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BackgroundAssembly of oocytes into primordial follicles is essential for establishing the ovarian reserve required for female fertility. In mice, this process begins during embryonic development. Primordial germ cells form cysts by incomplete mitosis until 13.5 days post coitum (dpc). These cysts break down just before birth. Some oocytes undergo apoptosis while surviving oocytes are enclosed by granulosa cells to form primordial follicles. Cyst breakdown and primordial follicle formation were previously shown to be inhibited by estradiol and estrogenic compounds in vitro, suggesting that estrogen is important for regulation of this process. MethodsTo determine the role of fetal estrogen in cyst breakdown and follicle formation these processes were quantified in aromatase deficient (ArKO) mice between 17.5 dpc and postnatal day (PND) 9. Ovaries of ArKO mice were also examined at 2-week intervals to determine if folliculogenesis is affected by lack of estrogen and the age at which the typical ArKO ovarian phenotype first appears. ResultsOocyte number, follicle assembly and follicle development in ArKO mice did not differ from controls between 17.5 dpc and PND9 except for a difference in the proportion of follicles at the primordial and primary stage at PND7. At 2 weeks, ArKO heterozygous and homozygous ovaries still had oocytes in cyst while all oocytes were enclosed in follicles in wildtype ovaries. From 2 to 8 weeks oocyte numbers were similar in all genotypes though there was a trend toward fewer total oocytes in ArKO homozygous females as compared to controls at 8 weeks and a significant reduction at 10 weeks. Abnormal structures such as hemorrhagic follicles and hemosiderin deposits were also observed starting at 6 weeks. ConclusionsThese results suggest that a lack of fetal estrogen does not affect the rate of cyst breakdown or primordial follicle formation perinatally, and maternal estrogen or other signals are the chief regulators. Furthermore, the typical ArKO ovarian phenotype occurs earlier than previously reported.

The surface patterning of a biomaterial, i.e. placing areas with different surface chemistries next to each other is an important step for various biomedical and biotechnological applications, e.g. in biosensor technology, drug screening or tissue engineering. In the current work we investigated the patterning of the surface termination of ultrananocrystalline diamond (UNCD) films prepared by microwave plasma chemical vapor deposition. In order to achieve a high wettability contrast in the neighboring areas O- and F-terminations were selected. The sequence of the modifications and the feasibility of different masking techniques were tested in advance. Based on the achieved results the following process of surface patterning was applied. Initially the whole UNCD surface was modified by UV/O3 treatment rendering the surface O-terminated and strongly hydrophilic (contact angle against water of 11°). The partial masking of this surface before the second modification was achieved with a gold hard mask photolithographically structured and wet etched in form of a grid. After the second modification with CHF3 plasma which provided F-termination and hydrophobic character of the surface (contact angle > 110°), the gold layer was removed. The results of the patterning of the surface termination were visualized by scanning electron microscopy revealing different contrasts of the areas with different terminations. Finally, human SH-SY5Y neuroblastoma cells were plated on the patterned UNCD surfaces and they exhibited preferential attachment and growth on the hydrophilic grid.

Ultrananocrystalline diamond films have been subjected to CHF3 and SF6 rf plasmas in order to achieve a fluorine terminated surface. Major parameter varied in this study was besides the nature of the process gas the exposure time. The treated surfaces have been investigated by contact angle and atomic force microscopy measurements; X-ray photoelectron spectroscopy has been used to determine the surface composition and to get insight into the bonding environments of the treated surfaces. Both plasma treatments lead to similar results. The surfaces are fluorinated by fluorine containing groups, namely CF, CF2, and CF3, with F concentrations of about 20%. This leads to high contact angles of 95–110°; the surfaces are slightly etched and became somewhat smoother. Nevertheless, there are distinct differences between the surface obtained by the CHF3 and SF6 treatments, respectively, the nature of which will be discussed in this paper, together with the underlaying mechanisms.

Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O-3 treatment or NH3 plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H-, O- and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability. (C) 2016 Elsevier B.V. All rights reserved.

Transplantation studies have pinpointed the circadian clock of the Madeira cockroach to the accessory medulla (AME) of the brain's optic lobes. The AME is innervated by approximately 240 adjacent neuropeptidergic neurons, including 12 pigment-dispersing factor (PDF)-expressing neurons anterior to the AME (aPDFMEs). Four of the aPDFMEs project contralaterally, controlling locomotor activity rhythms of the night-active cockroach. The present in vitro Ca2+ imaging analysis focuses on contralaterally projecting AME neurons and their responses to PDF, GABA, and acetylcholine (ACh). First, rhodamine-dextran backfills from the contralateral optic stalk identified contralaterally projecting AME neurons, which were then dispersed in primary cell cultures. After characterization of PDF, GABA, and ACh responses, PDF immunocytochemistry identified ipsilaterally and contralaterally projecting PDFMEs. All PDF-sensitive clock neurons, PDF-immunoreactive clock neurons, and the majority of ipsilaterally and contralaterally projecting cells were excited by ACh. GABA inhibited all PDF-expressing clock neurons, and about half of other ipsilaterally projecting and most contralaterally projecting clock neurons. For the first time, we identified PDF autoreceptors in PDF-secreting cockroach circadian pacemakers. The medium-sized aPDFMEs and all other contralaterally projecting PDF-sensitive clock cells were inhibited by PDF. The ipsilaterally remaining small PDF-sensitive clock cells were activated by PDF. Only the largest aPDFME did not express PDF autoreceptors. We hypothesize that opposing PDF signaling generates 2 different ensembles of clock cells with antiphasic activity, regulating and maintaining a constant phase relationship between rest and activity cycles of the night-active cockroach.

