excellent biocompatibility.A controlled release system composed of surface modified porous polycaprolactone (PCL) membranes combined with a layer of tetraorthosilicate (TEOS)–chitosan sol–gel was reported in this study. PCL is a hydrophobic, semi-crystalline, and biodegradable polymer with a relatively slow degradation rate. The drugs chosen for release experiments were silver-sulfadiazine (AgSD) and ketoprofen which were impregnated in the TEOS–chitosan sol–gel. The surface modification was achieved by O₂ plasma and the surfaces were characterized by water contact angle (WCA) measurements, atomic force microscope (AFM), scanning electron microscope and electron spectroscopy for chemical analysis (ESCA). The results showed that the release of AgSD on O₂ plasma treated porous PCL membranes was prolonged when compared with the pristine sample. On the contrary, the release rate of ketoprofen revealed no significant difference on pristine and plasma treated PCL membranes. The prepared PCL membranes showed good biocompatibility for the wound dressing biomaterial applications.

Keywords: Polycaprolactone; Silver-sulfadiazine; Ketoprofen; Sol–gel; Plasma modifications; Control release

Phytotoxicity of multi-walled carbon nanotubes assessed by selected plant species in the seedling stage by Parvin Begum; Refi Ikhtiari; Bunshi Fugetsu; Makoto Matsuoka; Tsukasa Akasaka; Fumio Watari (pp. 120-124).

► MWNTs showed no detrimental effects on seed germinations of seven elected species of plants. ► Induction in root and shoot growth of red spinach, lettuce, rice and cucumber were observed after the 15-days exposure to MWNTs. ► MWNTs induced phytotoxicity at the seedling stage at above 1000mg/L.Abundant experimental data have shown that multi-walled carbon nanotubes (MWNTs) are toxic to plants, but the potential impacts of exposure remain unclear. The objective of the present study was to evaluate possible phytotoxicity of MWNTs at 0, 20, 200, 1000, and 2000mg/L with red spinach, lettuce, rice, cucumber, chili, lady's finger, and soybean based on root and shoot growth, cell death, and electrolyte leakage at the seedling stage. After 15 days of hydroponic culture, the root and shoot lengths of red spinach, lettuce, and cucumber were significantly reduced following exposure to 1000mg/L and 2000mg/L MWNTs. Similar toxic effects occurred regarding cell death and electrolyte leakage. Red spinach and lettuce were most sensitive to MWNTs, followed by rice and cucumber. Very little or no toxic effects were observed for chili, lady's finger, and soybean.

Keywords: Abbreviations; MWNTs; multi-walled carbon nanotubes; ROS; reactive oxygen species; DNA; deoxyribo nucleic acid; SEM; scanning electron microscopy; TEM; transmission electron microscopy; SD; standard deviation; US EPA; United States of Environmental Protection AgencyPhytotoxicity; Multi-walled carbon nanotubes (MWNTs); Seedling stage; Cell death; Electrolyte leakage

Investigation of biodistribution behavior of platinum particles in mice: Correlation between inductively coupled plasma-atomic emission spectroscopy and X-ray scanning analytical microscopy by Shigeaki Abe; Chika Koyama; Mami Mutoh; Tsukasa Akasaka; Motohiro Uo; Fumio Watari (pp. 125-128).

► We investigated the biodistribution of platinum microparticles in mice. ► The biodistribution behavior was observed using inductively coupled plasma-atomic emission spectroscopy (ICP) and scanning X-ray analytical microscopy (XSAM). ► The administered particles quickly reached in spleen, liver and lung with constant ration. ► We also estimated the correlation ship between XSAM and ICP measurement. ► The relative ratio of XSAM intensity showed highly correlation with the relative ratio of Pt concentration in organs.In this study, we investigated the biodistribution of platinum (Pt) microparticles in mice. The particles were administered through the tail vein, and then the biodistribution behavior was observed using inductively coupled plasma-atomic emission spectroscopy (ICP) and scanning X-ray analytical microscopy (XSAM). The administered particles quickly reached the spleen, liver, and lung at a constant ratio, and the ratio remained constant for four weeks. We also estimated the correlation between XSAM and ICP measurement. The relative ratio of XSAM intensity showed strong correlation with the relative ratio of Pt concentration in organs.

Keywords: Biodistribution; Platinum microparticle; Quantitative analysis; Correlation coefficient

Self-assembly of gold nanoparticles on a single crystalline sapphire substrate by Hiroyuki Takahashi; Naoto Shirahata; Takashi Narushima; Tetsu Yonezawa (pp. 129-133).

► Functionalization of the surface of single crystalline sapphire substrate. ► Hot hydrogen peroxide makes the surface contact angle of the sapphire substrate smaller according to OH groups on the surface. ► Silan coupling reagent can be attached to the hydrophilic sapphire substrate surface. ► Negatively charged gold nanoparticles could be immobilized on the cationic sapphire substrate surface.Single crystalline sapphire is an atomically flat substrate with a high transparency in a wide wavelength region. However, its surface is chemically stable so that the modification by a self-assembled monolayer is somewhat difficult. We have used a H₂O₂ treatment at 70°C to activate the surface and modified with a silan coupling reagent. The modification of the surface is discussed with the water contact angle. Immobilization of citrate-stabilized anionic gold nanoparticles on a cationically modified sapphire surface was carried out.

Keywords: Sapphire; Gold; Nanoparticle; Immobilization; SAM

Induction of classical activation of macrophage in vitro by water soluble chitin by Dong-Won Jeon; Woong Shick Ahn; Su Jung You; Gue Tae Chae; Young Bock Shim; Heung Jae Chun (pp. 134-139).

