bluuebunny/crossref_metadata_2025 huggingface dataset:
A Short Review on the Microstructure, Transformation Behavior and Functional Properties of NiTi Shape Memory Alloys Fabricated by Selective Laser Melting
Authors: Wang Xiebin , Kustov Sergey , Van Humbeeck Jan
Published: September 2018

Due to unique functional and mechanical properties, NiTi shape memory alloys are one of the most promising metallic functional materials. However, the poor workability limits the extensive utilization of NiTi alloys as components of complex shapes. The emerging additive manufacturing techniques provide high degrees of freedom to fabricate complex structures. A freeform fabrication of complex structures by additive manufacturing combined with the unique functional properties (e.g., shape memory effect and superelasticity) provide great potential for material and structure design, and thus should lead to numerous applications. In this review, the unique microstructure that is generated by selective laser melting (SLM) is discussed first. Afterwards, the previously reported transformation behavior and mechanical properties of NiTi alloys produced under various SLM conditions are summarized.
Interlaminar Shear Behavior of Laminated Carbon Fiber Reinforced Plastic from Microscale Strain Distributions Measured by Sampling Moiré Technique
Authors: Wang Qinghua , Ri Shien , Tsuda Hiroshi , Takashita Yosuke , Kitamura Ryuta , Ogihara Shinji
Published: September 2018

In this article, the interlaminar shear behavior of a [±45°]4s laminated carbon fiber reinforced plastic (CFRP) specimen is investigated, by utilizing microscale strain mapping in a wide field of view. A three-point bending device is developed under a laser scanning microscope, and the full-field strain distributions, including normal, shear and principal strains on the cross section of CFRP, in a three-point bending test, are measured using a developed sampling Moiré technique. The microscale shear strain concentrations at interfaces between each two adjacent layers were successfully detected and found to be positive-negative alternately distributed before damage occurrence. The 45° layers slipped to the right relative to the −45° layers, visualized from the revised Moiré phases, and shear strain distributions of the angle-ply CFRP under different loads. The absolute values of the shear strain at interfaces gradually rose with the increase of the bending load, and the sudden decrease of the shear strain peak value implied the occurrence of interlaminar damage. The evolution of the shear strain concentrations is useful in the quantitative evaluation of the potential interlaminar shear failure.
Effect of Remelting Temperature and Soaking Time on Microstructure and Mechanical Properties of the Thixoformed Part of Nano-Sized SiCp/7075 Composite
Authors: Jiang Jufu , Xiao Guanfei , Liu Yingze , Wang Ying , Nie Xi
Published: September 2018

Semisolid billet of the 7075 aluminum matrix composite reinforced with nano-sized SiC particles was first fabricated by an ultrasonic-assisted semisolid stirring method and rheoforming technology. Then it was thixoformed into a cylinder part under different remelting temperatures and soak times. The effects of the remelting temperature and soaking time on the mechanical properties and microstructure of the thixoformed composite part were investigated. The results show that parts of good quality were thixoformed successfully. The microstructure of the top side wall of the thixoformed part consisted of near spheroidal grains. A large quantity of elongated grains occurred in the medium and bottom side walls and the bottom itself. With increasing remelting temperatures, the size of the solid grains of the thixoformed parts showed a trend of first to increase and afterwards to decrease. High density dislocations were found in the microstructure when the remelting temperatures were 590 °C and 600 °C. When the soaking time was 15 min, the severest deformation occurred in the thixoformed part. High mechanical properties of the thixoformed parts were achieved under conditions such as a remelting temperature between 590 °C and 600 °C and a soaking time between 10 min and 15 min. The fracture mode of the thixoformed part changed from transgranular fracture to intergranular fracture when the remelting temperature was elevated from 580 °C to 610 °C. After the thixoformed parts were treated by T6, the ultimate tensile strength (UTS) and elongation of the side wall were improved to 552 MPa and 7.9%, respectively. Dispersed MgZn2 precipitates created by T6 heat treatment led to an improvement of the mechanical properties.
Insights into Machining of a β Titanium Biomedical Alloy from Chip Microstructures
Authors: Kent Damon , Rahman Rashid Rizwan , Bermingham Michael , Attar Hooyar , Sun Shoujin , Dargusch Matthew
Published: September 2018

New metastable β titanium alloys are receiving increasing attention due to their excellent biomechanical properties and machinability is critical to their uptake. In this study, machining chip microstructure has been investigated to gain an understanding of strain and temperature fields during cutting. For higher cutting speeds, ≥60 m/min, the chips have segmented morphologies characterised by a serrated appearance. High levels of strain in the primary shear zone promote formation of expanded shear band regions between segments which exhibit intensive refinement of the β phase down to grain sizes below 100 nm. The presence of both α and β phases across the expanded shear band suggests that temperatures during cutting are in the range of 400–600 °C. For the secondary shear zone, very large strains at the cutting interface result in heavily refined and approximately equiaxed nanocrystalline β grains with sizes around 20–50 nm, while further from the interface the β grains become highly elongated in the shear direction. An absence of the α phase in the region immediately adjacent to the cutting interface indicates recrystallization during cutting and temperatures in excess of the 720 °C β transus temperature.
Using the Multi-Response Method with Desirability Functions to Optimize the Zinc Electroplating of Steel Screws
Authors: Lorza Ruben Lostado , Calvo María Ángeles Martínez , Labari Carlos Berlanga , Fuente Pedro J. Rivero
Published: September 2018