Diamond and diamond-like carbon (DLC) films possess a set of excellent physical and chemical properties which together with a high biocompatibility make them attractive candidates for a number of medical and biotechnological applications. In the current work thin ultrananocrystalline diamond (UNCD) and DLC films were comparatively investigated with respect to cell attachment and proliferation after different surface modifications. The UNCD films were prepared by microwave plasma enhanced chemical vapor deposition, the DLC films by pulsed laser deposition (PLD). The films were comprehensively characterized with respect to their basic properties, e.g. crystallinity, morphology, chemical bonding nature, etc. Afterwards the UNCD and DLC films were modified applying O2 or NH3/N2 plasmas and UV/O3 treatments to alter their surface termination. The surface composition of as-grown and modified samples was studied by X-ray photoelectron spectroscopy (XPS). Furthermore the films were characterized by contact angle measurements with water, formamide, 1-decanol and diiodomethane; from the results obtained the surface energy with its dispersive and polar components was calculated. The adhesion and proliferation of MG63 osteosarcoma cells on the different UNCD and DLC samples were assessed by measurement of the cell attachment efficiency and MTT assays. The determined cell densities were compared and correlated with the surface properties of as-deposited and modified UNCD and DLC films.

Two types of carbon-based films, namely diamond-like carbon (DLC) and ultrananocrystalline diamond (UNCD) films were comparatively investigated. The DLC films were prepared by pulsed laser deposition (PLD) without or with additional ion bombardment, the UNCD films by microwave plasma enhanced chemical vapour deposition. The films were characterized with respect to their topography and morphology (AFM, SEM) and bonding structure, e.g. fractions of sp3 and sp2 carbon atoms (XPS, FTIR and Raman spectroscopy). Finally, the wettability and the surface energies of both types of films were investigated with contact angle measurements as well as the antibacterial properties with two types of bacteria.

We have investigated the stability of the surface termination of ultrananocrystalline diamond/amorphous carbon (UNCD/a-C) composite films after their modification with UV/O 3 treatments or NH 3 -containing plasmas. The films were prepared by microwave plasma chemical vapor deposition from CH 4 /N 2 mixtures and possess the H-termination typical for all CVD grown diamond films. The treatment with UV/O 3 or NH 3 /N 2 plasma resulted in a change of the surface termination as revealed by contact angle measurements and X-ray photoelectron spectroscopy (XPS). Both processes rendered the as-grown hydrophobic UNCD surface hydrophilic; the influence of the treatment time as well as that of the carrier gas (N 2 or Ar) for the plasma process were investigated. The main task of this investigation was to determine the stability of the surface termination with storage time and storage conditions and upon exposure to standard organic solvents, like acetone, ethanol and i-propanol. It was found that for the UV/O 3 treated UNCD surfaces the storage in air, and after NH 3 /N 2 plasma modification package under reduced pressure preserved to a great extent the achieved hydrophilic properties in a period up to three months. The changes that occur predominantly during the first days after the modification step were due mostly to interchange of surface groups with the ambient. Similar processes were observed after exposure of the UNCD surfaces to standard organic solvents.

Ultrananocrystalline diamond (UNCD) films have been deposited by microwave plasma chemical vapor deposition from 17% CH4/N2 mixtures. In order to change the original hydrogen termination of the UNCD surfaces, the films have subsequently been subjected either to the so-called UV/O3 treatment which leads to OH-terminated surfaces, or to NH3/N2 plasmas which introduces NH2 groups but also a certain amount of OH groups. These three types of surfaces have been characterized by X-ray photoelectron spectroscopy, contact angle and ζ-potential measurements. The contact angle measurements have shown that as-grown UNCD surfaces are highly hydrophobic but became highly hydrophilic after both treatments. The ζ-potential measurements revealed that the isoelectric point of the H-terminated as-grown surface is distinctively higher than that of either UV/O3 or NH3/N2 plasma treated surfaces. Finally, the interactions of these surfaces with neurons of the cockroach Leucophaea maderae have been investigated. These studies have shown that especially the two treated surfaces allow for a fast, strong attachment of these cells without compromising their viability and without changing their normal physiological responses. These results will be discussed in terms of those obtained with the different surface characterization techniques.