Enhanced bone marrow stromal cell adhesion and growth on segmented poly(ether ester)s based on poly(ethylene oxide) and poly(butylene terephthalate)

In previous studies in rats and goats, hydrophilic compositions of the PEOT/PBT block copolymer family have shown in vivo calcification and bone bonding. These copolymers are therefore interesting candidates as scaffolding materials in bone tissue engineering applications. Model studies using goat bone marrow stromal cells, however, showed that it was not possible to culture bone marrow stromal cells in vitro on these hydrophilic copolymers. In this paper two ways of surface modifying these materials to improve in vitro bone marrow stromal cell attachment and growth are discussed. Two different approaches are described: (1) blending of hydroxyapatite (HA) followed by CO(2) gas plasma etching; (2) surface modification using CO(2) gas plasma treatments. It was observed that not only HA but also the CO(2) plasma treatment by itself has a positive effect on bone marrow stromal cell attachment and growth. Gas plasma treatment appeared to be the most successful approach, resulting in a large increase in the amount of bone marrow stromal cells present on the surface (determined by a DNA assay). The amount of DNA present on the plasma-treated copolymer 1000/70/30 PEOT/PBT, based on poly(ethylene oxide, M(w) = 1000, 70 m% soft segment), was comparable to the amount present on PDLLA and significantly higher than the amount present on PCL after 7 days of cell culturing. The fact that after gas plasma treatment bone marrow stromal cells do attach to PEOT/PBT copolymers, enables in vitro bone marrow stromal cell culturing, making bone tissue engineering applications of these materials possible.     

Functionalization of cellulose-containing fabrics by plasma and subsequent metal salt treatments

In order to upgrade the UV-protection and antibacterial functional properties of cotton/polyester (80/20), cotton/linen (50/50) and linen/viscose-polyester (50/50) fabric blends, they were treated with different plasma gases (oxygen, air, and argon) followed by subsequent treatment with certain metal salts namely Zn-acetate, Cu-acetate, Al-chloride, and Zr-oxychloride. The obtained results show that the type of plasma gas, the kind of metal salt as well as the nature of the treated substrate play an important role in the extent of enhancing the demanded functional properties. Oxygen plasma treatment followed by Cu-acetate or Zn-acetate treatment gives the best UV-protection or antibacterial activity respectively, keeping other parameters constant. The surface morphology of some untreated and plasma-treated samples was also analyzed by SEM.     

A novel heart‐cell line from brown‐marbled grouper Epinephelus fuscoguttatus and its susceptibility to iridovirus

A novel cell line (bmGH) was established from the heart of brown‐marbled grouper Epinephelus fuscoguttatus and its viral susceptibility was evaluated. The bmGH cells have been subcultured to passage 65 in Dulbecco's modified eagle medium:Ham's nutrient mixture F‐12 (1:1) medium (DMEM/F12) which was further supplemented with foetal bovine serum (FBS), carboxymethyl‐chitosan, basic fibroblast growth factor (bFGF) and insulin‐like growth factor‐I (IGF‐I) at 24° C. The heart cells have a fibroblastic morphology and proliferated to confluence 14 days later. The cells grew at a steady rate during subsequent subculture and had a population doubling time of 40·3 h at passage 60. Karyotype analysis showed that these cells exhibited chromosomal aneuploidy with a modal chromosome number of 48. The results of viral susceptibility characterization revealed that cytopathic effects (CPE) of bmGH cells appeared after infection by two iridoviruses, turbot reddish body iridovirus (TRBIV) and lymphocystis disease virus (LCDV). A large number of TRBIV and LCDV particles were also observed in the infected bmGH cells by electron microscope examination. All of these facts indicate that the bmGH cell line established here may serve as a valuable tool for studies of cell‐virus interactions and has potential applications in fish virus isolation, propagation and vaccine development.     

Determination of peptido-aminobenzophenone (2-o-chlorobenzoyl-4-chloro-N-methyl-N′-glycyl-glycinanilide) by electron-capture gas—liquid chromatography

A gas—liquid chromatographic method for the determination of peptido-aminobenzophenone (2-o-chlorobenzoyl-4-chloro-N-mehtyl-N′-glycyl-glycinanilide, I) in dog plasma was developed. Decomposition of compound I was observed during chromatography. In alkaline medium, compound I in plasma was submitted to ring closure to yield 3-amino-6-chloro-5-(2-chlorophenyl)-1-methylquinolin-2-one (aminoquinolone), and the hexane extract was assayed by gas—liquid chromatography using electron-capture detection. The concentration range of compound I studied was 10–90 ng per 0.5 ml of plasma. Interference from both endogenous and exogenous sources was negligible. The method could be applied to the determination of the plasma level of compound I in dogs following oral administration of a single 5 mg/kg dose.     

Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases

Formation of bacterial biofilms at solid–liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: (1) killing bacteria in biofilms by causing severe cell membrane damage, and (2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum. Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections.     

Embryoid and Plantlet Formation from Stock Cultures of Nigella Tissues

Embryogenesis could be induced within a short period in differentiated roots of Nigella sativa on solid Murashige and Skoog's medium containing casein hydrolysate (500 mg/1) and IAA (0.5 mg/1). Experimental root explants were taken from two different stocks: (i) previously differentiated from leaf callus and now being maintained through subcultures in liquid White's medium and (ii) freshly differentiated from leaf callus on solid Murashige and Skoog's medium + casein hydrolysate (100 mg/1) + IAA (0.5 mg/1). Fifty per cent of the embryoids produced 2n plantlets within 40–60 days.     

Improving the attachment and proliferation of umbilical cord mesenchymal stem cells on modified polystyrene by nitrogen-containing plasma

Wharton’s jelly mesenchymal stem cells (WJMSCs) are important alternative source of pluripotent cells for several therapeutic purposes. Understanding of adhesion properties of such cells is necessary to regulate the attachment, growth and proliferation on targeted culture surfaces. BCP-K1, a line of WJMSCs, and polystyrene (PS) culture dishes were used as membrane samples. A 13.56 MHz inductively coupled discharge plasma reactor with a mixture of N-containing gas and noble gas was used. This was expected to introduce the more hydrophilic groups on PS surface and enhance the cell adhesion. The plasma-treated PS dishes with the mixed gas of N₂ + He at 50 W and NH₃ + He at 100 W were reactive towards BCP-K1. Cellular adhesion and proliferation was significantly twice as efficient on the treated surfaces than on PS dishes. BCP-K1 also secreted more focal adhesion kinase to adhere and proliferate when cultured on N₂-treated PS dishes than on the NH₃-treated PS dishes. Stable stemness markers were detected, including CD105, CD9 and SSEA-4, expressed on BCP-K1 growing on the modified PS dish surfaces, during 7 days of culturing. The presence of –NH₂ groups on the PS dish surface were revealed by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. A large amount of oxygen- and nitrogen-containing functional groups, up to 9.0 %, were introduced by NH₃ plasma and N₂ plasma. The functional groups introduced on to the PS surfaces were clearly the key factors which enhanced WJMSCs attachment and stemness stability.     

Antimicrobial effects of microwave-induced plasma torch (MiniMIP) treatment on Candida albicans biofilms

The susceptibility of Candida albicans biofilms to a non-thermal plasma treatment has been investigated in terms of growth, survival and cell viability by a series of in vitro experiments. For different time periods, the C. albicans strain SC5314 was treated with a microwave-induced plasma torch (MiniMIP). The MiniMIP treatment had a strong effect (reduction factor (RF) = 2.97 after 50 s treatment) at a distance of 3 cm between the nozzle and the superior regions of the biofilms. In addition, a viability reduction of 77% after a 20 s plasma treatment and a metabolism reduction of 90% after a 40 s plasma treatment time were observed for C. albicans. After such a treatment, the biofilms revealed an altered morphology of their cells by atomic force microscopy (AFM). Additionally, fluorescence microscopy and confocal laser scanning microscopy (CLSM) analyses of plasma-treated biofilms showed that an inactivation of cells mainly appeared on the bottom side of the biofilms. Thus, the plasma inactivation of the overgrown surface reveals a new possibility to combat biofilms.     

A Comparative Laboratory Study of Liquid Nitrogen and Argon Gas Cryosurgery Systems

Cryotherapy can now be applied using a variety of delivery systems and cryogens. We compared the Cryotech LCS 3000 liquid nitrogen system (Spembly, Andover, UK) with the CRYOcare argon gas-based system (Irvine, CA, U.S.A.) using three different 3-mm cryoprobes: anoldliquid nitrogen probe (N-probe), anewN-probe featuring gas bypass and an argon gas probe. Each probe was tested in two models: (i) fresh sheep liver at 20°C—the probe was inserted to a depth of 1.5 cm; the rate of ice ball formation was monitored by recording radial temperatures every 15 s at 5, 10, 15, and 20 mm from the cryoprobe, and the ice-ball diameter was measured every 2.5 min. After 10 min, the probe was warmed and the time taken until it could be extracted from the liver was recorded. (ii) Warm water bath—the probe was immersed in warm water (42°C) for 15 min and the ice-ball diameter was measured at 5-min intervals. Radial temperatures in liver declined more rapidly (P< 0.001) and time to probe extraction was less (P< 0.01) when the argon gas system was used. ThenewN-probe performed better than its older counterpart, but was still slower than the argon gas system. In liver (20°C), ice-ball diameters were similar after 10 min, but in warm water, they were larger when thenewN-probe was used (P< 0.02). It would appear that the argon gas system is initially faster, but it does not achieve as large an ice ball in a warm environment as the liquid nitrogen system.     