Zinc electroplating is a coating process controlled by several input process parameters. However, the commonly used input parameters for setting the process of zinc deposition are current density, temperature of the coating solution, zinc concentration, deposition time, and concentration of additives (conditioner and brightener). The power consumed in the zinc plating process, coating thickness, increase in coating mass, and corrosion resistance are considered to be outputs or zinc coating parameters. They are widely used when the zinc coating requirements are based on the coating process cost, coating process speed, corrosion resistance, and coating thickness. This paper seeks to determine regression models by the response surface method (RSM) that relate the zinc coating parameters to the input parameters in steel screws. When considering the coating requirements of cost, coating process speed, corrosion resistance, and coating thickness, the optimal input parameters were found by using a multi-response surface (MRS). Input parameters of 0.3 amps/dm2, 20.0 °C, 13.9 g/L, 45 min, 28.5 mL/L, and 2.8 mL/L, respectively (relative to the commonly used input parameters detailed above), were obtained when considering the cost. Considering minimization of the deposition time, the input parameters obtained were 0.5 amps/dm2, 24.6 °C, 13.9 g/L, 45 min, 26.9 mL/L, and 1.1 mL/L, respectively. The optimal inputs to maximize the corrosion resistance were 0.6 amps/dm2, 32.4 °C, 14.0 g/L, 45 min, 28.7 mL/L, and 2.5 mL/L, respectively. Finally, when maximizing the coating thickness, the inputs were 0.7 amps/dm2, 38.4 °C, 12.2 g/L, 45 min, 26.5 mL/L, and 1.5 mL/L, respectively.
Design, Simulation and Experimental Study of the Linear Magnetic Microactuator
Authors: Feng Hanlin , Miao Xiaodan , Yang Zhuoqing
Published: September 2018

This paper reports the design, simulation and experimental study of a linear magnetic microactuator for portable electronic equipment and microsatellite high resolution remote sensing technology. The linear magnetic microactuator consists of a planar microcoil, a supporter and a microspring. Its bistable mechanism can be kept without current by external permanent magnetic force, and can be switched by the bidirectional electromagnetic force. The linearization and threshold of the bistable mechanism was optimized by topology structure design of the microspring. The linear microactuator was then fabricated based on non-silicon technology and the prototype was tested. The testing results indicated that the bistable mechanism was realized with a fast response of 0.96 ms, which verified the simulation and analysis.
Electrohydrodynamic Direct-Writing Micropatterns with Assisted Airflow
Authors: Jiang Jiaxin , Wang Xiang , Li Wenwang , Liu Juan , Liu Yifang , Zheng Gaofeng
Published: September 2018

Electrohydrodynamic direct-writing (EDW) is a developing technology for high-resolution printing. How to decrease the line width and improve the deposition accuracy of direct-written patterns has been the key to the promotion for the further application of EDW. In this paper, an airflow-assisted spinneret for electrohydrodynamic direct-writing was designed. An assisted laminar airflow was introduced to the EDW process, which provided an additional stretching and constraining force on the jet to reduce the surrounding interferences and enhance jet stability. The flow field and the electric field around the spinneret were simulated to direct the structure design of the airflow-assisted spinneret. Then, a series of experiments were conducted, and the results verified the spinneret design and demonstrated a stable ejection of jet in the EDW process. With assisted airflow, the uniformity of printed patterns and the deposition position accuracy of a charged jet can be improved. Complex patterns with positioning errors of less than 5% have been printed and characterized, which provide an effective way to promote the integration of micro/nanosystems.
Amino Acids in the Development of Prodrugs
Authors: Vale Nuno , Ferreira Abigail , Matos Joana , Fresco Paula , Gouveia Maria João
Published: September 2018

Although drugs currently used for the various types of diseases (e.g., antiparasitic, antiviral, antibacterial, etc.) are effective, they present several undesirable pharmacological and pharmaceutical properties. Most of the drugs have low bioavailability, lack of sensitivity, and do not target only the damaged cells, thus also affecting normal cells. Moreover, there is the risk of developing resistance against drugs upon chronic treatment. Consequently, their potential clinical applications might be limited and therefore, it is mandatory to find strategies that improve those properties of therapeutic agents. The development of prodrugs using amino acids as moieties has resulted in improvements in several properties, namely increased bioavailability, decreased toxicity of the parent drug, accurate delivery to target tissues or organs, and prevention of fast metabolism. Herein, we provide an overview of models currently in use of prodrug design with amino acids. Furthermore, we review the challenges related to the permeability of poorly absorbed drugs and transport and deliver on target organs.
Multi-Body Interactions in Molecular Docking Program Devised with Key Water Molecules in Protein Binding Sites
Authors: Xiao Wei , Wang Disha , Shen Zihao , Li Shiliang , Li Honglin
Published: September 2018

