Formation Of Three-dimensional Structures Research Articles

Effect of salinity and incubation time of planktonic cells on biofilm formation, motility, exoprotease production, and quorum sensing of Aeromonas hydrophila

The aim of this study was to determine the effect of salinity and age of cultures on quorum sensing, exoprotease production, and biofilm formation by Aeromonas hydrophila on stainless steel (SS) and crab shell as substrates. Biofilm formation was assessed at various salinities, from fresh (0%) to saline water (3.0%). For young and old cultures, planktonic cells were grown at 30 °C for 24 h and 96 h, respectively. Biofilm formation was assessed on SS, glass, and crab shell; viable counts were determined in R2A agar for SS and glass, but Aeromonas-selective media was used for crab shell samples to eliminate bacterial contamination. Exoprotease activity was assessed using a Fluoro™ protease assay kit. Quantification of acyl-homoserine lactone (AHL) was performed using the bioreporter strain Chromobacterium violaceum CV026 and the concentration was confirmed using high-performance liquid chromatography (HPLC). The concentration of autoinducer-2 (AI-2) was determined with Vibrio harveyi BB170. The biofilm structure at various salinities (0–3 %) was assessed using field emission electron microscopy (FESEM). Young cultures of A. hydrophila grown at 0–0.25% salinity showed gradual increasing of biofilm formation on SS, glass and crab shell; swarming and swimming motility; exoproteases production, AHL and AI-2 quorum sensing; while all these phenotypic characters reduced from 0.5 to 3.0% salinity. The FESEM images also showed that from 0 to 0.25% salinity stimulated formation of three-dimensional biofilm structures that also broke through the surface by utilizing the chitin surfaces of crab, while 3% salinity stimulated attachment only for young cultures. However, in marked contrast, salinity (0.1–3%) had no effect on the stimulation of biofilm formation or on phenotypic characters for old cultures. However, all concentrations reduced biofilm formation, motility, protease production and quorum sensing for old culture. Overall, 0–0.25% salinity enhanced biofilm formation and expression of quorum sensing regulatory genes in young cultures, whereas these responses were reduced when salinity was >0.25%. In old cultures, salinity at any concentrations (0.1–3%) induced stress in A. hydrophila. The present study provides insight into the ecology of A. hydrophila growing on fish and crustaceans such as shrimp and crabs in estuarine and seawater.

Coculture of stem cells from apical papilla and human umbilical vein endothelial cell under hypoxia increases the formation of three-dimensional vessel-like structures in vitro.

The success of bioengineered dental pulp depends on two principles, (1) whether the transplanted tissue can develop its own vascular endothelial tubule network and (2) whether the host vasculature can be induced to penetrate the bioengineered pulp replacement and conjoin. Major inductive molecules that participate in laying down blood vessels include vascular endothelial growth factor (VEGF), ephrinB2, and hypoxia-inducible factor 1α (HIF-1α). Being able to modulate the genes encoding these angiogenic molecules is a therapeutic target in pulp regeneration for endogenous blood vessel formation, prevention of graft rejection, and exclusion of infection. Once implanted inside the root canal, bioengineered pulp is subjected to severe hypoxia that causes tissue degeneration. However, short-term hypoxia is known to stimulate angiogenesis. Thus, it may be feasible to prime dental cells for angiogenic activity before implantation. Stem cells from apical papilla (SCAP) are arguably one of the most potent and versatile dental stem cell populations for bioengineering pulp in vitro. Our study aimed to investigate whether coculture of SCAP and human umbilical vein endothelial cells (HUVECs) under hypoxia promotes the formation of endothelial tubules and a blood vessel network. In addition, we clarified the interplay between the genes that orchestrate these important angiogenic molecules in SCAP under hypoxic conditions. We found that SCAP cocultured with HUVEC at a 1:5 ratio increased the number of endothelial tubules, tubule lengths, and branching points. Fluorescence staining showed that HUVEC formed the trunk of tubular structures, whereas SCAP located adjacent to the endothelial cell line, resembling the pericyte location. When we used CoCl2 (0.5 mM) to induce hypoxic environment, the expression of proteins, HIF-1α and VEGF, and transcript of ephrinB2 in SCAP was upregulated. However, minimal VEGF levels in supernatants of HUVEC and coculture Petri dishes were detected, suggesting that VEGF secreted by SCAP might be used by HUVEC to accelerate the formation of vessel-like structures. Taken together, we revealed that artificial hypoxia stimulates angiogenic responses in SCAP for possible use in engineering dental pulp replacements. Our results may help to delineate the optimal therapeutic target to promote angiogenesis so that future bioengineered pulp replacements integrate faster and permanently within the host.