A possible function of the chilamydospores of Candida albicans

Strains ofCandida albicans produce large numbers of chlamydospores in a liquid medium composed of 1% sodium taurocholate in distilled water. Studies made of these chlamydospores revealed that they do not contain endospores or ascospores but they do contain globules which are lipoid in character. These globules are extruded when chlamydospores split in the liquid medium or under slight pressure. Evidence was not obtained which could support van der Walt's (1967, 1969) statements to the effect that chlamydospores bud, germinate or change into other types of cells. Furthermore, no morphological or cytological evidence was found to substantiate the proposed life cycle ofC. albicans as outlined by van der Walt (1969). It is suggested that a possible function of the chlamydospores ofC. albicans is that of a storage cell.     

Hydrogen peroxide: A central player in physical plasma-induced oxidative stress in human blood cells

Abstract

Plasma medicine is an interdisciplinary field and recent clinical studies showed benefits of topical plasma application to chronic wounds. Whereas most investigations have focused on plasma–skin cell interaction, immune cells are omnipresent in most tissues as well. They not only elicit specific immune responses but also regulate inflammation, which is central in healing and regeneration. Plasma generates short-lived radicals and species in the gas phase. Mechanisms of plasma–cell interactions are not fully understood but it is hypothesized that reactive oxygen and nitrogen species (RONS) mediate effects of plasma on cells. In this study human blood cells were investigated after cold atmospheric plasma treatment with regard to oxidation and viability. Plasma generates hydrogen peroxide (H₂O₂) and the responses were similar in cells treated with concentration-matched H₂O₂. Both treatments gave an equivalent reduction in viability and this was completely abrogated if catalase was added prior to plasma exposure. Further, five oxidation probes were utilized and fluorescence increase was observed in plasma-treated cells. Dye-dependent addition of catalase diminished most but not all of the probe fluorescence, assigning H₂O₂ a dominant but not exclusive role in cellular oxidation by plasma. Investigations for other species revealed generation of nitrite and formation of 3-nitrotyrosine but not 3-chlorotyrosine after plasma treatment indicating presence of RNS which may contribute to cellular redox changes observed. Together, these results will help to clarify how oxidative stress associates with physical plasma treatment in wound relevant cells.     

Some Results from Studies of Microwave Discharges in Liquid Heavy Hydrocarbons

Some results from studies of microwave discharges in heavy hydrocarbons are presented. Microwave energy was introduced into liquid hydrocarbon via a coaxial line. The pressure above the liquid surface was equal to the atmospheric pressure. The discharge was ignited in a mixture of argon and hydrocarbon vapor. Argon was supplied through a channel in the central conductor of the coaxial line. The emission spectra of discharges in different liquid hydrocarbons were studied. It is shown that the emission spectra mainly consist of sequences of Swan bands, while radiation of other plasma components is on the noise level. Spectra of plasma emission are presented for discharges in liquid n-heptane, nefras, and C-9 oil used to produce chemical fibers. The rotational (gas) and vibrational temperatures are determined by processing the observed spectra.     

High frequency androgenesis from isolated microspores of maize

Anthers from a highly androgenic genotype of maize (139/39-02), when cultured in a modified, liquid YP medium, dehisced within 2–7 days resulting in a stationary suspension of microspores. After 12–15 days, the microspore suspension was found to contain multicellular masses which went on to produce macroscopic embryo-like structures within 20–25 days of culture initiation. Embryogenic callus could be obtained by transferring microspore-derived embryos onto a modified N6 medium supplemented with 2.5 mg/l dicamba and 0.1 mg/l 2,4-D. Subculture onto hormone-free medium resulted in plant regeneration. Over 400 embryo-like structures per 100 anthers cultured have been obtained from liquid induction medium as compared to 55 embryos per 100 anthers cultured on an agar-solidified medium. Approximately 5–25% of these embryo-like structures went on to produce callus from which plants could be recovered. Mechanical isolation of microspores from anthers precultured for 0, 3, and 7 days also resulted in embryo production and plant regeneration. This represents the first report of plant recovery from isolated maize microspores. The use of a liquid induction medium applied to a highly androgenic genotype allows for the production of large numbers of microspore-derived plants and provides a single, haploid cell regeneration system for maize.     