Water molecules play an important role in modeling protein-ligand interactions. However, traditional molecular docking methods often ignore the impact of the water molecules by removing them without any analysis or keeping them as a static part of the proteins or the ligands. Hence, the accuracy of the docking simulations will inevitably be damaged. Here, we introduce a multi-body docking program which incorporates the fixed or the variable number of the key water molecules in protein-ligand docking simulations. The program employed NSGA II, a multi-objective optimization algorithm, to identify the binding poses of the ligand and the key water molecules for a protein. To this end, a force-field-based hydration-specific scoring function was designed to favor estimate the binding affinity considering the key water molecules. The program was evaluated in aspects of the docking accuracy, cross-docking accuracy, and screening efficiency. When the numbers of the key water molecules were treated as fixed-length optimization variables, the docking accuracy of the multi-body docking program achieved a success rate of 80.58% for the best RMSD values for the recruit of the ligands smaller than 2.0 Å. The cross-docking accuracy was investigated on the presence and absence of the key water molecules by four protein targets. The screening efficiency was assessed against those protein targets. Results indicated that the proposed multi-body docking program was with good performance compared with the other programs. On the other side, when the numbers of the key water molecules were treated as variable-length optimization variables, the program obtained comparative performance under the same three evaluation criterions. These results indicated that the multi-body docking with the variable numbers of the water molecules was also efficient. Above all, the multi-body docking program developed in this study was capable of dealing with the problem of the water molecules that explicitly participating in protein-ligand binding.
Novel Titania Nanocoatings Produced by Anodic Oxidation with the Use of Cyclically Changing Potential: Their Photocatalytic Activity and Biocompatibility
Authors: Radtke Aleksandra , Bal Monika , Jędrzejewski Tomasz
Published: September 2018

The anodic oxidation of Ti6Al4V substrate surfaces with the use of cyclically changing potential enabled the production of novel titania nanocoatings. Morphologically different coatings were prepared in the ethylene glycol-based electrolyte with diluted HF and various amounts of water. The structure of the produced surface materials was characterized by X-ray diffraction, Raman spectroscopy, and diffuse reflectance infrared Fourier transform spectroscopy. Their morphology was visualized by scanning electron microscopy. The photocatalytic efficiency of produced materials was studied by the observation of methylene blue degradation under UV irradiation. Their biointegration properties were established on the basis of immunological assays, which checked murine fibroblasts adhesion and proliferation on novel coatings. The obtained results pointed out to both high biocompatibility, as well as the photoactivity of one of the obtained nanocoatings, allowing trusting in the applicative nature of this material.
Bidisperse Magnetic Particles Coated with Gelatin and Graphite Oxide: Magnetorheology, Dispersion Stability, and the Nanoparticle-Enhancing Effect
Authors: Fu Yu , Yao Jianjun , Zhao Honghao , Zhao Gang , Wan Zhenshuai , Qiu Ying
Published: September 2018

The magnetorheology and dispersion stability of bidisperse magnetic particles (BMP)-based magnetorheological (MR) fluids were improved by applying a novel functional coating composed of gelatin and graphite oxide (GO) to the surfaces of the micron-sized carbonyl iron (CI) and nanoparticles Fe3O4. Gelatin acted as a grafting agent to reduce the aggregation and sedimentation of CI particles and prevent nanoparticles Fe3O4 from oxidation. In addition, a dense GO network on the surface of gelatin-coated BMP was synthesized by self-assembly to possess a better MR performance and redispersibility. The rheological properties of MR fluids containing dual-coated BMP were measured by a rotational rheometer under the presence of magnetic field and their dispersion stability was examined through sedimentation tests. The results showed that CI@Fe3O4@Gelatin@GO (CI@Fe3O4@G@GO) particles possessed enhanced MR properties and dispersion stability. In addition, the nanoparticle-enhancing effects on the dispersion stability of BMP-based MR fluids were investigated using Monte Carlo simulations.
Fish Oil Supplementation Reduces Inflammation but Does Not Restore Renal Function and Klotho Expression in an Adenine-Induced CKD Model
Authors: Henao Agudelo Juan S. , Baia Leandro C. , Ormanji Milene S. , Santos Amandda R. P. , Machado Juliana R. , Saraiva Câmara Niels O. , Navis Gerjan J. , De Borst Martin H. , Heilberg Ita P.
Published: September 2018

Background: Chronic kidney disease and inflammation promote loss of Klotho expression. Given the well-established anti-inflammatory effects of omega-3 fatty acids, we aimed to investigate the effect of fish oil supplementation in a model of CKD. Methods: Male C57BL/6 mice received supplementation with an adenine-enriched diet (AD, n = 5) or standard diet (CTL, n = 5) for 10 days. Two other experimental groups were kept under the adenine diet for 10 days. Following adenine withdrawal on the 11th day, the animals returned to a standard diet supplemented with fish oil (Post AD-Fish oil, n = 9) or not (Post AD-CTL, n = 9) for an additional period of 7 days. Results: Adenine mice exhibited significantly higher mean serum urea, creatinine, and renal expression of the pro-inflammatory markers Interleukin-6 (IL-6), C-X-C motif chemokine 10 (CXCL10), and Interleukin-1β (IL-1β), in addition to prominent renal fibrosis and reduced renal Klotho gene expression compared to the control. Post AD-Fish oil animals demonstrated a significant reduction of IL-6, C-X-C motif chemokine 9 (CXCL9), and IL-1β compared to Post AD-CTL animals. However, serum creatinine, renal fibrosis, and Klotho were not significantly different in the fish oil-treated group. Furthermore, renal histomorphological changes such as tubular dilatation and interstitial infiltration persisted despite treatment. Conclusions: Fish oil supplementation reduced renal pro-inflammatory markers but was not able to restore renal function nor Klotho expression in an adenine-induced CKD model.
Cadaverous Decomposition as a Representation of the Soul’s Journey. Anthropological Evidence of a Funerary Ritual
Authors: Licata Marta , Borgo Melania , Badino Paola , Iorio Silvia
Published: September 2018

