To Compare the Effects of a Standard Versus Hydrophilic Polymer Coated Airway Stent in a Porcine Model: A Randomized, Single-Blinded Study.

BACKGROUND: Stent encrustation with debris and mucostasis is a significant cause of airway injury and comorbidity, leading to ~25% of stent exchanges (1-3). Previous work from our group has shown that the experimental coating can reduce mucous adhesion in bench testing and demonstrated a signal for reducing airway injury and mucostasis in a feasibility study. OBJECTIVES: The aim of this study is to continue our inquiry in a randomized, single-blinded multi-animal trial to investigate the degree of airway injury and mucostasis using silicone stents with and without this specialized coating. METHODS: We modified commercially available silicone stents with a hydrophilic polymer from Toray Industries. We conducted an in vivo survival study in 6 mainstem airways (3 coated and 3 uncoated) of 3 pigs to compare the degree of airway injury and mucostasis between coated versus noncoated stented airways. Both stents were randomized to either left or right mainstem bronchus. The pathologist was blinded to the stent type. RESULTS: We implanted a total of six 14×15 mm silicone stents (1 per mainstem bronchi) into 3 pigs. All animals survived to termination at 4 weeks. All stents were intact; however, 1 uncoated stent migrated out. On average, all the coated stents demonstrated reduced pathology and tissue injury scores (75 vs. 68.3, respectively). The average total dried mucous weight was slightly higher in the coated stents (0.07 g vs. 0.05 g; respectively). CONCLUSION: Coated stents had lower airway injury compared with uncoated stents in this study. Of all the stents, 1 uncoated stent migrated out and was not included in the dried mucous weight totals. This could explain the slightly higher mucous weight in the coated stents. Nevertheless, this current study demonstrates promising results in lowering airway injury in stents incorporated with the hydrophilic coating, and future studies, including a larger number of subjects, would be needed to corroborate our findings.

Development of Microfluidic Systems for Fabricating Cellular Multilayers.

We designed a microfluidic system comprising microfluidic channels, pumps, and valves to enable the fabrication of cellular multilayers in order to reduce labor inputs for coating extracellular matrices onto adhesive cells (e.g., centrifugation). Our system was used to fabricate nanometer-sized, layer-by-layer films of the extracellular matrices on a monolayer of C2C12 myoblasts. The use of this microfluidic system allowed the fabrication of cellular multilayers in designed microfluidic channels and on commercial culture dishes. The thickness of the fabricated multilayer using this microfluidic system was higher than that of the multilayer that was formed by centrifugation. Because cellular multilayer fabrication is less laborious and the mechanical force to the cell is reduced, this novel system can be applied to tissue modeling for cell biology studies, pharmaceutical assays, and quantitative analyses of mechanical or chemical stimuli applied to multilayers.

Evaluation and simplification of the occupational slip, trip and fall risk-assessment test.

OBJECTIVE: The purpose of this investigation is to evaluate the efficacy of the occupational slip, trip and fall (STF) risk assessment test developed by the Japan Industrial Safety and Health Association (JISHA). We further intended to simplify the test to improve efficiency. METHODS: A previous cohort study was performed using 540 employees aged ≥50 years who took the JISHA's STF risk assessment test. We conducted multivariate analysis using these previous results as baseline values and answers to questionnaire items or score on physical fitness tests as variables. The screening efficiency of each model was evaluated based on the obtained receiver operating characteristic (ROC) curve. RESULTS: The area under the ROC obtained in multivariate analysis was 0.79 when using all items. Six of the 25 questionnaire items were selected for stepwise analysis, giving an area under the ROC curve of 0.77. CONCLUSION: Based on the results of follow-up performed one year after the initial examination, we successfully determined the usefulness of the STF risk assessment test. Administering a questionnaire alone is sufficient for screening subjects at risk of STF during the subsequent one-year period.

3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications.

Three-dimensional multilayers of smooth muscle cells as a new experimental model for vascular elastic fiber formation studies.

