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Graphical abstract Abstract The preclinical drug screening of pancreatic cancer treatments suffers from the absence of appropriate models capable to reproduce in vitro the heterogeneous tumor microenvironment and its stiff desmoplasia. Driven by this pressing need, we describe in this paper the conception and the characterization of a novel 3D tumor model consisting of a triple co-culture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC), which assembled to form a hetero-type multicellular tumor spheroid (MCTS). By histological analyses and Selective Plain Illumination Microscopy (SPIM) we have monitored the spatial distribution of each cell type and the evolution of the spheroid composition. Results revealed the presence of a core rich in fibroblasts and fibronectin in which endothelial cells were homogeneously distributed. The integration of the three cell types enabled to reproduce in vitro with fidelity the influence of the surrounding environment on the sensitivity of cancer cells to chemotherapy. To our knowledge, this is the first time that a scaffold-free pancreatic cancer spheroid model combining both tumor and multiple stromal components has been designed. It holds the possibility to become an advantageous tool for a pertinent assessment of the efficacy of various therapeutic strategies. Statement of Significance Pancreatic tumor microenvironment is characterized by abundant fibrosis and aberrant vasculature. Aiming to reproduce in vitro these features, cancer cells have been already co-cultured with fibroblasts or endothelial cells separately but the integration of both these essential components of the pancreatic tumor microenvironment in a unique system, although urgently needed, was still missing. In this study, we successfully integrated cellular and acellular microenvironment components (i.e. , fibroblasts, endothelial cells, fibronectin) in a hetero-type scaffold-free multicellular tumor spheroid. This new 3D triple co-culture model closely mimicked the resistance to treatments observed in vivo , resulting in a reduction of cancer cell sensitivity to the anticancer treatment. [ABSTRACT FROM AUTHOR]

In vitro development of three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has long been desired in tissue regeneration and pharmaceutical assays. In particular, in vitro construction of 3D-iPSC–CM tissues with blood capillary networks have attracted much attention because blood capillaries are crucial for nutrient and oxygen supplies for CMs. Blood capillaries in 3D-iPSC–CM tissues will also be important for in vitro toxicity assay of prodrugs because of the signaling interaction between cardiomyocytes and endothelial cells. Here, we report construction of vascularized 3D-iPSC–CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique for cells, instead of our previous centrifugation-LbL technique. The filtration-LbL allowed us to fabricate nanometer-sized extracellular matrices (ECM), fibronectin and gelatin (FN–G), films onto iPSC–CM surfaces without any damage and with high yield, although centrifugation-LbL induced physical stress and a lower yield. The fabricated FN–G nanofilms interacted with integrin molecules on the cell membrane to construct 3D-tissues. We found that the introduction of normal human cardiac fibroblasts (NHCFs) into the iPSC–CM tissues modulated organization and synchronous beating depending on NHCF ratios. Moreover, co-culture with normal human cardiac microvascular endothelial cells (NHCMECs) successfully provided blood capillary-like networks in 3D-iPSC–CM tissues, depending on NHCF ratios. The vascularized 3D-iPSC–CM tissues indicated significantly different toxicity responses as compared to 2D-iPSC–CM cells by addition of doxorubicin as a model of a toxic drug. The constructed vascularized 3D-iPSC–CM tissues would be a promising tool for tissue regeneration and drug development. Statement of Significance In vitro fabrication of vascularized three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has attracted much attention owing to their requirement of much amount of nutrition and oxygen, but not yet published. In this manuscript, we report construction of vascularized 3D-iPSC–CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique. The filtration-LbL fabricates nanometer-sized fibronectin and gelatin (FN–G) films onto iPSC–CM surfaces. The FN–G nanofilms induce cell–cell interactions via integrin molecules on cell surfaces, leading to construction of 3D-tissues. The constructed vascularized 3D-iPSC–CM tissues would be a promising tool for tissue regeneration and drug development. We believe that this manuscript has a strong impact and offers important suggestions to researchers concerned with biomaterials and tissue engineering. [ABSTRACT FROM AUTHOR]