Our archaeological and anthropological investigations carried out inside the Crypt of the Franciscan Monastery in Azzio (Varese, Northern Italy) allowed us to discover a singular funerary practice of Franciscan friars. It consisted of a secondary burial practice.
The Development of Highly Flexible Stretch Sensors for a Robotic Hand
Authors: Devaraj Harish , Giffney Tim , Petit Adeline , Assadian Mahtab , Aw Kean
Published: September 2018

Demand for highly compliant mechanical sensors for use in the fields of robotics and wearable electronics has been constantly rising in recent times. Carbon based materials, and especially, carbon nanotubes, have been widely studied as a candidate piezoresistive sensing medium in these devices due to their favorable structural morphology. In this paper three different carbon based materials, namely carbon black, graphene nano-platelets, and multi-walled carbon nanotubes, were utilized as large stretch sensors capable of measuring stretches over 250%. These stretch sensors can be used in robotic hands/arms to determine the angular position of joints. Analysis was also carried out to understand the effect of the morphologies of the carbon particles on the electromechanical response of the sensors. Sensors with gauge factors ranging from one to 1.75 for strain up to 200% were obtained. Among these sensors, the stretch sensors with carbon black/silicone composite were found to have the highest gauge factor while demonstrating acceptable hysteresis in most robotic hand applications. The highly flexible stretch sensors demonstrated in this work show high levels of compliance and conformance making them ideal candidates as sensors for soft robotics.
On Linking of Task Analysis in the HRA Procedure: The Case of HRA in Offshore Drilling Activities
Authors: Strand Geir-Ove , Haskins Cecilia
Published: September 2018

Human reliability analysis (HRA) has become an increasingly important element in many industries for the purpose of risk management and major accident prevention; for example, recently to perform and maintain probabilistic risk assessments of offshore drilling activities, where human reliability plays a vital role. HRA experience studies, however, continue to warn about potential serious quality assurance issues associated with HRA methods, such as too much variability in comparable analysis results between analysts. A literature review highlights that this lack of HRA consistency can be traced in part to the HRA procedure and a lack of explicit application of task analysis relevant to a wide set of activity task requirements. As such, the need for early identification of and consistent focus on important human performance factors among analysts may suffer, and consequently, so does the ability to achieve continuous enhancements of the safety level related to offshore drilling activities. In this article, we propose a method that clarifies a drilling HRA procedure. More precisely, this article presents a novel method for the explicit integration of a generic task analysis framework into the probabilistic basis of a drilling HRA method. The method is developed and demonstrated under specific considerations of multidisciplinary task and well safety analysis, using well accident data, an HRA causal model, and principles of barrier management in offshore regulations to secure an acceptable risk level in the activities from its application.
Association between Subjective Indicators of Recovery Status and Heart Rate Variability among Divison-1 Sprint-Swimmers
Authors: Flatt Andrew A. , Esco Michael R. , Nakamura Fabio Y.
Published: September 2018

Heart rate variability (HRV) is a physiological marker of training adaptation among athletes. However, HRV interpretation is challenging when assessed in isolation due to its sensitivity to various training and non-training-related factors. The purpose of this study was to determine the association between athlete-self report measures of recovery (ASRM) and HRV throughout a preparatory training period. Ultra-short natural logarithm of the root mean square of successive differences (LnRMSSD) and subjective ratings of sleep quality, fatigue, muscle soreness, stress and mood were acquired daily for 4 weeks among Division-1 sprint-swimmers (n = 17 males). ASRM were converted to z-scores and classified as average (z-score −0.5–0.5), better than average (z-score > 0.5) or worse than average (z-score < −0.5). Linear mixed models were used to evaluate differences in LnRMSSD based on ASRM classifications. LnRMSSD was higher (p < 0.05) when perceived sleep quality, fatigue, stress and mood were better than average versus worse than average. Within-subject correlations revealed that 15 of 17 subjects demonstrated at least one relationship (p < 0.05) between LnRMSSD and ASRM variables. Changes in HRV may be the result of non-training related factors and thus practitioners are encouraged to include subjective measures to facilitate targeted interventions to support training adaptations.
The Influence of Cognitive Dimensions on the Consumer-SME Relationship: A Sustainability-Oriented View
Authors: Caputo Francesco , Carrubbo Luca , Sarno Debora
Published: September 2018