► Water soluble chitin (WSC) was prepared by re-acetylation of chitosan and was treated with the murine RAW 264.7 macrophage cell lines (ATCC TIB-71). ► WSC induced classical activation in the RAW 264.7 cells, accompanied by the induction of associated genes. ► The association of histone H2A gene perturbation was found.The purpose of this study is to understand the effect of chitin on macrophage mediated immunity, which is a significant factor to wound healing and tissue regeneration. In this work, water soluble chitin (WSC) was prepared by re-acetylation of chitosan and was treated with the murine RAW 264.7 macrophage cell lines (ATCC TIB-71). WSC induced classical activation in the RAW 264.7 cells, accompanied by the induction of associated genes. The results suggest that WSC is one of the functional chitin molecules that are responsible for the immune response, especially present in macrophage classical activation.

Keywords: Water soluble chitin; Macrophage; RAW 264.7; Classical activation

Effect of nano-hydroxyapatite on bone morphogenetic protein-2-induced hard tissue formation and dentin resorption on a dentin surface by Hiroki Tamagawa; Taichi Tenkumo; Tsutomu Sugaya; Masamitsu Kawanami (pp. 140-145).

► A nano-hydroxyapatite collagen composite with bone morphogenetic protein was studied. ► Implanted nano-hydroxyapatite enhanced hard tissue formation on a dentin surface. ► Implanted nano-hydroxyapatite did not accelerate dentin resorption.The purpose of this study was to evaluate the effects of the addition of nano-hydroxyapatite to a collagen membrane-carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) on hard tissue formation and dentin resorption on dentin surfaces in vivo.Nano-hydroxyapatite collagen composite (nHAC) membranes or collagen (C) membranes were each immersed in either 100 or 400μg/ml rhBMP-2 and placed on dentin chips that were implanted into rat thigh muscle. The implants were analyzed at 2 or 4weeks after surgery by histological observation and histomorphometric analysis.The percentage of the hard tissue formed by each nHAC group was significantly higher than that formed by any of the C groups, except for that formed by the group loaded with 400μg/ml rhBMP-2 at 4weeks after implantation. No significant differences were observed in the percentage of dentin resorption between the nHAC groups and C groups at any stage or at any rhBMP-2 concentration.These findings showed that addition of nano-hydroxyapatite to a collagen membrane accelerated the formation of hard tissue induced by a low dose of rhBMP-2 on dentin surfaces at an early stage after implantation into rat thigh muscle, without increasing dentin resorption.

Keywords: Nano-hydroxyapatite; rhBMP-2; Hard tissue formation; Dentin resorption

Instrumental studies on silicone oil adsorption to the surface of intraocular lenses by Chun Ho Kim; Choun-Ki Joo; Heung Jae Chun; Bok Ryul Yoo; Dong Il Noh; Young Bock Shim (pp. 146-152).

► It was found that PHEMA and Acrysof IOLs possess silicone oil repellant ability. ► The residual silicone oil was detected on the surfaces of PMMA and silicone IOLs. ► XPS studies showed that silicone oil coverage of PMMA lenses was 12%. ► Silicone oil covered the entire surface of the silicone IOLs.The purpose of this study was to examine the degree of adherence of silicone oil to various intraocular lenses (IOLs) through comparison of the physico-chemical properties of the oil and IOLs. Four kinds of IOLs comprising various biomaterials were examined: PMMA (720A™), PHEMA (IOGEL 1103™), Acrysof (MA60BM™), and silicone (SI30NB™). Each lens was immersed in silicone oil or carboxylated silicone (CS-PDMS) oil for 72h. For determination of the changes in chemical and elemental compositions on the surfaces of IOLs caused by the contact with silicone oil, IOLs were washed and rinsed with n-pentane to remove as much of the adsorbed silicone oil as possible, then subjected to Fourier transform infrared spectroscopic (FTIR) and X-ray photoelectron spectroscopic (XPS) analyses.The results of FTIR studies strongly indicate that washing with n-pentane completely removed the adhered silicone oil on the surfaces of PHEMA and Acrysof IOLs, whereas the residual silicone oil was detected on the surfaces of PMMA and silicone IOLs. XPS studies showed that silicone oil coverage of PMMA lenses was 12%, even after washing with n-pentane. In the case of silicone IOLs, the relative O1s peak area of carboxyl group in the residual CS-PDMS oil was found to be ∼2.7%. Considering that 2.8% carboxyl group-substituted silicone oil was used in the present study, CS-PDMS oil covered the entire surface of the silicone IOLs.

Keywords: Intraocular lens (IOL); Silicone oil; Polymethylmethacrylate; Polyhydroxyethylmethacrylate; Polydimethylsiloxane; XPS; FTIR; AFM

Thermodynamic aspects of calcium binding by poly( α-L-guluronate) chains. A molecular simulation study by Wojciech Plazinski; Mateusz Drach (pp. 153-155).

► The molecular dynamics studies on binding of calcium ions by poly( α-l-guluronate) chains were carried out. ► The Gibbs free energy landscapes corresponding to the process of calcium binding were calculated. ► The Effective coordination number parameter was introduced in order to describe the dynamic changes in the arrangement of water molecules coordinating calcium ions.The theoretical studies on binding of calcium ions by poly( α-l-guluronate) chains were carried out to provide the insight into the molecular basis of this process. The three local minima of the Gibbs free energy (corresponding to the two distinct stable states and to the one short living, meta-stable state) were distinguished. The results emphasize the important role of water molecules. The ECN (effective coordination number) parameter was introduced in order to describe the dynamic changes in the arrangement of solvent molecules coordinating calcium ion.

Keywords: Alginate; Calcium; Molecular modeling; Molecular dynamics; GROMOS; Free energy; Coordination number

Deposition of TiC film on titanium for abrasion resistant implant material by ion-enhanced triode plasma CVD by Yuhe Zhu; Wei Wang; Xingya Jia; Tsukasa Akasaka; Susan Liao; Fumio Watari (pp. 156-158).