Three-dimensional in vitro co-culture model of breast tumor using magnetic levitation.

In this study, we investigate a novel in vitro model to mimic heterogeneous breast tumors without the use of a scaffold while allowing for cell-cell and tumor-fibroblast interactions. Previous studies have shown that magnetic levitation system under conventional culturing conditions results in the formation of three-dimensional (3D) structures, closely resembling in vivo tissues (fat tissue, vasculature, etc.). Three-dimensional heterogeneous tumor models for breast cancer were designed to effectively model the influences of the tumor microenvironment on drug efficiency. Various breast cancer cells were co-cultured with fibroblasts and then magnetically levitated. Size and cell density of the resulting tumors were measured. The model was phenotypically compared to in vivo tumors and examined for the presence of ECM proteins. Lastly, the effects of tumor stroma in the 3D in vitro model on drug transport and efficiency were assessed. Our data suggest that the proposed 3D in vitro breast tumor is advantageous due to the ability to: (1) form large-sized (millimeter in diameter) breast tumor models within 24 h; (2) control tumor cell composition and density; (3) accurately mimic the in vivo tumor microenvironment; and (4) test drug efficiency in an in vitro model that is comparable to in vivo tumors.

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Generation of myospheres from hESCs by epigenetic reprogramming.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Implantable microfluidic device for the formation of three-dimensional vasculature by human endothelial progenitor cells

Vasculogenesis is an important morphogenetic event for vascular tissue engineering and ischemic disease treatment. Stem and progenitor cells can contribute to vasculogenesis via endothelial differentiation and direct participation in blood vessel formation. In this study, we developed an implantable microfluidic device to facilitate formation of three-dimensional (3D) vascular structures by human endothelial progenitor cells (hEPCs). The microfluidic device was made of biodegradable poly(lactic-co-glycolic acid) (PLGA) using a microchannel patterned silicon wafer made by soft lithography. A collagen type I (Col I) hydrogel containing hEPCs filled the microfluidic channels to reconstitute a 3D microenvironment for facilitating vascular structure formation by hEPCs. The device seeded with hEPCs was implanted into the subcutaneous space of athymic mice and retrieved one and four weeks after implantation. Histology and immunohistochemistry revealed that hEPCs formed a 3D capillary network expressing endothelial cell-specific proteins in the channel of the PLGA microfluidic device. This result indicates that a 3D microscale extracellular matrix reconstituted in the microchannel can promote the endothelial differentiation of hEPCs and in turn hEPC-mediated vasculogenesis. The PLGA microfluidic device reported herein may be useful as an implantable tissue-engineering scaffold for vascularized tissue reconstruction and therapeutic angiogenesis.

Formation of three-dimensional Parylene C structures via thermoforming

The thermoplastic nature of Parylene C is leveraged to enable the formation of three-dimensional structures using a thermal forming (thermoforming) technique. Thermoforming involves the heating of Parylene films above its glass transition temperature while they are physically confined in the final desired conformation. Micro and macro scale three-dimensional structures composed of Parylene thin films were developed using the thermoforming process, and the resulting chemical and mechanical changes to the films were characterized. No large changes to the surface and bulk chemistries of the polymer were observed following the thermoforming process conducted in vacuum. Heat treated structures exhibited increased stiffness by a maximum of 37% depending on the treatment temperature, due to an increase in crystallinity of the Parylene polymer. This study revealed important property changes resulting from the process, namely (1) the development of high strains in thermoformed areas of small radii of curvature (30–90 µm) and (2) ∼1.5% bulk material shrinkage in thermoformed multilayered Parylene–Parylene and Parylene–metal–Parylene films. Thermoforming is a simple process whereby three-dimensional structures can be achieved from Parylene C-based thin film structures with tunable mechanical properties as a function of treatment temperature.