While companies are investing increasingly in sustainability measures, the effects of firms’ sustainability actions on consumer perceptions should not be taken for granted and deserve to be investigated. By adopting the interpretative lens of consumer culture theory, this paper defines a set of cognitive dimensions which can describe consumer perceptions of sustainability actions and the behaviors of small–medium enterprises (SMEs), with the aim of identifying the relationships between firms’ sustainability actions and the economic performance of SMEs. To this extent, the cognitive dimensions proposed by Green & Petre (1996) are re-elaborated to define a set of customer cognitive dimensions in the sustainability domain (resilience, wholeness, mapping, coding, and engagement). Thus, the relationships between SMEs’ sustainability actions and behaviors, the defined cognitive influencers and SME performance is empirically tested. In particular, a survey of a sample of 1137 customers of 175 Italian SMEs is analyzed using structural equation modelling (SEM). The findings show that the adoption of certain sustainability actions influences consumer perceptions, which in turn impacts the economic performance of SMEs.
“One Belt, One Road” Initiative to Stimulate Trade in China: A Counter-Factual Analysis
Authors: Chen Shih-Chih , Hou Jianing , Xiao De
Published: September 2018

This research implements the panel data control method to evaluate the stimulative effects of the “One Belt, One Road” initiative on trade performance in China. We constructed a counterfactual of China’s trade surplus by exploiting the unobservable common factors that create observable trade balances among other countries. We also modified the traditional control group selection by extending it to the Elastic-Net method. This study found the following: (i) China’s annual trade surplus increased sharply by 10.69% on average since 2015. In contrast, analysis of the counterfactual showed that the net exports of China would have remained constant without the stimulation of the “One Belt, One Road” initiative; (ii) These results are robust to exports growth rates and checking by various control group selections; (iii) Although the analysis shows return to an average trading balance, we should not underestimate the benefits of the initiative in the long run.
Malting of Fusarium Head Blight-Infected Rye (Secale cereale): Growth of Fusarium graminearum, Trichothecene Production, and the Impact on Malt Quality
Authors: Jin Zhao , Gillespie James , Barr John , Wiersma Jochum J. , Sorrells Mark E. , Zwinger Steve , Gross Thomas , Cumming Jaime , Bergstrom Gary C. , Brueggeman Robert , Horsley Richard D. , Schwarz Paul B.
Published: September 2018

This project was initiated with the goal of investigating the malt quality of winter rye cultivars and hybrids grown in the United States in 2014 and 2015, but high levels of deoxynivalenol (DON) were subsequently found in many of the malt samples. DON levels in 75% of the investigated rye samples (n = 117) were actually below 1.0 mg/kg, as quantified by a gas chromatography combined with electron capture detector (GC-ECD). However, 83% of the samples had DON in excess of 1.0 mg/kg following malting, and the average DON level in malted rye was 10.6 mg/kg. In addition, relatively high levels of 3-acetate DON (3-ADON), 15-acetate DON (15-ADON), nivalenol (NIV), and DON-3-glucoside (D3G) were observed in some rye malts. Our results show that rye grain DON is likely a poor predicator of type B trichothecenes in malt in practice, because high levels of malt DON, 15-ADONm and D3G were produced, even when the rye samples with DON levels below 0.50 mg/kg were processed. Fusarium Tri5 DNA content in rye was highly associated with malt DON levels (r = 0.83) in a small subset of samples (n = 55). The impact of Fusarium infection on malt quality was demonstrated by the significant correlations between malt DON levels and wort viscosity, β-glucan content, wort color, wort p-coumaric acid content, and total phenolic content. Additional correlations of rye Fusarium Tri5 DNA contents with malt diastatic power (DP), wort free amino nitrogen (FAN) content, and arabinoxylan content were observed.
Cartilage Produced after Transplantation of Syngeneic Chondrocytes is Rejected in Rats Presensitized with Allogeneic Chondrocytes
Authors: Moskalewski Stanislaw , Osiecka-Iwan Anna , Hyc Anna
Published: September 2018

Cartilage produced in 2-week-old intramuscular transplants of syngeneic chondrocytes in rats did not display any signs of rejection. Cartilage produced by similar transplants in animals presensitized with intramuscular transplants of allogeneic chondrocytes was surrounded by infiltrations composed mainly of lymphocytes and was partially resorbed. Spleen mononuclear cells (SMC) from recipients of syngeneic transplants alone were not stimulated in mixed splenocyte–chondrocyte cultures by syngeneic or allogeneic chondrocytes. SMC from recipients of allogeneic and subsequent syngeneic transplants were strongly stimulated by both syngeneic and allogeneic chondrocytes, although stimulation by the latter was significantly more pronounced. Sera from naive rats usually contained cytotoxic antichondrocyte antibodies but their level varied considerably in various individuals. In rats chosen as transplant recipients on the basis of low antichondrocyte cytotoxicity of their sera, this toxicity was markedly raised after sensitization with allo- and syngeneic chondrocytes. Absorption with thymocytes or fibroblasts decreased but did not abrogate cytotoxicity. These observations support previous reports suggesting expression of tissue-specific antigen(s) by chondrocytes.
Cryopreservation of Porcine Fetal Ventral Mesencephalic Tissue for Intrastriatal Transplantation in Parkinson's Disease
Authors: Koopmans Jan , HogenEsch Ineke , Copray Sjef , Middel Berrie , van Dijk Henk , Go Kian-Gwan , Staal Michiel
Published: September 2018