► Deposition of Titanium Carbide (TiC) layer on titanium (Ti) surface has been demonstrated by an ion-enhanced triode plasma chemical vapor deposition (CVD) method. ► The Vickers hardness of surface carbide was more than 2000, which confirmed its high abrasion resistance. ► Physical and mechanical properties of the deposited TiC film on Ti were investigated to examine its potential application as an abrasion resistant implant material.Deposition of titanium carbide (TiC) layer on titanium (Ti) surface has been demonstrated by an ion-enhanced triode plasma chemical vapor deposition (CVD) method using a TiCl₄+CH₄+H₂ gas mixture. Physical and mechanical properties of the deposited TiC film on Ti were investigated to examine its potential application as an abrasion resistant implant material. X-ray diffraction (XRD) showed that the specimen was consisted of TiC and Ti. Carbide layer of about 6μm thickness was observed on the cross section of the specimen by scanning electron microscopy (SEM). The Vickers hardness of surface carbide was more than 2000, which confirmed its high abrasion resistance.

Keywords: Titanium carbide (TiC); Hard coating; Ion-enhanced triode plasma CVD; Abrasion resistance

Choice of biomaterials—Do soft occlusal splints influence jaw-muscle activity during sleep? A preliminary report by Taro Arima; Tamiyo Takeuchi; Akio Tomonaga; Wataru Yachida; Noboru Ohata; Peter Svensson (pp. 159-162).

► Tested if choice of biomaterials for occlusal splints influences biological outcome such as jaw-muscle activity during sleep. ► Jaw-muscle activity during sleep was decreased by wearing hard-material occlusal splint. ► Jaw-muscle activity during sleep was not influenced by wearing soft-material occlusal splint. ► This specific choice of biomaterials may have impact on the neurobiological regulation of jaw-muscle activity during sleep.The choice of biomaterials for occlusal splints may significantly influence biological outcome. In dentistry, hard acrylic occlusal splints (OS) have been shown to have a temporary and inhibitory effect on jaw-muscle activity, such as tooth clenching and grinding during sleep, i.e., sleep bruxism (SB). Traditionally, this inhibitory effect has been explained by changes in the intraoral condition rather than the specific effects of changes in occlusion. The aim of this preliminary study was to investigate the effect of another type of occlusal surface, such as a soft-material OS in addition to a hard-type OS in terms of changes in jaw-muscle activity during sleep.Seven healthy subjects (mean±SD, six men and one woman: 28.9±2.7 year old), participated in this study. A soft-material OS (ethylene vinyl acetate copolymer) was fabricated for each subject and the subjects used the OS for five continuous nights. The EMG activity during sleep was compared to baseline (no OS). Furthermore, the EMG activity during the use of a hard-type OS (Michigan-type OS, acrylic resin), and hard-type OS combined with contingent electrical stimulation (CES) was compared to baseline values. Each session was separated by at least two weeks (washout). Jaw-muscle activity during sleep was recorded with single-channel ambulatory devices (GrindCare, MedoTech, Herlev, Denmark) in all sessions for five nights.Jaw-muscle activity during sleep was 46.6±29.8 EMG events/hour at baseline and significantly decreased during the hard-type OS (17.4±10.5, P=0.007) and the hard-type OS+CES (10.8±7.1, P=0.002), but not soft-material OS (36.3±24.5, P=0.055). Interestingly, the soft-material OS (coefficient of variance=98.6±35.3%) was associated with greater night-to-night variations than baseline (39.0±11.8%) and the hard-type OS+CES (53.3±13.7%, P<0.013).The present pilot study in small sample showed that a soft-material occlusal splint does not seem to inhibit jaw-muscle activity during sleep. Within the limitation of the study, it appears that the choice of biomaterials for occlusal splints may have a significant impact on the neurobiological regulation of jaw-muscle activity during sleep.

Keywords: Biomaterials; Electromyography; Occlusal splint; Sleep bruxism; Temporalis muscle

Ti–6Al–4V alloy surface modification for medical applications by Jaroslav Fojt (pp. 163-167).

► Nanostructuring of titanium–aluminium–vanadium alloy. ► Interaction of nanostructure and diamond-like carbon with simulated body fluid. ► Electrochemical response of different surface treatments.The objective of the presented work was to compare the properties of the two surface modifications of Ti–6Al–4V alloy. In the first case, a nanostructure of a tube character was constructed electrochemically under defined conditions and in the second case the alloy was coated with a DLC layer. Surfaces were analyzed in terms of their composition and morphology. Electrochemical impedance spectroscopy study of the specimens’ interaction with a simulated body fluid followed. The interaction of the modified surfaces with the cell environment was evaluated using heteronuclear cell line MG 63. Both surface treatments lead to improve of biological properties of studied alloy.

Keywords: Nanostructure; Diamond-like carbon; Surface analysis; Impedance spectroscopy; Titanium alloy

Study on drug release of and biological response to UHMWPE wear debris carrying estradiol by Shuxin Qu; Aiqin Liu; Xiaomin Liu; Yinlong Bai; Jie Weng (pp. 168-175).

► We prepared ultra-high molecular weight polyethylene (UHMWPE) loaded with 17β-estradiol (E2) to treat osteolysis after artificial joint replacement. ► We investigate the in vitro release of E2 and the cell biological response to UHMWPE-E2 wear debris. ► The in vitro E2 release included three stages during the release process: initial burst release, celerity release and steady release. ► The UHMWPE-E2 wear debris could promote the proliferation and ALP activity of osteoblasts and inhibit the expression of IL-6 of osteoblasts. ► The E2 in UHMWPE-E2 would play a role in the treatment of the osteolysis after artificial hip joint replacement.The aim of this study is to investigate in vitro release of 17β-estradiol (E2), the potential drug to treat osteolysis, and the biological response to ultra-high molecular weight polyethylene loaded with E2 (UHMWPE-E2) wear debris. The osteoblasts (MC3T3-E1) and macrophages (RAW264.7) were co-cultured with UHMWPE-E2 wear debris via inversion culture technique, respectively. MTT, ALP and ELISA assay were employed to evaluate the cell proliferation, ALP activity and the expression of interleukin-6 (IL-6). In vitro E2 release included: initial burst release, celerity release and steady release. The E2 released steadily after 40d and lasted more than 60d. The E2 in UHMWPE-E2 wear debris promoted the proliferation and ALP activity of MC3T3-E1 cells at the high debris dosages of 8–10mg. In particular, the UHMWPE-E2 wear debris inhibited the expression of IL-6 of osteoblasts at all dosages in the present study. RAW264.7 cells cultured with UHMWPE-E2 and UHMWPE wear debris exhibited large sizes about 100μm in diameter. The small size wear debris presented inside of cells indicated that the wear debris activated the phagocytosis of macrophages. The results indicated the E2 in UHMWPE-E2 would play a role in the treatment of the debris induced-osteolysis after artificial hip joint replacement.