OBJECTIVE: Elastic fiber formation is disrupted with age and by health conditions including aneurysms and atherosclerosis. Despite considerable progress in the understanding of elastogenesis using the planar culture system and genetically modified animals, it remains difficult to restore elastic fibers in diseased vessels. To further study the molecular mechanisms, in vitro three-dimensional vascular constructs need to be established. We previously fabricated vascular smooth muscle cells (SMCs) into three-dimensional cellular multilayers (3DCMs) using a hierarchical cell manipulation technique, in which cells were coated with fibronectin-gelatin nanofilms to provide adhesive nano-scaffolds. Since fibronectin is known to assemble and activate elastic fiber-related molecules, we further optimized culture conditions. METHODS AND RESULTS: Elastica stain, immunofluorescence, and electron microscopic analysis demonstrated that 3DCMs, which consisted of seven layers of neonatal rat aortic SMCs cultured in 1% fetal bovine serum (FBS) in Dulbecco's modified Eagle's medium, exhibited layered elastic fibers within seven days of being in a static culture condition. In contrast, the application of adult SMCs, 10% FBS, ε-poly(lysine) as an alternative adhesive for fibronectin, or four-layered SMCs, failed to generate layered elastic fiber formation. Radioimmunoassay using [(3)H]valine further confirmed the greater amount of cross-linked elastic fibers in 3DCMs than in monolayered SMCs. Layered elastic fiber formation in 3DCMs was inhibited by the lysyl oxidase inhibitor ß-aminopropionitrile, or prostaglandin E2. Furthermore, infiltration of THP-1-derived macrophages decreased the surrounding elastic fiber formation in 3DCMs. CONCLUSION: 3DCMs may offer a new experimental vascular model to explore pharmacological therapeutic strategies for disordered elastic fiber homeostasis.

An industry-based cohort study of the association between weight gain and hypertension risk among rotating shift workers.

OBJECTIVES: We investigated whether the association between shift work and hypertension is independent of weight gain. METHODS: Subjects were 10,173 male employees (9209 daytime workers, 964 three-shift workers; mean follow-up period: 12.7 years). Hypertension was defined as systolic blood pressure 140 mm Hg or more or diastolic blood pressure 90 mm Hg or more. The risk of developing hypertension among shift workers was estimated using a Cox proportional hazards model with adjustment for several factors. RESULTS: Analysis revealed that "shift work" (hazard ratio [HR]: 1.85; 95% confidence interval [CI]: 1.68, 2.03), "baseline body mass index" (HR: 1.13; 95% CI: 1.12, 1.15), and "increase in body mass index during follow-up" (HR: 1.15; 95% CI: 1.13, 1.17) were significant independent risk factors for hypertension. CONCLUSIONS: Shift work is a significant risk factor for hypertension that is independent of both starting weight and weight gained over years of work.

A cross-sectional study of workplace social capital and blood pressure: a multilevel analysis at Japanese manufacturing companies.

OBJECTIVES: We examined the contextual effect of workplace social capital on systolic blood pressure (SBP). DESIGN: Cross-sectional. SETTING: A conglomerate from 58 workplaces in Japan. PARTICIPANTS: Of the 5844 workers at a Japanese conglomerate from 58 workplaces, 5368 were recruited. Individuals who received drugs for hypertension (n=531) and who lacked information on any variable (n=167) were excluded from the analyses, leaving 4735 individuals (3281 men and 1454 women) for inclusion. PRIMARY AND SECONDARY OUTCOME MEASURES: Systolic blood pressure. RESULTS: The contextual effect of workplace social capital on SBP was examined using a multilevel regression analysis with a random intercept. Coworker support had a contextual effect at the workplace level (coefficient=-1.97, p=0.043), while a lack of trust for coworkers (coefficient=0.27, p=0.039) and lack of helpfulness from coworkers were associated with SBP (coefficient=0.28, p=0.002). CONCLUSIONS: The present study suggested that social capital at the workplace level has beneficial effects on SBP.

Survival and structural evaluations of three-dimensional tissues fabricated by the hierarchical cell manipulation technique.

Mouse L929 fibroblasts and normal human dermal fibroblasts (NHDFs) were constructed into three-dimensional (3-D) multilayered tissues by directly coating them with nano-films consisting of fibronectin (FN) and gelatin (G) onto the surface of the cell layer using layer-by-layer assembly. The one-, two- and five-layered (1L, 2L and 5L) tissues were cultured for 1 month in order to evaluate their long-term survival and structural changes. L929 cells in the 3-D tissues showed excessive proliferation throughout the culture period, regardless of the starting layer number. The cross-sectional images stained with hematoxylin and eosin revealed heterogeneous and random increases in the thickness of their layered structures, probably due to the immortalized property of L929 fibroblasts. On the other hand, NHDFs, which are primary cells, showed high stability in their amount of DNA in the multilayered structures, and their thicknesses were completely maintained even after 1 month of incubation. To evaluate the living cell density in each layer of 5L tissues during the culture period, 5L NHDFs were fluorescently labeled with LIVE/DEAD reagent and analyzed by confocal laser scanning microscopy. Although the upper layers did not show any dead cells, the bottom layers showed pieces of dead cell nuclei depending on culture time. However, the living cell densities in all layers were almost the same, even after 1 month of culture, suggesting that the 5L structures were completely filled with living cells. These findings from the multilayered tissue constructs will provide important information not only for the construction of 3-D engineered tissues in tissue engineering but also on 3-D cell culture in biological science generally.