In this study we examined the efficacy of cryopreserving porcine fetal mesencephalic tissue. After micro-scopical dissection of the ventral mesencephalon (VM) from E28 pig fetuses, the collection of explants was randomly divided into two equal parts. One part was directly prepared as cell suspension. The other part was stored in hibernation medium for less than 2 days and then cryopreserved as tissue fragments and stored in liquid nitrogen. After 2 weeks up to 1 year, these tissue fragments were thawed and processed as cell suspensions. After cell counting and assessment of viability, these cell suspensions were used to examine survival, morphology, and neurite formation of the dopaminergic neurons in cell culture as well as after intrastriatal implantation in 6-OHDA-lesioned rats. Comparison of cryopreserved with fresh VM cell suspensions showed no significant difference with respect to cell viability and the average number of living cells per VM explant. The morphology of cultured dopaminergic neurons after cryopreservation was identical to that of fresh cells. After intrastriatal implantation, survival and outgrowth of cryopreserved dopaminergic neurons as well as functional effects did not differ from those of fresh cells. In conclusion, the cryopreservation technique we used proves to be a reliably effective method for storing porcine fetal VM tissue.
Regulation of Cell Proliferation Using Tissue Engineering in MIN6 Cells
Authors: Kinoshita Naoko , Echigo Yoshiya , Shinohara Sigeo , Gu Yuanjun , Miyazaki Junichi , Inoue Kazutomo , Imamura Masayuki
Published: September 2018

Pancreatic islet transplantation for patients with diabetes mellitus has been hindered by the problem of donor shortage, as is the case for transplantation of other organs. Among several measures to overcome this problem, cell transplantation using xenogenic cell lines has been considered. For the treatment of diabetic patients, a murine pancreatic β-cell line MIN6 is a potential source of cell transplant. In order to restrict otherwise unlimited proliferation of transplanted MIN6 cells, cells are rendered to form spheroidal aggregates (SMIN6) on nonadherent culture dishes. SMIN6 stopped its growth around day 7 with a diameter of 220 ± 40 μm and kept its size almost constant at least until day 28. SMIN6 cells, however, had reduced responsiveness of insulin secretion to glucose concentration compared with MIN6 cells cultured in a monolayer. On the other hand, spheroid MIN6 cells formed in the presence of extracellular matrix gel (SMIN6E) possessed the capacity for glucose-dependent insulin secretion comparable with conventional MIN6 cells. SMIN6E encapsulated in agarose beads (SMIN6E-B) was also viable for at least 1 month in vitro with a constant diameter and favorable glucose responsiveness. The development of spheroid-type MIN6 may contribute to the future clinical application of MIN6 or other β-cell lines for treatment of diabetes mellitus.
Rapidly Functional Immobilization of Immortalized Human Hepatocytes Using Cell Adhesive GRGDS Peptide-Carrying Cellulose Microspheres
Authors: Kobayashi Naoya , Taguchi Takehito , Noguchi Hirofumi , Okitsu Teru , Totsugawa Toshinori , Watanabe Takamasa , Matsumura Toshihisa , Fujiwara Toshiyoshi , Urata Haruo , Kishimoto Nobuyuki , Hayashi Nobuyuki , Nakaji Shuhei , Murakami Takuro , Tanaka Noriaki
Published: September 2018

With the development of biotechnology, hepatic support by a hybrid artificial liver (HAL) using hepatocytes has been given much attention. Because the availability of human livers is limited, we have established a tightly regulated immortal human hepatocyte cell line, NKNT-3, for developing HAL. Because high-density cell culture allows the compactness of the HAL device and its easy use under emergency circumstances, we have developed cell adhesive GRGDS peptide-containing cellulose microspheres (GRGDS/CMS). The GRGDS/CMS efficiently immobilized NKNT-3 cells within 24 h in a stirred suspension culture. Electron microscopic examinations demonstrated glycogen granules and well-developed endoplasmic reticulum and mitochondria in NKNT-3 cells attached to the GRGDS/CMS. The cells showed ammonia clearance activity, whereas HepG2-transformed human liver cells did not remove the loaded ammonia. An efficient adenoviral delivery of the lacZ reporter gene was performed in GRGDS/CMS-immobilized NKNT-3 cells. In this study we present rapid immobilization of NKNT-3 immortal human hepatocytes using cellulose microspheres carrying GRGDS peptides. These microspheres satisfied immediate preparation of NKNT-3 cells in sufficient quantity and of adequate quality.
Clonal Expansion of Hepatic Stem/Progenitor Cells following Flow Cytometric Cell Sorting
Authors: Suzuki Atsushi , Zheng Yun-Wen , Fukao Katashi , Nakauchi Hiromitsu , Taniguchi Hideki
Published: September 2018