Keywords: UHMWPE; Wear debris; Estradiol; In vitro; drug release; Cell response

Preparation and characterization of hydroxyapatite/gelatin composite membranes for immunoisolation by Jyh-Ping Chen; Feng-Nian Chang (pp. 176-183).

► Cross-linking gelatin in the presence of hydroxyapatite forms composite membranes. ► The membrane was used for immunoisolation and encapsulation of cells. ► Encapsulated islet cells secrete insulin in response to glucose concentrations. ► The membrane is a good candidate for bioartificial pancreas development.Composite membranes are fabricated from hydroxyapatite (HAP) and gelatin for immunoisolation of cells. The films were fabricated by crosslinking 5wt%, 10wt%, and 20wt% gelatin with 1wt% glutaraldehyde (GA) in the presence of HAP. Fourier transform infrared spectroscopy analysis confirms imide bond formation between GA and gelatin, while the crystal structure of HAP powder remains unchanged from X-ray diffraction analysis. The degree of crosslinking depends on crosslinking time and gelatin concentration. For 5% and 10% gelatin, the degree of crosslinking levels off at 90% within 48h. From scanning electron microscopy micrographs, the microstructure of the composite membrane depends on the amount of gelatin used in the crosslinking reaction. The mechanical strength of the composite membrane could be enhanced by increasing the gelatin concentration. BET analysis indicates that pore size of the micropores on the surface HAP/gelatin agglomerates decreases with increasing gelatin concentration. However, the macropore, through which diffusion of molecules occurs, is larger at higher gelatin concentrations. The permeability coefficients of different molecules through a HAP/gelatin composite membrane increase with increasing gelatin concentration and is inversely correlated with the molecular weight of the molecule. For immunoisolation of cells, the diffusion of large molecules stimulated by the immune system can be rejected by a chamber constructed from the HAP/gelatin membrane. Insulinoma cells were encapsulated in alginate-poly-l-lysine-alginate microcapsules and enclosed in a HAP/gelatin chamber. The chamber did not impair the viability and function of insulinoma cells and cells can secrete insulin in response to glucose concentration change. The chamber is therefore useful for the physiologically controlled secretion of insulin in response to the blood glucose level. Intraperitoneal transplantation of the chamber into streptozotocin-induced diabetic SD rats could maintain normal blood glucose levels in test animals for up to 60days without immunosuppression.

Keywords: Composite membranes; Biomedical application; Hydroxyapatite; Gelatin; Immunoisolation

Aggregation mechanism of Pd nanoparticles inl-cysteine aqueous solution studied by NEXAFS and AFM by C. Tsukada; S. Ogawa; T. Mizutani; G. Kutluk; H. Namatame; M. Taniguchi; S. Yagi (pp. 184-187).

Display Omitted► We focus on the biocompatibility of Pd nanoparticles (NPs) forl-cysteine under water environment. ► The Pd NPs have been fabricated and deposited on Si wafer by gas evaporation method. ► When the Pd NPs/Si has been dipped intol-cysteine aqueous solution, thel-cysteine has selectively adsorbed on Pd NPs surface and existed as thel-cysteine thiolate, atomic S andl-cystine. ► Moreover, the aggregation of Pd NPs occurs by the migration of Pd NPs on Si and the cross-linked reaction betweenl-cysteine thiolate molecules adsorbed on Pd NPs.We focus on the biocompatibility of Pd nanoparticles (NPs) from the point of microscopic view. Thus, as the basic research for the biocompatibility, we have investigated the adsorbates on the Pd NPs surface and the aggregation mechanism for the Pd NPs on Si substrate after dipping intol-cysteine aqueous solution by means of NEXAFS measurement and AFM observation. The Pd NPs have been fabricated and deposited on the Si wafer by the gas evaporation method. Judging from the results of NEXAFS measurement, it is clear that thel-cysteine thiolate and atomic S exist on the Pd NPs surface. The results of AFM observation show that the Pd NPs aggregate. It is thought that the aggregation of the Pd NPs occurs by both the migration of the Pd NPs on Si wafer and the cross-linked reaction.

Keywords: Pd nanoparticle; l; -cysteine; Aggregation; Cross-linked reaction; Migration; NEXAFS

Repair of segmental bone defects with bone marrow and BMP-2 adenovirus in the rabbit radius by Lijia Cheng; Xiaofeng Lu; Yujun Shi; Li Li; Jing Xue; Li Zhang; Jie Xia; Yujia Wang; Xingdong Zhang; Hong Bu (pp. 188-193).