Purpose Hydroxyapatite agarose composite gel (HAp gel) is a radiolucent and absorbable bone–graft material that differs from hydroxyapatite ceramic; it is more rapidly absorbed and replaced by new bone than existing materials. This study examined the usefulness of HAp gel in the repair of alveolar bone defects due to cleft lip and palate. Materials and methods HAp gel was prepared by an alternate soaking technique. HAp gel was mixed with an equal volume of autologous bone and applied to bone defects due to cleft lip and palate (mixture group). As a reference group, 10 cases were treated with autologous bone graft alone (bone group). To determine the presence of osteogenesis, bone density on X-ray film was measured at the transplanted site and surrounding normal bone, and the ratio was calculated. The marginal bone level was assessed by intraoral radiography. Results In the mixture group, the bone density of the transplanted site increased on radiography 1 month postoperatively and was maintained thereafter. A similar increase in bone density was observed in the bone group. The marginal bone level in the alveolar cleft was similar between groups. There were no complications such as infection and prolonged inflammation of the transplanted bone in association with the use of HAp gel. Conclusions The mixture of HAp gel and autologous bone is as effective as autologous bone in the treatment of bone defects due to cleft lip and palate. HAp gel can reduce the weight of autologous bone required. [ABSTRACT FROM AUTHOR]

Advanced bioactive systems with defined macroscopic properties and spatio-temporal sequestration of extracellular biomacromolecules are highly desirable for next generation therapeutics. Here, chitosan (CT) hydrogels were prepared with neutral or negatively charged cross-linkers in order to promote selective electrostatic complexation with charged drugs. CT was functionalized with varied dicarboxylic acids, such as tartaric acid, poly(ethylene glycol) bis(carboxymethyl) ether, 1,4-phenylenediacetic acid and 5-sulfoisophthalic acid monosodium salt (PhS), whereby PhS was hypothesized to act as a simple mimetic of heparin. Attenuated total reflectance Fourier transform infrared spectroscopy showed the presence of C O amide I, N–H amide II and C O ester bands, providing evidence of covalent network formation. The cross-linker content was reversely quantified by proton nuclear magnetic resonance on partially degraded network oligomers, so that 18mol.% PhS was exemplarily determined. Swellability (SR: 299±65–1054±121wt.%), compressibility (E: 2.1±0.9–9.2±2.3kPa), material morphology and drug-loading capability were successfully adjusted based on the selected network architecture. Here, hydrogel incubation with model drugs of varied electrostatic charge, i.e. allura red (AR, doubly negatively charged), methyl orange (MO, negatively charged) or methylene blue (MB, positively charged), resulted in direct hydrogel–dye electrostatic complexation. Importantly, the cationic compound, MB, showed different incorporation behaviours, depending on the electrostatic character of the selected cross-linker. In light of this tunable drug-loading capability, these CT hydrogels would be highly attractive as drug reservoirs towards e.g. the fabrication of tissue models in vitro. [ABSTRACT FROM AUTHOR]

Abstract: We prepared size-regulated nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid) (γ-PGA). In this study, 40, 100 and 200nm γ-PGA-graft-l-phenylalanine ethylester (γ-PGA-Phe) NPs were employed. The size of NPs significantly influenced the uptake and activation behaviors of antigen-presenting cells (APCs). When 40nm γ-PGA-Phe NPs were applied to these cells in vitro, they were highly activated compared with 100 and 200nm NPs, while cellular uptake was size dependent. The size of the γ-PGA-Phe NPs also significantly affected their migration to the lymph nodes and uptake behavior of NPs by dendritic cells (DCs) in vivo. The 40nm γ-PGA-Phe NPs migrated more rapidly to the lymph nodes and were taken up by a greater number of DCs compared with 100 and 200nm NPs. On the other hand, when the amount of γ-PGA-Phe NPs taken up per DC was evaluated, it was higher for 100 and 200nm NPs than for 40nm NPs, which suggests that the larger γ-PGA-Phe NPs can deliver a large amount of antigen to a single DC compared with smaller NPs. Furthermore, when examined the maturation of DCs in lymph nodes, 40nm γ-PGA-Phe NPs efficiently stimulated DCs. These results suggest that the activation, uptake behavior by APCs, migration to lymph nodes, and DC maturation can be controlled by the size of γ-PGA-Phe NPs. [Copyright &y& Elsevier]

Abstract: 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 1month 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 1month 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 1month 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. [Copyright &y& Elsevier]