Although hepatic stem cells are believed to exist and play a critical role in developing and regenerating liver, little is known about their cell surface specificity or differentiation capabilities. To make prospective studies of hepatic stem cells possible, we established an in vitro culture system for identification and characterization of hepatic stem/progenitor cells. By combining this culture system with fluorescence activated cell sorting (FACS), a population of cells that were capable of forming large colonies and providing their descendants for relative longer period was isolated from fetal mouse livers. These data suggest that hepatic stem/progenitor cells with high proliferative potential are existent in the developing mouse liver, and that they are enriched by using flow cytometry.
Effect of the Extracellular Matrix on Pancreatic Endocrine Cell Function and its Biocompatibility in Dogs
Authors: Edamura Kazuya , Ohgawara Hisako , Nasu Koko , Iwami Yukiko , Sato Ayako , Ishikawa Shiho , Matsuki Naoaki , Ono Kenichiro , Ogawa Hiroyuki , Sasaki Nobuo
Published: September 2018

The effect of the synthetic extracellular matrix (ECM) in a diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP) on pancreatic endocrine cells in vitro and its biocompatibility in dogs were investigated. Two different types of ECM were used: type I collagen treated with low antigen (type I LA), and reconstituted basement membrane matrix (Matrigel) derived from Englbreth-Holm-Swarm (EHS) mouse sarcoma. Matrigel contains growth and differentiation factors and cell adhesion molecules such as laminin, heparan sulfate proteoglycan, and entactin. Purified porcine pancreatic endocrine (PE) cells were suspended in type I LA or Matrigel and then placed into a 12-well culture plate (4 × 107 cells/ml · gel/well). The insulin accumulation from PE cells in Matrigel was significantly greater than that in type I LA (9.3 ± 3.6 mU/well vs. 2.3 ± 1.3 mU/well). When Bio-AEP with Matrigel and PE cells was implanted into the abdominal cavity of a pancreatectomized diabetic dog, the exogenous insulin requirement for maintaining normoglycemia was reduced for the first 4 weeks. However, after 6 weeks of implantation, fasting blood glucose levels suddenly increased. Laparotomy revealed encapsulated Bio-AEP with thick fibrous tissue. Following removal of the Bio-AEP from the abdominal cavity, another Bio-AEP containing type I LA and PE cells was implanted into the same dog. The exogenous insulin requirement was gradually decreased to almost half that of preim-plantation levels. Bio-AEPs containing type I LA or Matrigel, but not PE cells, were implanted into the abdominal cavities of four healthy dogs. After 4 weeks of implantation, the Bio-AEP with Matrigel was encapsulated with fibrous tissue similar to that in the diabetic dog, but the Bio-AEP with type I LA was not. These results indicate that Matrigel may be incompatible with dogs and that the type I LA is more suitable for Bio-AEP.
Insertion of a Suicide Gene into an Immortalized Human Hepatocyte Cell Line
Authors: Kobayashi Naoya , Noguchi Hirofumi , Totsugawa Toshinori , Watanabe Takamasa , Matsumura Toshihisa , Fujiwara Toshiyoshi , Miyazaki Masahiro , Fukaya Kenichi , Namba Masayoshi , Tanaka Noriaki
Published: September 2018

For developing a bioartificial liver (BAL) device, an attractive alternative to the primary human hepatocytes would be the use of highly differentiated immortalized human hepatocytes with a safeguard. To test the feasibility, the primary human hepatocytes were immortalized by a plasmid SV3neo encoding simian virus 40 large T antigen (SV40Tag) gene. A highly differentiated hepatocyte line OUMS-29 was established. A suicide gene of herpes simplex virus-thymidine kinase (HSV-TK) was retrovirally introduced into OUMS-29 cells as a safeguard for clinical application. One of the resulting HSV-TK-positive cell lines, OUMS-29/ tk, grew in chemically defined serum-free medium with the gene expression of differentiated liver functions. OUMS-29/tk cells were 100 times more sensitive to ganciclovir compared with unmodified OUMS-29 cells in in vitro experiments. We have established a tightly regulated immortalized human hepatocyte cell line. Essentially unlimited availability of OUMS-29/tk cells may be clinically useful for BAL therapy.
Isolation, Culture, and Characterization of Endocrine Cells from 6-Month-Old Porcine Pancreas
Authors: Hori Hiroshi , Gu Yuan Jun , Nagata Natsuki , Balamurugan Appakaalai N. , Satake Akira , Morimoto Yoshihiko , Wang Wen Jing , Misawa Yuko , Nozawa Yuka , Nembai Tomoko , Miyamoto Masaaki , Nozawa Masumi , Inoue Kazutomo
Published: September 2018