► Firstly, bone marrow combined with BMP-2 and porous ceramics could signiﬁcantly increase the amount of newly formed bone. ► Secondly, adding BMP-2 leaded to more bone formation than ceramics alone, suggesting both BM and virus play key roles. ► At last, our study might provide a new strategy for carrying out bone tissue engineering in the future clinical application.Bone tissue engineering (BTE) is approached via implantation of autogenous mesenchymal stem cells (MSCs), marrow cells, or platelet-rich plasma, etc. To the contrary, gene therapy combining with the bone marrow (BM) has not been often reported. This study was performed to investigate whether a modified BTE method, that is, the BM and a recombinant human bone morphogenetic protein-2 adenovirus (Ad.hBMP-2) gene administering in hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramics could accelerate the healing of segmental defects in the rabbit radius. In our study, ceramics were immersed in the adenovirus overnight, and half an hour before surgery, autologous BM aspirates were thoroughly mixed with the ceramics; at the same time, a 15-mm radius defect was introduced in the bilateral forelimbs of all animals, after that, this defect was filled with the following: (1) Ad.hBMP-2+HA/β-TCP+autologous BM (group 1); (2) HA/β-TCP+Ad.hBMP-2 (group 2); (3) HA/β-TCP alone (group 3); (4) an empty defect as a control (group 4). Histological observation and μ-CT analyses were performed on the specimens at weeks 2, 4, 8, and 12, respectively. In group 1, new bone was observed at week 4 and BM appeared at week 12, in groups 2 and 3, new bone was observed at week 8 and it was more mature at week 12, in contrast, the defect was not bridged in group 4 at week 12. The new bone area percentage in group 1 was significantly higher than that in groups 2 and 3. Our study indicated that BM combined with hBMP-2 adenovirus and porous ceramics could significantly increase the amount of newly formed bone. And this modified BTE method thus might have potentials in future clinical application.

Keywords: Ad.hBMP-2; Bone marrow; HA/β-TCP; Bone tissue engineering

Carbon nanotubes/hydroxyapatite nanocomposites fabricated by spark plasma sintering for bonegraft applications by Wei Wang; Yuhe Zhu; Fumio Watari; Susan Liao; Atsuro Yokoyama; Mamoru Omori; Hongjun Ai; FuZhai Cui (pp. 194-199).

► Multi-wall carbon nanotubes/hydroxyapatite (MWCNTs/HA) composites with 20%, 40% and 60% HA were fabricated successfully by spark plasma sintering (SPS). ► MWCNTs/HA composites kept the nanostructure of MWCNTs after SPS treatment. ► The in vivo reaction of four weeks showed that the MWCNTs/HA composites possessed better osseointegration compared to pure MWCNTs.Multi-wall carbon nanotubes/hydroxyapatite (MWCNTs/HA) composites with 20%, 40% and 60% HA were successfully fabricated at 1200°C and 120MPa by spark plasma sintering (SPS). Scanning electron microscope (SEM) observations showed that MWCNTs/HA composites kept the nanostructure of MWCNTs after SPS treatment. The bulk density of the MWCNTs/HA composites was increased by the addition of HA. However, the Vickers hardness, porosity, Young's modulus, flexure strength and compression strength of the MWCNTs/HA composites were decreased with increasing content of HA. To test their biocompatibility, the MWCNTs/40% HA composites were inserted into rat femur. The in vivo reaction of four weeks showed that the composites possessed better osseointegration compared to pure MWCNTs. The results indicated that the MWCNTs/HA composites have potential for a wide variety of bonegraft applications in the future.

Keywords: Carbon nanotube; Hydroxyapatite; Spark plasma sintering; Mechanical properties; Biocompatibility

Ectopic bone formation cannot occur by hydroxyapatite/β-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice by Lijia Cheng; Xin Duan; Zhou Xiang; Yujun Shi; Xiaofeng Lu; Feng Ye; Hong Bu (pp. 200-206).

► Firstly, chimeric mouse model could be established successfully by bone marrow transplantation after irradiation. ► Secondly, bone induction can occur in wild-type mice 90 days after implantation, but not occur in chimeric mice. ► Thirdly, destruction of immune function will block osteoinduction by calcium phosphate ceramics.Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups ( n=10), in group 1, a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/β-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede osteoinduction by CP. Overall, our results may shed light on clear mechanism study of bone induction in the future.

Keywords: Calcium phosphate ceramics; GFP; Chimeric model; Osteoinduction; BM transplantation

Preliminary study on the effect of wear process on drug release of ALN-loaded UHMWPE by Dan Yang; Shuxin Qu; Sunzhong Lin; Jie Huang; Rong Fu; Zhongrong Zhou (pp. 207-211).

► Ultra-high molecular weight polyethylene (UHMWPE) was loaded with alendronate sodium (ALN) to treat osteolysis. ► The ALN release of ALN-loaded UHMWPE during wear process was investigated and compared with that without wear process. ► The presence of ALN gave rise to the different wear behavior of ALN-loaded UHMWPE compared with that of UHMWPE in water. ► The ALN release of ALN-loaded UHMWPE was greatly promoted by the wear process.Ultra-high molecular weight polyethylene (UHMWPE) loaded with alendronate sodium (ALN) for anti-osteolysis was developed in our previous study. As a potential material of artificial joints, ALN-loaded UHMWPE is subjected to friction and wear which probably affect the ALN release in vivo. This study aims to explore the influence of friction and wear on the ALN release rate. For comparison, the specimens of control group, immersed motionlessly in distilled water, were not applied any friction. The morphological change of worn surface of ALN-loaded UHMWPE was observed through an independent wear test and was compared with that of control UHMWPE. The ALN release rate in the friction and wear process was higher than that of non-friction test. The cumulative mass of ALN increased slowly at the onset of wear process and then speeded up. The fibrils-like wear debris accumulated on the worn surface of ALN-loaded UHMWPE but did not appear on that of UHMWPE. The micro-pores formed during wear process, were probably favorable of the dissolution of ALN. It indicated that the ALN release of ALN-loaded UHMWPE was affected by the friction and wear. The frictional factors should be taken into account in predicting the ALN release rate of ALN-loaded UHMWPE.

Keywords: UHMWPE; Alendronate sodium (ALN); Drug release; Friction and wear

Preparation of Ag nanoparticle dispersed silk fibroin compact by Weidong Yu; Toshihiro Kuzuya; Shinji Hirai; Yasushi Tamada; Ken Sawada; Tatsuo Iwasa (pp. 212-217).