Abstract: Calcium carbonate formation on a porous membrane was accelerated by using an alternating current system. A hydrophilic-treated polytetrafluoroethylene (PTFE) porous membrane was utilized for the calcium carbonate formation. The PTFE membrane was interposed with two pairs of glass cells. Calcium chloride and sodium carbonate solutions were filled in each glass cell, and an alternating current of 10Hz was applied for 30min. The resulting membrane was characterized in terms of crystal morphology and polymorph using scanning electron microscopy and X-ray diffraction. The major component of the resulting calcium carbonate was spheroidal vaterite, along with rhombohedral calcite. The composition of polymorphs was slight different on the two sides of the membrane. In the absence of the alternating current, only calcite was formed on the membrane. It is considered that the alteration of calcium carbonate formation is regulated by supersaturation, including electric migration, diffusion and electrolysis, during application of the alternating current. The resulting supersaturation was different on the two sides of the membrane, and different quantities of the polymorphs were formed. Taking this result into account, the alternating current catalyzed the development of supersaturation around the membrane, enabling calcium carbonate crystal to be formed. [Copyright &y& Elsevier]

Abstract: The grafting of l-phenylalanine onto low molecular weight chitosan is accomplished by using carbodiimide as a coupling agent. As increase in the amount of phenylalanine in feed, the grafting chain length increases, while a number of grafting chains hardly change. The obtained product, LMWCts-g-Phe, performs sphere with an average size of ∼80nm when the % grafting is less than 123. The complexes of the LMWCts-g-Phe and DNA (LMWCts-g-Phe/DNA) prepared by a complex coacervation method possess various shapes with an average size of ∼50–150nm and a negatively charged surface. The LMWCts-g-Phe and its complex show very reduced toxicity to fibroblast cells. The release of DNA from the complex is very fast in high pH media (tris buffer, pH 8.0 and carbonate buffer, pH 9.5), and relatively slow or more sustainable in neutral and low pH ones (PBS, pH 7.4 and citric acid/trisodium citrate buffer, pH 3.0). The results suggest that the LMWCts-g-Phe be an alternative promising carrier for negatively charged active molecules. [Copyright &y& Elsevier]

Abstract: Polyelectrolyte multilayers were formed on a quartz crystal microbalance (QCM) substrate by using polyelectrolyte droplets. In this study, poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrene sulfonate) (PSS) were used. The formation of the multilayers was evaluated in terms of changes in the frequency of the QCM. The polymer droplet could be used to fabricate a polyelectrolyte multilayer. Model protein adsorption such as that in bovine serum albumin and its release behavior were compared with the ionic strength in the media. Moreover, the secondary structure of the adsorbed protein was evaluated by analyzing its circular dichroism spectrum. The adsorbed protein maintained its secondary structure on the layer-by-layer (LbL) membrane. This result strongly supports the fact that the multilayers fabricated by the polyelectrolyte droplet were similar in quality to those fabricated via an alternating adsorption process. In summary, the polyelectrolyte droplet method is a good candidate for the preparation of LbL assemblies in the biomedical field. [Copyright &y& Elsevier]

In the creation of controllable selective adhesive materials, we have utilized a stereocomplex (SC) on an adhesive interface. We selected poly(L,L-lactide) (PLLA) and poly(D,D-lactide) (PDLA) initiated benzyl alcohol (PLLAb and PDLAb, respectively) as a substrate and gold nanoparticle-coated PLLA and PDLA (PLLA-Au and PDLA-Au, respectively) as an injection material. Before performing measurements, SC formation was examined via Fourier Transform Infrared Spectroscopy (FT-IR), X-ray diffraction (XRD), and Differential Scanning Calorimetry (DSC). Subsequently, the force acting on the interface after drying additional chloroform between two substrates was measured using lap-shear testing by changing the molecular weight of the PLLA and PDLA adsorbed on gold nanoparticles or the gold nanoparticle concentration. As a result, it was found that optimal conditions exist to show differences. Additionally, until present SC formation has required an organic solvent such as chloroform or acetonitrile. We have discovered that SC formation can be induced using novel organic solvents approved by the Food and Drug Administration (FDA). We anticipate applications for this study in the creation of biodegradable medical adhesive materials where these materials can be released based on environmental considerations using the selective adhesion behavior and induce SC formation through the use of novel solvents. [ABSTRACT FROM AUTHOR]