Retrospective cohort study of the risk of impaired glucose tolerance among shift workers.

OBJECTIVES: The aim of this study was to investigate the effect of shift working on the risk of developing impaired glucose tolerance (IGT). METHODS: This study comprised 6413 male employees (5608 daytime workers, 512 3-shift workers and 293 2-shift workers) whose work schedules remained constant during the follow-up period (mean follow-up period 9.9 years). IGT was defined as Hemoglobin A1c ≥ 5.9%. The Cox proportional hazards model was used to estimate the risk of developing IGT, after making adjustments for age, smoking, alcohol consumption, leisure-time physical activity, body mass index (BMI) and Hemoglobin A1c (HbA1c) at baseline. Analysis was additionally performed for a sub-cohort of 1625 workers with BMI of 20.0-25.0 kg/m² that did not change by >2.0 kg/m² during the follow up period. RESULTS: The risk of developing IGT was significantly elevated among both 3-shift workers [hazard ratio (HR) 1.78, 95% confidence interval (95% CI) 1.49- 2.14] and 2-shift workers (HR 2.62, 95% CI 2.17-3.17). Significant elevations of the risk were still observed at the additional analysis on the sub-cohort (3-shift work: HR 3.55, 95% CI 2.02-6.25; 2-shift work: HR 4.86, 95% CI 2.62-9.01). CONCLUSIONS: Both 2- and 3-shift workers were associated with a high risk factor of developing IGT compared to day workers. Moreover, 2-shift workers had a higher risk than 3-shift workers. The risk was observed even among workers whose body mass remained within the normal range.

Engineering fibrotic tissue in pancreatic cancer: a novel three-dimensional model to investigate nanoparticle delivery.

Pancreatic cancer contains both fibrotic tissue and tumor cells with embedded vasculature. Therefore anti-cancer nanoparticles need to extravasate from tumor vasculature and permeate thick fibrotic tissue to target tumor cells. To date, permeation of drugs has been investigated in vitro using monolayer models. Since three-dimensional migration of nanoparticles cannot be analyzed in a monolayer model, we established a novel, three-dimensional, multilayered, in vitro model of tumor fibrotic tissue, using our hierarchical cell manipulation technique with K643f fibroblasts derived from a murine pancreatic tumor model. NIH3T3 normal fibroblasts were used in comparison. We analyzed the size-dependent effect of nanoparticles on permeation in this experimental model using fluorescent dextran molecules of different molecular weights. The system revealed permeation decreased as number of layers of cultured cells increased, or as molecule size increased. Furthermore, we showed changes in permeation depended on the source of the fibroblasts. Observations of this sort cannot be made in conventional monolayer culture systems. Thus our novel technique provides a promising in vitro means to investigate permeation of nanoparticles in fibrotic tissue, when both type and number of fibroblasts can be regulated.

Morphological and histological evaluations of 3D-layered blood vessel constructs prepared by hierarchical cell manipulation.

Three-dimensional (3D)-layered blood vessel constructs consisting of human umbilical artery smooth muscle cells (SMCs) and human umbilical vascular endothelial cells (ECs) were fabricated by hierarchical cell manipulation, and their basic morphology, histology and blood compatibility were evaluated in relation to the EC layers. For the hierarchical cell manipulation, fibronectin-gelatin (FN-G) nanofilms were prepared on the surface of SMC layers to provide a cell adhesive nano-scaffold for the second layer of cells. The layer number of blood vessel constructs was easily controllable from 2 to 7 layers, and the histological evaluation, scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated a hierarchical blood vessel analogous morphology. The immunefluorescence staining revealed homogeneous and dense tight-junction of the uppermost EC layer. Furthermore, the nano-meshwork morphology of the FN-G films like a native extracellular matrix was observed inside the blood vessel constructs by SEM. Moreover, a close association between actin microfilaments and the nano-meshworks was observed on the SMC surface by TEM. The blood compatibility of the blood vessel constructs, 4-layered SMC/1-layered EC (4L-SMC/1L-EC), was clearly confirmed by inhibition of platelet adhesion, whereas the blood vessel constructs without EC layers (4L-SMC) showed high adhesion and activation of the platelet. The 3D-blood vessel constructs prepared by hierarchical cell manipulation technique will be valuable as a blood vessel model in the tissue engineering or pharmaceutical fields.

