Cord like-structure formation in testicular cell cultures (see also Video S1).

<p>2A) Two series of three micrographs presenting the stages of cord-like structure formation in testicular cells cultured on 350 nm and flat PDMS substrates in DMEM at days 14, 14.5 and 15 of culture. Primary rat (7-day-old) Sertoli and peritubular cells were seeded at an initial density of 10⁶ cells/cm². At two weeks, cultures consisted of confluent mixed monolayers with several Sertoli cell clusters. Imaging was started at day 14 when first indications of cellular migration were observed. During the observation period active movements triggered a change of the confluent monolayers into cord-like structures on both 350 nm (upper panel) and flat PDMS substrates (lower panel). The orientation of cord-like structures did not follow the direction of nanogratings (red arrows). 2B) Testicular cell cultures performed under identical conditions as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060054#pone-0060054-g002" target="_blank">Fig. 2A</a> except that cells were seeded at a lower initial density of 10⁵ cells/cm² either on nanograting (left panel) or flat (right panel) PDMS substrates. Micrographs were taken at day 15 of culture. The orientation of cord-like structures was following the direction of nanogratings (red arrow). Scale bar = 200 um. 2C) Analysis of changes in the direction of cord-like structures seeded at high (10⁶ cells/cm²) or low (10⁵ cells/cm²) initial density on 350 nm PDMS substrates at day 15. 2D) Analysis of the size of individual cord-like structures seeded at high (10⁶ cells/cm²) or low (10⁵ cells/cm²) initial density on 350 nm and flat PDMS substrates at day 15.</p

Effects of Nanostructures and Mouse Embryonic Stem Cells on <i>In Vitro</i> Morphogenesis of Rat Testicular Cords

<div><p>Morphogenesis of tubular structures is a common event during embryonic development. The signals providing cells with topographical cues to define a cord axis and to form new compartments surrounded by a basement membrane are poorly understood. Male gonadal differentiation is a late event during organogenesis and continues into postnatal life. The cellular changes resemble the mechanisms during embryonic life leading to tubular structures in other organs. Testicular cord formation is dependent on and first recognized by SRY-dependent aggregation of Sertoli cells leading to the appearance of testis-specific cord-like structures. Here we explored whether testicular cells use topographical cues in the form of nanostructures to direct or stimulate cord formation and whether embryonic stem cells (ES) or soluble factors released from those cells have an impact on this process. Using primary cell cultures of immature rats we first revealed that variable nanogratings exerted effects on peritubular cells and on Sertoli cells (at less than <1000 cells/mm²) by aligning the cell bodies towards the direction of the nanogratings. After two weeks of culture testicular cells assembled into a network of cord-like structures. We revealed that Sertoli cells actively migrate towards existing clusters. Contractions of peritubular cells lead to the transformation of isolated clusters into cord-like structures. The addition of mouse ES cells or conditioned medium from ES cells accelerated this process. Our studies show that epithelial (Sertoli cell) and mesenchymal (peritubular cells) cells crosstalk and orchestrate the formation of cords in response to physical features of the underlying matrix as well as secretory factors from ES cells. We consider these data on testicular morphogenesis relevant for the better understanding of mechanisms in cord formation also in other organs which may help to create optimized in vitro tools for artificial organogenesis.</p> </div

Combined effects of nanostructures and OG2 cell conditioned medium on cord formation of testicular cells.

<p>5A) Phase contrast micrographs of primary rat (7-day-old) testicular cells seeded at an initial density of 10⁶ cells/cm² on 350 nm (right panel) and flat (left panel) PDMS substrates in conditioned medium (Three day culture of OG2 cells at a density of 10⁵ cells/cm²) at day 1 (upper panel) and day 3 (lower panel) of culture. At day 1 orientation of cells in the direction of nanogrids was visible when compared to flat PDMS substrates. At day 3 of culture cellular aggregation and cord-like structure formation occurred on both flat and 350 nm PDMS substrates. The direction of cord-like structure was aligned with nanogratings on 350 nm PDMS substrates but not on flat PDMS substrates. Red arrows indicate the direction of nanogratings. Scale bar = 200 um. 5B) Analysis of the direction of cord-like structures in conditioned medium and after exposure to 350 nm or flat PDMS substrates at day 3 of culture. The quantitative results confirm the microscopic observation that nanogratings have an impact on the orientation of cord-like structures. Cord-like structures on flat PDMS show a random orientation. 5C) Analysis of the size of cord-like structures at day 3 of culture. Cord-like structures were of similar size irrespective of the exposure to nanogratings.</p

Effects of mouse embryonic stem cells (OG2 cells expressing the GFP-transgene) on cord formation of rat testicular cells.