► We reported the facile synthesis of Ag-nanoparticle (Ag-NP) dispersed silk fibroin compact. ► The fibroin protein itself served as the reducing agent for AgNO₃. ► Ag NPs dispersed silk compact exhibited a characteristic color, corresponding to the surface plasmon resonance of Ag NPs. ► Ag NPs did not affect the thermal stability of silk fibroin. ► The antimicrobial test indicated that Ag-NPs dispersed silk compact have an excellent antimicrobial activity.We prepared a silk fibroin compact in which Ag nanoparticles (Ag NPs) were dispersed, by adding only silver nitrate (AgNO₃) to a silk fibroin solution without using any reducing agent. FT-IR and DSC revealed that the formation of Ag NPs distorted the conformation of the silk fibroin. However, the Ag NPs did not affect the thermal stability of the silk fibroin compact. An antimicrobial test against Escherichia coli ( E. coli) was performed using the silk fibroin compact in which Ag NPs were dispersed. The compact showed sufficient antimicrobial activity because the average survival of E. coli was 1.6% after 8h incubation even when the silver content was 0.01wt%. The addition of Ag NPs into silk fibroin compact can provide color and an antimicrobial function without spoiling the function of the silk fibroin compact.

Keywords: Ag; Nanoparticle; Silk fibroin resin

Porous chitosan scaffold cross-linked by chemical and natural procedure applied to investigate cell regeneration by Chih-Kai Yao; Jiunn-Der Liao; Chia-Wei Chung; Wei-I. Sung; Nai-Jen Chang (pp. 218-221).

► Polymeric scaffolds, made from chitosan-based films fixed by chemical (citrate) or natural method (genipin), were developed. ► Nano-indentation with a constant harmonic frequency was applied on porous scaffolds to explore their surface mechanics. ► The relationship between surface mechanical property and cell-surface interactions of scaffold materials was demonstrated. ► Porous scaffolds cross-linked by genipin showed adequate cell affinity, non-toxicity, and suitable mechanical properties.Porous chitosan scaffold is used for tissue engineering and drug delivery, but is limited as a scaffold material due to its mechanical weakness, which restrains cell adhesion on the surface. In this study, a chemical reagent (citrate) and a natural reagent (genipin) are used as cross-linkers for the formation of chitosan-based films. Nanoindentation technique with a continuous stiffness measurement system is particularly applied on the porous scaffold surface to examine the characteristic modulus and nanohardness of a porous scaffold surface. The characteristic modulus of a genipin-cross-linked chitosan surface is ≈2.325GPa, which is significantly higher than that of an uncross-linked one (≈1.292GPa). The cell-scaffold surface interaction is assessed. The cell morphology and results of an MTS assay of 3T3-fibroblast cells of a genipin-cross-linked chitosan surface indicate that the enhancement of mechanical properties induced cell adhesion and proliferation on the modified porous scaffold surface. The pore size and mechanical properties of porous chitosan film can be tuned for specific applications such as tissue regeneration.

Keywords: Porous chitosan film; Natural cross-linking agent; Scaffold; Genipin; Nano-indentation

Quantitative analysis on orientation of human bone integrated with midpalatal implant by micro X-ray diffractometer by Masaru Murata; Toshiyuki Akazawa; Toshihiro Yuasa; Miki Okayama; Junichi Tazaki; Takao Hanawa; Makoto Arisue; Itaru Mizoguchi (pp. 222-226).

► A titanium fixture is implanted into palatal bone of an 18-year-old patient as the unmoved anchorage for the orthodontic treatment. ► The fixture is integrated with compact bone with cortical bone-like osteon. ► Microbeam X-ray diffraction denotes the crystallinity and orientation of HAp. ► X-ray images of c-face in HAp reveal functionally graded distribution of bone quality. ► The crystal growth of c-face is caused by propagation of the continuous lateral stress.A midpalatal implant system has been used as the unmoved anchorage for teeth movement. An 18-year-old male patient presented with reversed occlusion and was diagnosed as malocclusion. A pure titanium fixture (lengths: 4mm, diameter: 3.3mm, Orthosystem^®, Institute Straumann, Switzerland) was implanted into the palatal bone of the patient as the orthodontic anchorage. The implant anchorage was connected with the upper left and right first molars, and had been used for 3 years. After dynamic treatments, the titanium fixture connected with bone was removed surgically, fixed in formalin solution, and embedded in resin. Specimens were cut along the frontal section of face and the direction of longitudinal axis of the implant, stained, and observed histologically. The titanium fixture was integrated directly with compact bone showing cortical bone-like structure such as lamella and osteon. In addition, to qualitatively characterize the implant-supported human bone, the crystallinity and orientation of hydroxyapatite (HAp) phase were evaluated by the microbeam X-ray diffraction analysis. Preferential alignment of c-axis of HAp crystals was represented by the relative intensity ratio of (002)-face diffraction peak to (310)-face one. The values decreased monotonously along the direction of the lateral stress from the site near the implant thread to the distant site in all horizontal lines of the map. These results indicated that the X-ray images for the intensity of c-face in HAp revealed functionally graded distribution of cortical bone quality. The micro-scale measurements of HAp structure could be a useful method for evaluating the mechanical stress distribution in human hard tissues.

Keywords: Midpalatal implant; Titanium; Hydroxyapatite; Crystallinity; Bone quality; Microbeam-X-ray diffraction

Preparation of foam-like carbon nanotubes/hydroxyapatite composite scaffolds with superparamagnetic properties by X.Y. Lu; T. Qiu; X.F. Wang; M. Zhang; X.L. Gao; R.X. Li; X. Lu; J. Weng (pp. 227-230).