Industry-based retrospective cohort study of the risk of prostate cancer among rotating-shift workers.

OBJECTIVES: The risk of prostate cancer among shift workers was examined in the present industry-based retrospective cohort study. METHODS: The study was established based on a health-care database from a Japanese manufacturing corporation. Work schedules of 4995 male workers (mean age = 55.5 years) were followed up retrospectively; mean follow-up period = 25.0 years. Of the subjects, 4168 had previously undertaken only daytime work (daytime workers), whereas 827 had undertaken rotating three-shift work for >80% of their career (shift workers). All subjects had undergone prostate-specific antigen (PSA) testing. Prostate cancer incidence data were obtained from the health insurance records of 13 daytime and four shift workers. Multivariate logistic regression was used to estimate the relative risk of prostate cancer for the two groups with adjustments for age, body mass index, alcohol intake, smoking, exercise and marital status. RESULTS: Compared with daytime workers, shift workers showed a non-significant increase in the risk of prostate cancer (odds ratio = 1.79; 95% confidence interval = 0.57, 5.68; P-value = 0.32). CONCLUSIONS: Some increase in the risk of prostate cancer for shift workers was observed, although the result was not statistically significant as a result of the small number of cases. To identify the prostate cancer risk among shift workers, longer-term follow up, including the period after retirement, is required.

Retrospective cohort study of the risk of obesity among shift workers: findings from the Industry-based Shift Workers' Health study, Japan.

OBJECTIVES: The authors investigated the effect of shift working on the risk of obesity using data from the Industry-based Shift Workers' Health (IbSH) study, a retrospective cohort study based on a health care database system belonging to a manufacturing corporation in Japan. METHODS: The study database contains data on annual health check-ups and work schedules for every worker in the corporation in Japan since 1981. Study subjects consisted of 9912 male employees (8892 daytime workers and 920 rotating three-shift workers; mean age at first check-up was 23.7 years) whose work schedules were consistent during the follow-up period. Obesity was defined as a body mass index ≥ 25.0. RESULTS: 3319 cases of obesity were recorded over the 27.5 years of retrospective follow-up. Kaplan-Meier survival analysis visually demonstrated an increased risk of obesity among shift workers. The risk becomes particularly obvious after 10 years of follow-up. Cox proportional-hazards model analysis revealed a significantly increased risk among shift workers (RR 1.14, 95% CI 1.01 to 1.28). CONCLUSION: The risk of obesity among male shift workers was visually and statistically demonstrated.

Three-dimensional constructs induce high cellular activity: Structural stability and the specific production of proteins and cytokines.

The specific properties responsible for the stability and function induced by three-dimensional (3D) cellular constructs were evaluated and compared to a monolayer structure. 3D-cellular multilayers composed of human fibroblast cells (FCs) and human umbilical vascular endothelial cells (ECs) were fabricated by a hierarchical cell manipulation technique. Interestingly, the ECs adhered homogeneously onto four-layered (4L) FCs, and tight-junction formation was widely observed at the centimeter scale, while heterogeneous EC domain structures were observed on the monolayered (1L) FCs. The production of heat shock protein70 (Hsp70) and interleukin-6 (IL-6) from the cellular structures were investigated to elucidate any 3D-structural effect on cellular function. The Hsp70 expression of the ECs decreased after adhesion onto the 4L-FC structure as compared with the EC monolayer. Surprisingly, the Hsp70 production response to heat shock increased drastically by approximately 10-fold as compared with a non-heat shock by 3D structure formation, whereas the monolayer structures showed no change. Moreover, the production of the inflammatory cytokine IL-6 decreased significantly depending on the layer number of FCs. To the best of our knowledge, this is the first report on a basic, 3D-structural effect on cellular stability and function. These findings could be important for not only tissue engineering, but also for basic cell biology.