Equilibrium spreading pressure and Langmuir–Blodgett film formation of omega-substituted palmitic acids

Langmuir–Blodgett isotherms and equilibrium spreading pressures were measured for compounds of the series X–(CH2)15COOH, X=CH3, SH, OH, F, Cl, Br. Only the CH3 and F terminated compounds formed monolayers with sufficient stability for accurate isotherm measurement, film transfer and X-ray photoelectron spectroscopic analysis. The presence of the terminal heteroatom substituents significantly diminished the stability of the L–B film and depressed the equilibrium spreading pressures (20°C) from 15.4mN/m for the CH3 terminated compound to a range of 0.95 to 0.08mN/m for the other members of the series. These characteristics are attributed to the monolayer film being in a metastable state and the dipole moment of the heteroatom terminal group increasing the monolayer film kinetic instability by facilitating the formation of three-dimensional structures.

Cluster self-organization theory for crystal-forming systems: The geometrical and topological model of formation, selection, and evolution of precursor nanoclusters of molecular and framework compounds

This review concerns the contemporary state of the problem of self-organization in crystal-forming systems, where a long-range order appears spontaneously in the arrangement of nanoscale structural units of any nature (atomic clusters and molecules), which initially existed as a chaotic mixture. Examples are provided where combinatorial topological analysis algorithms are used to restore, from structure data, the convergent matrix self-assembly code of crystal structures in the form of the sequence of significant elementary events e i . For a cyclic six-node cluster S 3 0 , the geometrical and topological modeling of various self-organization levels of hierarchic structures was carried out for six types of primary chains S 3 1 , fifteen types of networks S 3 2 , and thirty types of frameworks S 3 3 . The model is universal and has been used to model the self-assembly of the following crystal structures: monomolecular compound S6 and bimolecular compound S6 + S10, ozone O3, benzene C6H6, cubane C8H8, Zn4O4 (NaCl structure type), carbon oxides C6-GRA (graphite), C6-DIA (diamond), and C6-LON (lonsdaleite), boron nitrides B3N3-GRB, B3N3-DIA, and B3N3-LON, Ni3As3-NIC, B6(OH)16, zeolite K3(Al2Si4O12(OH)-LIT, Na2ZrSi2O7-PKL (parakeldyshite), La3Ga5GeO14 (LGG), and La3GaGe5O16 (LAN). It is for the first time that structural invariants are recognized in topologically different crystal structures of chemical systems. Bifurcations in the evolution pathways of precursor clusters (structural branching points) have been determined for the formation of three-dimensional periodic structures. Frequency analysis carried out for the topological and symmetry pathways in the formation and evolution of clusters (primary chain S 3 1 -microlayer S 3 2 -microframework S 3 3 ) elucidated new crystal-formation trends in diverse chemical systems at the microscopic level.

Heat-induced denaturation and aggregation of actomyosin and myosin from yellowcheek carp during setting

Thermal inactivation kinetics of Ca2+-ATPase, changes in turbidity and rheological properties of actomyosin and myosin from yellowcheek carp during setting at different temperatures were investigated. Actomyosin and myosin setting at 40–45°C exhibited greater extent and more rapid Ca2+-ATPase inactivation compared to at 25–30°C. Formation of protein aggregates and three-dimensional network structures of actomyosin and myosin at 25–30°C was far less than those at 40–45°C. Thermal stability of actomyosin was higher than that of myosin as revealed by its higher activation energy for the inactivation of Ca2+-ATPase. Actomyosin and myosin also exhibited different dynamic rheological properties, especially when the setting temperatures were 40 and 45°C.

Bioinspired material surfaces – Science or engineering?

Natural systems are able to produce material surfaces with unique properties. The formation of complex three-dimensional structures in nature is of fundamental importance to break the limits imposed by available construction elements. Structuring into ordered, and especially into irregular or disordered, systems is the key to define new roadmaps to innovative engineering materials. Basic consideration on the importance of disordered structures in biology can be most efficiently demonstrated on natural optical materials, which is the focus of this viewpoint.