<p>3A) Micrographs showing immunofluorescence of GFP-signal (upper panels) and merged images (phase contrast plus immunofluorescence) of primary rat (7-day-old) Sertoli cells and peritubular cells seeded at an initial density of 10⁶ cells/cm² on flat PDMS substrates after culture for one day (upper two panels), three days (middle two panels) and six days (lower two panels) in DMEM. A variable number of OG2 cells (10, 10², 10³, 10⁴, 10⁵ cells/cm²) was added to the primary cells at the initiation of cell cultures. No OG2 cells were added to controls. No obvious change of cellular arrangements was observed at day 1. On days 3 and 6 of culture cord-like structure formation is observed which intensified with increasing numbers of OG2 cells. OG2 cells formed expanding colonies in contact with the cord-like structures. 3B) Analysis of the size of cord-like structures at day 6 of culture. Cord-like structures were significantly larger compared to all other experimental groups depending on the initial density of OG2 cells on flat PDMS substrates at day 6. In the control group and after addition of only 10 OG2 cells no cord like structures were encountered.</p

Cell specific and density dependent response of testicular cells to nanogratings.

<p>Micrographs showing immunostaining of primary rat (7-day-old) Sertoli cells and peritubular cells. The cells were seeded at an initial density of 10⁶ cells/cm² in DMEM and cultured for one week. Cells were fixed at day 7. Peritubular cells are marked for α-smooth muscle actin (brown precipitate). Nuclei were stained blue with hematoxylin. 1A–D) Cells cultured on flat PDMS substrate (1A), and substrates carrying nanogratings of 200 nm (1B), 350 nm (1C) and 5 um (1D) dimensions. Red arrows indicate the direction of nanogratings. 1E) Quantitatitive analysis of directional changes in peritubular cells on different substrates. All nanogratings evoked changes in the orientation of peritubular cells. No visible influence on Sertoli cells was noted. 1F–H) Cells cultured on 350 nm PDMS substrate after inhomogeneous seeding creating diversity in plating density. Areas of high cell density (1F) revealed a random distribution of Sertoli cells in contrast to an aligned orientation towards nanogratings in low cell density zones (1G) on the same substrate in the same well. Peritubular cells were always oriented in accordance with the direction of the nanogratings. Quantification of cellular orientation in Sertoli cells (1H). Sertoli cell density was determined in randomly selected microscopic frames (as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060054#pone-0060054-g001" target="_blank">Figs. 1F–G</a>) and the predominant cellular characteristic (either randomly shaped or spindle shaped (aligned)) was recorded for each frame. Seven recordings were performed per experiment and seven independent experiments were analyzed. We established that the threshold to respond to the nanogratings occurred at a density of approximately 1000 cells/mm². Scale bar = 50 um.</p

Controlled Assembly and Release of Retinoic Acid Based on the Layer-by-Layer Method

All-<i>trans</i> retinoic acid (RA) has been proved to play important roles in regulating cell growth in various types of cells. Yet most experiments were performed by adding RA in solution previously. In this Article, we focus on the incorporation of RA, as a negatively charged moiety, into layered polyelectrolyte films on surfaces by means of layer-by-layer (LbL) deposition, followed by adding of capping layers to regulate the release of RA from the films. The incorporated RA was designed to release over 5 days in buffer solution. The assembly and release of RA were verified by UV and QCM results. The controlled release of RA from multilayer films can serve as a model system to study the influence of small molecules on cell growth

Imparting Catalytic Activity to a Covalent Organic Framework Material by Nanoparticle Encapsulation

Integrating covalent organic frameworks (COFs) with other functional materials is a useful route to enhancing their performances and extending their applications. We report herein a simple encapsulation method for incorporating catalytically active Au nanoparticles with different sizes, shapes, and contents in a two-dimensional (2D) COF material constructed by condensing 1,3,5-tris­(4-amino­phenyl)­benzene (TAPB) with 2,5-dimethoxy­terephthaldehyde (DMTP). The encapsulation is assisted by the surface functionalization of Au nanoparticles with polyvinyl­pyrrolidone (PVP) and follows a mechanism based on the adsorption of nanoparticles onto surfaces of the initially formed polymeric precursor of COF. The incorporation of nanoparticles does not alter obviously the crystallinity, thermal stability, and pore structures of the framework matrices. The obtained COF composites with embedded but accessible Au nanoparticles possess large surface areas and highly open mesopores and display recyclable catalytic performance for reduction of 4-nitrophenol, which cannot be catalyzed by the pure COF material, with activities relevant to contents and geometric structures of the incorporated nanoparticles

Metal-Mediated Assembly of 1,<i>N</i>⁶‑Ethenoadenine: From Surfaces to DNA Duplexes