► A new method has been employed to prepare the foam-like CNTs/HA composite scaffolds. ► The scaffolds have a withstanding mechanical property for their novel structures. ► About 2wt% MWNTs with a diameter of 60–100nm grow in situ from D-HA grains. ► These porous CNTs/HA composite scaffolds have a superparamagnetic property.In this paper, the foam-like composite scaffolds composed of hydroxyapatite (HA) and carbon nanotubes (CNTs) were prepared by a new method, where a polymer impregnating method was used for porous HA-based scaffold and a chemical vapor deposition (CVD) method was used for the growth of CNTs from the HA-based scaffold. The process produces the CNTs/HA scaffolds that have a foam-like structure with better mechanical property, better microstructure and a high degree of interconnection. A favorable pore size with big pores of 1–2mm and small pores of 20–300μm for osteoconduction and bone ingrowth is presented in these scaffolds. About 2wt% multi-walled CNTs with the diameter of 60–100nm are observed to be in situ grown from deficient nano-HA crystallites. Magnetic measurement exhibits these scaffolds are superparamagnetic with a saturation magnetization of 1.14emug⁻¹ at a room temperature, benefiting the scaffolds to take up growth factors in vivo, stem cell or other bioactive molecules easily. This new type of CNTs/HA scaffolds is expected to have a promising applications in bone tissue engineering, targeted drug delivery system and other biomedical fields.

Keywords: Scaffolds; Carbon nanotube; Hydroxyapatite; Composites; Superparamagnetism

Adsorption behavior ofl-cysteine on Ag nanoparticles under water environment studied by S K-edge NEXAFS by C. Tsukada; S. Yagi; T. Nomoto; T. Mizutani; S. Ogawa; H. Nameki; Y. Nakanishi; G. Kutluk; H. Namatame; M. Taniguchi (pp. 231-233).

Display Omitted► We focus on the biocompatibility of Ag nanoparticles fabricated by solution plasma. ► We investigated L-cysteine adsorption on Ag nanoparticle under water environment. ► Cystine is synthesized by disulfide bonding reaction of adsorbed L-cysteine thiolate. ► The synthesis reaction is much faster than that for previous metal nanoparticles.We have focused on the biocompatibility of the Ag nanoparticles. In this paper, we have fabricated the Ag nanoparticles by means of the solution plasma method. Since this method does not need to use the surfactant molecule, such as the water soluble polymer, the fabricated nanoparticle might possess the clean surface. The average diameter of the Ag nanoparticles is estimated to be 5.0±1.1nm. NEXAFS spectra showl-cysteine thiolate adsorbs on the Ag nanoparticle surface andl-cystine is synthesized. Though this reaction behavior is similar to the result of Rh(PVP, polyvinylpyrrolidone) system, in our previous study, the reaction speed for the Ag nanoparticle is much faster than that for the Rh(PVP) nanoparticle.

Keywords: Ag nanoparticle; l; -Cysteine; Solution plasma method; He-path; NEXAFS

The effect of porosity on barrier properties of DLC layers for dental implants by Ludek Joska; Jaroslav Fojt (pp. 234-239).

► Determination of the influence of porosity on titanium – DLC (a:CH) system corrosion behaviour. ► Characterisation of the layered systems corrosion behaviour by EIS is not always unambiguous. ► The results obtained in the study suggest that having the surface jet-blasted before application of coating substantially limits the layer's barrier function.

Keywords: DLC layers; Porosity; Corrosion; Electrochemical impedance spectroscopy

Enhanced compatibility of chemically modified titanium surface with periodontal ligament cells by T. Kado; T. Hidaka; H. Aita; K. Endo; Y. Furuichi (pp. 240-247).

► Cell-adhesive molecules were covalently immobilized on a Ti surface. ► Immobilized cell-adhesive molecules maintained native function on the Ti surface. ► Immobilized collagen enhanced adhesion of periodontal ligament cells to the Ti.A simple chemical modification method was developed to immobilize cell-adhesive molecules on a titanium surface to improve its compatibility with human periodontal ligament cells (HPDLCs).The polished titanium disk was immersed in 1% (v/v) p-vinylbenzoic acid solution for 2h to introduce carboxyl groups onto the surface. After rinsing with distilled deionized water, the titanium disk was dipped into 1.47% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution containing 0.1mg/ml Gly-Arg-Gly-Asp-Ser (GRGDS), human plasma fibronectin (pFN), or type I collagen from calf skin (Col) to covalently immobilize the cell-adhesive molecules on the titanium surface via formation of peptide bonds.X-ray photoelectron spectroscopy analyses revealed that cell-adhesive molecules were successfully immobilized on the titanium surfaces. The Col-immobilized titanium surface revealed higher values regarding nano rough characteristics than the as-polished titanium surface under scanning probe microscopy. The number of HPDLCs attached to both the pFN- and Col-immobilized titanium surfaces was twice that attached to the as-polished titanium surfaces. The cells were larger with the cellular processes that stretched to a greater extent on the pFN- and Col-immobilized titanium surfaces than on the as-polished titanium surface ( p<0.05). HPDLCs on the Col-immobilized titanium surfaces showed more extensive expression of vinculin at the tips of cell projections and more contiguously along the cell outline than on the as-polished, GRGDS-immobilized and pFN-immobilized titanium surfaces.It was concluded that cell-adhesive molecules successfully immobilized on the titanium surface and improved the compatibility of the surface with HPDLCs. The Col-immobilized titanium surface could be used for forming ligament-like tissues around titanium dental implants.

Keywords: Titanium; Implant; Chemical modification; Cell-adhesive molecules; Human periodontal ligament cells

Morphology, size distribution and elemental composition of several dental debris by Shigeaki Abe; Nobuki Iwadera; Mitsue Esaki; Ken-Ichi Aoyama; Tsukasa Akasaka; Motohiro Uo; Manabu Morita; Yasutaka Yawaka; Fumio Watari (pp. 248-252).