Chemical vapor deposition of ordered TiOx nanostructures on Au(111)

The deposition of TiOx (x≤2) structures on Au(111) by chemical vapor deposition (CVD) in ultrahigh vacuum (UHV) has been investigated with high-resolution core level photoelectron spectroscopy (PES), low-energy electron diffraction (LEED) and scanning tunneling microscopy (STM). Using titanium tetra-isopropoxide as single source precursor it is possible to form different TiOx phases on the surface after deposition: at low coverages, we observe large two-dimensional (2D) honeycomb-lattice Ti2O3 islands with a (2×2) registry with the substrate. Higher coverages are dominated by the formation of three-dimensional (3D) TiO2 structures. The TiO2 structures are atomically well ordered provided that the deposition temperature is high enough (500°C). The ordered structure exhibits a LEED pattern characteristic for a rectangular surface unit cell. By performing the deposition at different temperatures it is possible to tune the balance between the 2D and 3D phases: Growth at 500°C significantly favors the formation of 3D TiO2 islands as compared to growth at 200°C and 300°C.

Genomic Approach to Identify Factors That Drive the Formation of Three-Dimensional Structures by EA.hy926 Endothelial Cells

Understanding the mechanisms responsible for tube formation by endothelial cells (ECs) is of major interest and importance in medicine and tissue engineering. Endothelial cells of the human cell line EA.hy926 behave ambivalently when cultured on a random positioning machine (RPM) simulating microgravity. Some cells form tube-like three-dimensional (3D) aggregates, while other cells (AD) continue to grow adherently. Between the fifth and seventh day of culturing, the two types of cell growth achieve the greatest balance. We harvested ECs that grew either adherently or as 3D aggregates separately after five and seven days of incubation on the RPM, and applied gene array analysis and PCR techniques to investigate their gene expression profiles in comparison to ECs growing adherently under normal static 1 g laboratory conditions for equal periods of time. Using gene arrays, 1,625 differentially expressed genes were identified. A strong overrepresentation of transient expression differences was found in the five-day, RPM-treated samples, where the number of genes being differentially expressed in comparison to 1 g cells was highest as well as the degree of alteration regarding distinct genes. We found 27 genes whose levels of expression were changed at least 4-fold in RPM-treated cells as compared to 1 g controls. These genes code for signal transduction and angiogenic factors, cell adhesion, membrane transport proteins or enzymes involved in serine biosynthesis. Fifteen of them, with IL8 (interleukin 8) and VWF (von Willebrand factor) the most prominently affected, showed linkages to genes of another 20 proteins that are important in cell structure maintenance and angiogenesis and extended their network of interaction. Thus, the study reveals numerous genes, which mutually influence each other during initiation of 3D growth of endothelial cells.

Epigenetic Reprogramming of Human Embryonic Stem Cells into Skeletal Muscle Cells and Generation of Contractile Myospheres

Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for "disease in a dish" models of muscular physiology and dysfunction.

Three-dimensional characterization of bainitic microstructures in low-carbon high-strength low-alloy steel studied by electron backscatter diffraction

Abstract We investigated the microstructural evolution of high strength low alloy steel, Fe–2.0Mn–0.15Si–0.05C (wt.%), by varying the continuous cooling rates from 1 K/s to 50 K/s using three-dimensional electron backscatter diffraction and transmission electron microscopy. Granular bainitic microstructure was prevalent under a slow cooling rate of 1–10 K/s, while lath-type bainite was dominant at a high cooling rate of 50 K/s. The acicular ferrite that was the major microstructure under the intermediate ranges of cooling rates between 10 K/s and 30 K/s was tangled with each other, leading to a three-dimensional interwoven structure with highly misoriented grains. Because of the formation of three-dimensional structures, we propose that the terms “acicular ferrite” and “bainitic ferrite,” which are currently used in steel, be replaced by the terms “interwoven acicular bainite” and “lath bainite,” respectively. Moreover, we also confirmed that the cooling rate is an important factor in determining whether bainitic microstructures occur in the form of granular bainite, interwoven bainite, or lath bainite.