The design of multinuclear metal complexes requires a match of the ligand-to-metal vectors and the preferred coordination geometries of the metal ions. Only a few ligands are known with a parallel orientation of N→M vectors that brings the metal ions into close proximity. We establish here the adenine derivative 1,<i>N</i>⁶-ethenoadenine (εA) as an ideal bis­(monodentate) ligand. Scanning tunneling microscope images of alkylated εA on graphite surface clearly indicate that these ligands bind to Ag­(I) ions. The molecular structures of [Ag₂(<b>1</b>)₂]­(ClO₄)₂ and [Ag₂(<b>2</b>)₂]­(ClO₄)₂ (<b>1</b>, 9-ethyl-1,<i>N</i>⁶-ethenoadenine; <b>2</b>, 9-propyl-1,<i>N</i>⁶-propylenoadenine) confirm that dinuclear complexes with short Ag···Ag distances are formed (3.0256(3) and 2.984(1) Å, respectively). The structural motif can be extended to divalent metal ions, as was shown by determining the molecular structure of [Cu₂(<b>1</b>)₂(CHO₂)₂(OH₂)₂]­(NO₃)₂·2H₂O with a Cu···Cu distance of 3.162(2) Å. Moreover, when introducing the 1,<i>N</i>⁶-ethenoadenine deoxyribonucleoside into parallel-stranded DNA duplexes, even dinuclear Ag­(I)-mediated base pairs are formed, featuring the same transoid orientation of the glycosidic bonds as the model complexes. Hence, 1,<i>N</i>⁶-ethenoadenine and its derivatives are ideally suited as bis­(monodentate) ligands with a parallel alignment of the N→M vectors for the construction of supramolecular metal complexes that require two metal ions at close distance

Metal-Mediated Assembly of 1,<i>N</i>⁶‑Ethenoadenine: From Surfaces to DNA Duplexes

The design of multinuclear metal complexes requires a match of the ligand-to-metal vectors and the preferred coordination geometries of the metal ions. Only a few ligands are known with a parallel orientation of N→M vectors that brings the metal ions into close proximity. We establish here the adenine derivative 1,<i>N</i>⁶-ethenoadenine (εA) as an ideal bis­(monodentate) ligand. Scanning tunneling microscope images of alkylated εA on graphite surface clearly indicate that these ligands bind to Ag­(I) ions. The molecular structures of [Ag₂(<b>1</b>)₂]­(ClO₄)₂ and [Ag₂(<b>2</b>)₂]­(ClO₄)₂ (<b>1</b>, 9-ethyl-1,<i>N</i>⁶-ethenoadenine; <b>2</b>, 9-propyl-1,<i>N</i>⁶-propylenoadenine) confirm that dinuclear complexes with short Ag···Ag distances are formed (3.0256(3) and 2.984(1) Å, respectively). The structural motif can be extended to divalent metal ions, as was shown by determining the molecular structure of [Cu₂(<b>1</b>)₂(CHO₂)₂(OH₂)₂]­(NO₃)₂·2H₂O with a Cu···Cu distance of 3.162(2) Å. Moreover, when introducing the 1,<i>N</i>⁶-ethenoadenine deoxyribonucleoside into parallel-stranded DNA duplexes, even dinuclear Ag­(I)-mediated base pairs are formed, featuring the same transoid orientation of the glycosidic bonds as the model complexes. Hence, 1,<i>N</i>⁶-ethenoadenine and its derivatives are ideally suited as bis­(monodentate) ligands with a parallel alignment of the N→M vectors for the construction of supramolecular metal complexes that require two metal ions at close distance

Phase Behavior and Molecular Packing of Octadecyl Phenols and their Methyl Ethers at the Air/Water Interface

Noncovalent molecular interactions, such as hydrogen bonding and van der Waals forces, play an important role in self-assembling to supramolecular structures. To study these forces, we chose monolayers at the air/water interface to limit the possible arrangements of the interacting molecules. Furthermore, monolayers provide useful tools to understand and study interactions between molecules in a controlled and fundamental way. The phase behavior and molecular packing of the phenols 1-(4-hydroxyphenyl)-octadecane (<b>5a</b>), 1-(3,4-dihydroxyphenyl)-octadecane (<b>6</b>), and 1-(2,3,4-trihydroxyphenyl)-octadecane (<b>3</b>) and their methyl ethers in monolayers at the air/water interface have been examined by π/A isotherms, Brewster angle microscopy (BAM), grazing incidence X-ray diffraction (GIXD) measurements, and density functional theory (DFT) calculations. The phenols are synthesized by Friedel–Crafts acylation of methoxybenzenes, hydrogenation of the resulting aryl ketones, and cleavage of the aryl methyl ethers. In the π/<i>A</i> isotherms and in BAM, the phenols show patches of the solid condensed phase at large molecular areas and the monolayers collapse at high pressures. Furthermore, the dimensions of the unit cell obtained by GIXD measurements are compatible with an arrangement of the phenyl rings that allows one aryl ring to interact with four adjacent phenyl rings in an edge-to-face arrangement, which leads to a significant binding energy. The experimental data are in good agreement with DFT calculations of 2D crystalline benzene and <i>p</i>-cresol arrangements. The enhanced monolayer stability of phenol <b>5a</b> can be explained by hydrogen bonds of the hydroxyl group with water and van der Waals forces between the alkyl chains and aryl–aryl interactions