► We investigated morphologies, size distributions and elemental compositions of dental debris. ► These characteristics of the debris depended on the combination of the workpiece and the cutting/grinding materials. ► In some cases, the debris consisted of both the workpiece and the cutting/grinding materials. ► In the case of teeth as the workpiece, teeth debris was smaller than 2μm in diameter and had a potential to induce inflammation.We investigated morphologies, size distributions and elemental compositions of dental debris formed by cutting/grinding teeth or dental alloys. The average size of debris formed by cutting/grinding dental alloy was around 100μm and that of teeth was 20μm. The debris formed by grinding with diamond or carborundum point had isotropic irregular shape, while the debris formed by cutting with carbide bar had characteristic lathe-cut shape. The elemental analysis indicated that the debris formed by grinding dental alloy with carborundum point consisted of not only the particles of the alloy but also the particles of Si compounds with the size of around 10μm. The particles of Si compounds would be formed by abrasion of the grinding instrument (carborundum, SiC). Similarly, the debris formed by grinding with diamond point also contained submicro-sized particles consisting of C compounds. The results indicate that the morphology and composition of dental debris are varied depending on the combination between the workpiece and the cutting/grinding materials and that the dental debris consist of both the workpiece and the cutting/grinding materials in some combination. In addition, some of the debris of tooth had the size less than 2μm, which has a potential to induce inflammation. Though the inflammation can be expected at low level, it is required to investigate the details in future.

Keywords: Dental working debris; Dental alloy; Tooth; Morphology; Size distribution

SEM observation and wettability of variously processed and fractured surface of dental zirconia by Naoyoshi Tarumi; Motohiro Uo; Eiji Yamaga; Fumio Watari (pp. 253-257).

► Zirconia surface after various treatments was analyzed by SEM, contact angle and surface roughness measurement, and compared. ► Both mirror polishing and porcelain layering look similarly smooth. ► 24-h acid treatment induced much more surface roughening with the 50–100nm particulate roughness than tribochemical treatments. ► Experimentally attained fracture surfaces cracks were formed straight in most part and in some places curved or twisted into S or Ω form. ► Clinically occurred fracture exhibited the scab-like surface.Current dental zirconia has several problems in clinical application such as chipping, fracture and detachment. To reduce these problems the surface after various treatments was analyzed by SEM observation, contact angle measurement and surface roughness measurement, and compared. The surface after mirror polishing was smooth. Porcelain layering was smooth except large formed grooves by bubbles. After sandblast and tribochemical treatments, the surfaces showed several micron-sized caving with micron to submicron-level irregularities. Sandblast and tribochemical treatments with the lager roughness had the smaller water contact angle than silicone wheel polishing. Clinically fractured surface of zirconia showed a more complex structure than manually fractured surface, which may be due to the various mode of stress to be imposed repetitively to various direction.

Keywords: Zirconia; Fracture; Surface treatments; Wettability; Dental prostheses; CAD/CAM

A useful and non-invasive microanalysis method for dental restoration materials by M. Hosoki; T. Satsuma; K. Nishigawa; H. Takeuchi; K. Asaoka (pp. 258-262).

► This method for the microanalysis of dental alloys is beneficial for patients with allergies to dental materials. ► This metal sample is easy to mail it for inspection at specialist institutes. ► This method can be also be used in general dental clinics.The elemental analysis of intraoral dental restorations provides considerable information for the treatment of dental metal allergy. Elemental analyses require specific instruments and complicated procedures, so this examination is not commonly carried out in private dental clinics. We describe a novel, simple and useful micro-analytical method for dental metal restorations.Micro metal dust was obtained by polishing the surface of restorative metal material with an unused silicone point (SUPER-SNAP). The metal dust on the silicone point was then rubbed onto adhesive tape, and this tape was covered with polyethylene film. The amount of metal dust material was <20μg. An energy dispersive X-ray fluorescence spectrometer was used to carry out the elementary analysis of the metal dust on the polyethylene film.Three types of dental metal alloy materials of known components were examined. The results of elementary analyses were compared with the specifications provided by the manufacturer. The same procedure was carried out for three dental metal restorations of an adult female volunteer in vivo.The results of elemental analyses for five alloy materials exactly matched the product specification. Three metal samples obtained from intraoral restoration were also available for elemental analyses.The distinct advantage of this method is that it enables sample extraction without an invasive effect for the restoration. The metal sample is in a polyethylene film, so it is easy to mail it for inspection at specialist institutes yet it can be also be used in general dental clinics.

Keywords: Dental alloy; Allergy; X-ray fluorescence spectroscopy (XRFS); Dental restoration

Novel low temperature processing techniques for apatite ceramics and chitosan polymer composite bulk materials and its mechanical properties by Takamasa Onoki; Atsushi Nakahira; Tomoyuki Tago; Yoshiyuki Hasegawa; Tomoaki Kuno (pp. 263-266).

Display Omitted► CHI and DCPD were mixed in nano-scale by co-precipitation method. ► Bulk CHI/HA materials derived from the CHI/DCPD hybrid material were obtained by hydrothermal hot-pressing. ► Fracture toughness of the obtained bulk materials was enhanced from 0.30 to 0.40MPam^1/2 by the CHI hybridization.A co-precipitation method was used for processing chitosan (CHI)/calcium hydrogen phosphate dehydrate (DCPD) hybrid material. CHI solution was mixed into 1.0-M calcium nitrate solution. CHI/DCPD hybrid material was prepared by the above explained addition of CHI and Ca ion source to 1.0-M diammonium hydrogen phosphate solution. It was observed by transmission electron microscopy that CHI and DCPD were mixed within submicron meter scale. CHI/HA bulk materials derived from the CHI/DCPD hybrid materials were obtained by using a hydrothermal hot-pressing (HHP) method. A pressure of 40MPa was initially applied to the sample. An HHP autoclave was heated up to 150°C for 2h. Modified 3-point bending tests were conducted to obtain an easy estimate of the fracture toughness for the CHI/HA bulk materials made with the HHP method. The critical stress intensity factor K_c of the fabricated CHI/HA bulk materials was enhanced from 0.30 to 0.40MPam^1/2 by the hybridization of CHI into DCPD.

Keywords: Hydrothermal; Co-precipitation; Nanocomposite

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Applied Surface Science (v.262, #)