Immunomodulatory nanoparticles as adjuvants and allergen-delivery system to human dendritic cells: Implications for specific immunotherapy.

Novel adjuvants and antigen-delivery systems with immunomodulatory properties that shift the allergenic Th2 response towards a Th1 or regulatory T cell response are desired for allergen-specific immunotherapy. This study demonstrates that 200-nm sized biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) are activators of human monocyte-derived dendritic cells (MoDCs). Gamma-PGA NPs are efficiently internalized by immature MoDCs and strongly stimulate production of chemokines and inflammatory cytokines as well as up-regulation of co-stimulatory molecules and immunomodulatory mediators involved in efficient T cell priming. Furthermore, MoDCs from allergic subjects stimulated in vitro with a mixture of gamma-PGA NPs and extract of grass pollen allergen Phleum pratense (Phl p) augment allergen-specific IL-10 production and proliferation of autologous CD4(+) memory T cells. Thus, gamma-PGA NPs are promising as sophisticated adjuvants and allergen-delivery systems in allergen-specific immunotherapy.

Protein nanoarrays on a highly-oriented lamellar surface.

Well-aligned nanopatterns of various serum, antithrombogenic and cell adhesive proteins, such as gamma-globulin, fibrinogen, thrombomodulin, fibronectin and type I collagen, were fabricated on a highly-oriented block copolymer lamellar surface, and these bioactive protein nanoarrays will be useful in biological research.

Control of cell surface and functions by layer-by-layer nanofilms.

Various nanometer-sized multilayers were directly prepared onto the surface of mouse L929 fibroblast cells by a layer-by-layer (LbL) assembly technique to control the cell surface microenvironment and cell functions, such as viability, morphology, and proliferation. The species of LbL nanofilms strongly affected the cell morphology and growth. Polyelectrolyte (PE) multilayers induced a round-shaped morphology of the adhered cells, although each component of the multilayers had high cytocompatibility, whereas fibronectin (FN)-gelatin (G) and -dextran sulfate (DS) multilayers with FN-binding domain interactions (FN films) showed extended morphologies of the cells similar to that of control cells (without films). A clear difference in cell proliferation was observed for PE and FN films. The cells with FN films on their surfaces showed good proliferation profiles independent of the film thickness, but cell proliferation was not observed using the PE films although the cells survived during the culture period. Fluorescence microscopic and scanning electron microscopic observations clearly suggested a nanometer-sized meshwork morphology of the FN films on the cell surface after 24 h of incubation, whereas the PE films showed homogeneous film morphologies on the cell surface. These nanomeshwork morphologies seemed to be similar to the fibrous structure of the natural extracellular matrix. The results of this study demonstrated that the components, charge, and morphology of LbL nanofilms prepared directly on the cell surface strongly affected cell functions, and the effects of these LbL nanofilms on cell functions differed vastly as compared to PE films prepared on a substrate. The preparation of LbL nanofilms onto a cell surface might be a novel and interesting technique to control cell functions.

Scaffold-free tissue-engineered construct-hydroxyapatite composites generated by an alternate soaking process: potential for repair of bone defects.

Mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC)-hydroxyapatite (HAp) composites were developed by an alternate soaking process. The TEC derived from cultured synovial MSCs was alternately immersed in varying concentrations of CaCl(2)/Tris-HCl and Na(2)HPO(4)/Tris-HCl buffers, and HAp formation was analyzed by Fourier transform infrared spectroscopy (FT-IR), wide-angle X-ray diffraction, and scanning electron microscopy (SEM). These analyses clearly demonstrated HAp formation in the TEC. Specifically, SEM assessments showed that spherical HAp crystals of approximately 1 mum were directly formed on the surfaces of the cells and extracellular matrix (ECM) fibers. Cytotoxicity from exposure to calcium or phosphate buffers of >100 mM concentrations as assessed by LIVE/DEAD staining and total DNA assays was detected, but such cytotoxicity was not detected following exposure to concentrations of <50 mM. The HAp nanocrystals (ca. approximately 500 nm) were formed after 20 cycles in 10 mM calcium or phosphate buffers, and cell survival in the composites was confirmed. Moreover, preliminary implantation of TEC-HAp composites derived from rabbit synovial MSCs to rabbit osteochondral defects exhibited accelerated osteoinduction. These composites may be the first example of a hybrid material that consists of ECM, HAp nanocrystals, and living MSCs, and the TEC-HAp composite could be a unique and useful material for bone tissue engineering.