Editorial: Aggregation of Small Molecules: From Dimers to Crystals

Editorial: Aggregation of Small Molecules: From Dimers to Crystals

Molecular dynamics study of the structural modification of graphene by electron irradiation

Molecular dynamics simulations have been used to study the structural modification of graphene by electron irradiation. The authors used the Monte Carlo method to introduce the interaction between incident electrons and carbon atoms in graphene. Then, the effects of electron energy and incident angle on irradiation defects in single-layer graphene were studied, and the cutting of single-layer graphene using different methods of electron irradiation was compared. Following this, the authors simulated the process of single atom chain formation from single-layer graphene using electron irradiation. They also demonstrated the formation of three-dimensional structures, such as tubular structures and nanotube junctions, in bilayer graphene by electron irradiation. The simulations show the capability of structural modification of graphene to a variety of nanostructures by electron irradiation.

Formation of three-dimensional carbon nanotube structures by controllable vapor densification

A new and versatile vapor densification method is developed to fabricate various three-dimensional and dual-porosity carbon nanotube (CNT) structures. By introducing the exposure time of CNTs to the solvent vapor as a control parameter to vary the amount of collected solvent, this method is capable of producing different degrees of densification with high controllability and uniformity. It is also found that the densification effect is highly dependent on the original CNT volume fractions in their forest form, low volume fraction causing shape distortion in densification. An O2 plasma etching of the entangled nanotubes can help maintain the shapes of CNT forests, but only for those in a limited range of CNT volume fraction.

Epithelial and Mesenchymal Subpopulations Within Normal Basal Breast Cell Lines Exhibit Distinct Stem Cell/Progenitor Properties

It has been proposed that epithelial-mesenchymal transition (EMT) in mammary epithelial cells and breast cancer cells generates stem cell features, and that the presence of EMT characteristics in claudin-low breast tumors reveals their origin in basal stem cells. It remains to be determined, however, whether EMT is an inherent property of normal basal stem cells, and if the presence of a mesenchymal-like phenotype is required for the maintenance of all their stem cell properties. We used nontumorigenic basal cell lines as models of normal stem cells/progenitors and demonstrate that these cell lines contain an epithelial subpopulation ("EpCAM+," epithelial cell adhesion molecule positive [EpCAM(pos)]/CD49f(high)) that spontaneously generates mesenchymal-like cells ("Fibros," EpCAM(neg)/CD49f(med/low)) through EMT. Importantly, stem cell/progenitor properties such as regenerative potential, high aldehyde dehydrogenase 1 activity, and formation of three-dimensional acini-like structures predominantly reside within EpCAM+ cells, while Fibros exhibit invasive behavior and mammosphere-forming ability. A gene expression profiling meta-analysis established that EpCAM+ cells show a luminal progenitor-like expression pattern, while Fibros most closely resemble stromal fibroblasts but not stem cells. Moreover, Fibros exhibit partial myoepithelial traits and strong similarities with claudin-low breast cancer cells. Finally, we demonstrate that Slug and Zeb1 EMT-inducers control the progenitor and mesenchymal-like phenotype in EpCAM+ cells and Fibros, respectively, by inhibiting luminal differentiation. In conclusion, nontumorigenic basal cell lines have intrinsic capacity for EMT, but a mesenchymal-like phenotype does not correlate with the acquisition of global stem cell/progenitor features. Based on our findings, we propose that EMT in normal basal cells and claudin-low breast cancers reflects aberrant/incomplete myoepithelial differentiation.

Hollow structures with bilayer graphene walls

The formation of three-dimensional shell-like structures with bilayer graphene walls is described. The structures are produced by the passage of an electric current through graphite in an arc-discharge apparatus. High resolution transmission electron microscopy is used to characterize the carbon, and provides evidence that the structures are three-dimensional rather than flat. A striking feature of the material is that it contains bilayer nanotubes seamlessly joined to larger shell-like regions. The possible growth mechanism of the carbon is discussed, and potential applications considered.