Porcine endocrine cells were isolated from pancreas of 6-month-old pigs by two-step enzymatic digestion procedures. They were separated by the density gradient (isopycnic) centrifugation method using Histopaque-1077. Isolated cells were cultured and divided into two groups: suspension cells and adhesion cells. Suspension cells maintained their cell numbers on and after 7 days in culture. Approximately 1 107 cells were obtained from single pancreas of a 6-month-old pig. The cultured suspension cells took up dithizone (DTZ) staining 14 days after isolation in culture and indicated the presence of β-cells. In in vitro study, the suspension cells were capable of secreting insulin into the culture medium. The suspension cells were tested for insulin and glucagon staining by Western blot analysis. These results indicated the maintenance of endocrine cell function after isolation. However, cultured adhesion cells failed to maintain their function during culture. In in vivo study, the suspension cells were transplanted into diabetes-induced nude mice. Reduction in blood glucose level was obtained after transplantation. Intraperitoneal glucose tolerance test (IPGTT) results showed a normal pattern of blood glucose clearance. After 1 week, the transplanted endocrine cells were detected with anti-insulin antibody by immunostaining and it showed the presence of viable β-cells under the renal capsule of nude mice. Collectively, our results suggest that isolated and cultured suspension porcine endocrine cells maintained their endocrine function. These endocrine cells can be used as isolated islets for further study, including transplantation experiments.
Tissue-Engineered Pancreatic Islets: Culturing Rat Islets in the Chitosan Sponge
Authors: Cui Wanxing , Kim Do-Hoon , Imamura Masayuki , Hyon Suong-Hyu , Inoue Kazutomo
Published: September 2018

Subcutaneous islet transplantation has become an attractive modality. With development of tissue-engineering techniques, it is possible to rectify the disadvantage of poor blood supply in the subcutaneous site by reconstruction of the capillary network. According to reports, the Chitosan sponge (CS) could be used for reconstruction of in vitro capillary-like network and could be used in artificial skin equivalent. In this study, we cultured the islets in CS for future application. CSs, having 200–500 μm pore size, were prepared by freeze-drying method. Rat islets were isolated from the pancreas of Lewis rats (10 weeks old, 280–300 g, male) by collagenase digestion followed by discontinuous dextran gradient centrifugation method. Each 20 islets were seeded equally into the CSs and were cultured for 62 days with various culture media such as RPMI-1640, Dulbecco's modified Eagle's medium (DMEM), and Eagle's MEM. They contained 10% fetal bovine serum (FBS) and 5 ml/L antibiotic-antimycotic mixed stock solution in the culture dishes. Insulin concentration both inside and outside of the islet-seeded CS was measured during culture. Changes in the morphology of islets were also observed in this study. Freshly isolated islets had a loose appearance with an irregular border, and most were seen as a single islet. Occasionally a cluster, consisting of 2–4 islets ranging mainly from 150 to 250 μm in diameter, was observed. Islets cultured in the CSs in different culture media retained initial morphology, which had well-delineated smooth borders for at least 53 days. The insulin release behavior of islets cultured in the CS showed constant secretory capacities for 49 days. After that they exhibited a rapid and definitive decline from the initial insulin release. Until this stage, insulin concentration in the CS was well maintained. The properties were dependent on culture medium used and insulin diffusion released from islets. This experiment is a new study model for establishment of islet culture in a three-dimensional matrix. Also extension of this observation will provide new insights for islet transplantation at the subcutaneous site by a tissue-engineering approach.
Selective Repopulation of Mice Liver after Fas-Resistant Hepatocyte Transplantation
Authors: Fujino Masayuki , Li Xiao-Kang , Kitazawa Yusuke , Funeshima Naoko , Guo Lei , Okuyama Torayuki , Amano Takashi , Amemiya Hiroshi , Suzuki Seiichi
Published: September 2018

Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopulation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 × 106 cells in female MRL-+/+ (wildtype mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intravenously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The pathologic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver.
Comparative Functional Study of Clonal Insulin-Secreting Cells Cultured in Five Commercially Available Tissue Culture Media
Authors: Hamid Muhajir , McCluskey Jane T. , McClenaghan Neville H. , Flatt Peter R.
Published: September 2018

The electrofusion-derived rat insulin-secreting cell line BRIN-BD11 was cultured in five different commercially available media to determine the optimum medium for the in vitro maintenance of such clonal cell lines. Cells were cultured in RPMI-1640, DMEM, McCOY'S, F-12K, or MEM culture medium supplemented with 10% (v/v) fetal bovine serum and antibiotics (100 U/ml penicillin and 0.1 g/L streptomycin). Insulin secretion studies performed after 10 days revealed RPMI-1640 to be the best performing medium in terms of insulin secretory responsiveness to a range of stimuli including glucose, l-alanine, l-arginine, carbachol, and glibenclamide. Insulin release was significantly decreased (p < 0.01 to p < 0.05) in all other media compared to RPMI-1640. Only the cells cultured in RPMI-1640 and DMEM showed a significant glucose-induced insulin secretory response (p < 0.01 and p < 0.05). McCOY'S gave the next best result followed by F-12K and MEM. After the 10-day culture period, the highest insulin content was found in cells cultured in RPMI-1640 and DMEM with significantly lower levels of insulin in cells cultured in McCOY'S, F-12K, and MEM (p < 0.01 to p < 0.001). RPMI-1640 was used for further studies to investigate the effects of 5.6 – 16.7 mmol/L glucose in culture on the secretory responsiveness of BRIN-BD11 cells. Significant responses to a number of nonglucidic secretagogues were seen following culture at 5.6 and 16.7 mmol/L glucose, although responsiveness was less than after culture with 11.1 mmol/L glucose. At 16.7 mmol/L glucose culture, glucose-stimulated insulin release was abolished.