生姜离体叶片愈伤组织悬浮细胞系建立与植株再生

以‘莱芜大姜’为试材,研究了生姜离体叶片愈伤组织的诱导以及细胞悬浮系建立与植株再生。结果表明,以生姜试管苗叶片为外植体,接种到MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到MS+0.15 mg/L 2,4-D+6.0 mg/L 6-BA+30 g/L蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30 d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0-1.5 g,继代培养的适宜间隔期为15 d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基MS+0.2 mg/L NAA+10.0 mg/L 6-BA+30 g/L蔗糖上可获得再生植株。     

枸杞原生质体培养及高效成株体系的建立

由枸杞髓部组织诱导出胚性愈伤组织,并由此愈伤组织建立起稳定的细胞悬浮系。从悬浮细胞游离的原生质体在改良KM培养基(1.5 mg/L 6_BA,0.5 mg/L NAA和0.5 mg/L 2,4_D)中进行液体浅层培养,3～4 d后出现第一次分裂,第7 d统计分裂频率为50.3%,15 d左右可形成细胞团,3～4周后形成肉眼可见的愈伤组织,愈伤组织植板率为1.25%。将细胞团转移到液体分化培养基(MS+6_BA 1.5 mg/L+2,4_D 0.2 mg/L) 8～10 d可形成大量胚状体,及时将胚性愈伤组织块转移到固体分化培养基上(MS+6_BA 0.2 mg/L),可形成大量绿芽,分化率54.17%。绿芽在生根培养基（MS+NAA 0.2 mg/L）可形成完整植株,移栽后成活良好。     

Enhanced reconstruction of rat calvarial defects achieved by plasma-treated electrospun scaffolds and induced pluripotent stem cells

Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.     

Effect of biologically produced sulfur on gas absorption in a biotechnological hydrogen sulfide removal process

Absorption of hydrogen sulfide in aqueous suspensions of biologically produced sulfur particles was studied in a batch stirred cell reactor, and in a continuous set-up, consisting of a lab-scale gas absorber column and a bioreactor. Presence of biosulfur particles was found to enhance the absorption rate of H(2)S gas in the mildly alkaline liquid. The mechanism for this enhancement was however found to depend on the type of particles used. In the gently stirred cell reactor only small hydrophilic particles were present (d(p) < 3 microm) and the enhancement of the H(2)S absorption rate can be explained from the heterogeneous reaction between dissolved H(2)S and solid elemental sulfur to polysulfide ions, S(x) (2-). Conditions favoring enhanced H(2)S absorption for these hydrophilic particles are: low liquid side mass transfer (k(L)), high sulfur content, and presence of polysulfide ions. In the set-up of gas absorber column and bioreactor, both small hydrophilic particles and larger, more hydrophobic particles were continuously produced (d(p) up to 20 microm). Here, observed enhancement could not be explained by the heterogeneous reaction between sulfide and sulfur, due to the relatively low specific particle surface area, high k(L), and low [S(x) (2-)]. A more likely explanation for enhancement here is the more hydrophobic behavior of the larger particles. A local increase of the hydrophobic sulfur particle concentration near the gas/liquid interface and specific adsorption of H(2)S at the particle surface can result in an increase in the H(2)S absorption rate.     

Virucidal Effect of Cold Atmospheric Gaseous Plasma on Feline Calicivirus,a Surrogate for Human Norovirus

Minimal food-processing methods are not effective against foodborne viruses, such as human norovirus (NV). It is important, therefore, to explore novel nonthermal technologies for decontamination of foods eaten fresh, minimally processed and ready-to-eat foods, and food contact surfaces. We studied the in vitro virucidal activity of cold atmospheric gaseous plasma (CGP) against feline calicivirus (FCV), a surrogate of NV. Factors affecting the virucidal activity of CGP (a so-called radio frequency atmospheric pressure plasma jet) were the plasma generation power, the exposure time and distance, the plasma feed gas mixture, and the virus suspension medium. Exposure to 2.5-W argon (Ar) plasma caused a 5.55 log₁₀ unit reduction in the FCV titer within 120 s. The reduction in the virus titer increased with increasing exposure time and decreasing exposure distance. Of the four plasma gas mixtures studied (Ar, Ar plus 1% O₂, Ar plus 1% dry air, and Ar plus 0.27% water), Ar plus 1% O₂ plasma treatment had the highest virucidal effect: more than 6.0 log₁₀ units of the virus after 15 s of exposure. The lowest virus reduction was observed with Ar plus 0.27% water plasma treatment (5 log₁₀ unit reduction after 120 s). The highest reduction in titer was observed when the virus was suspended in distilled water. Changes in temperature and pH and formation of H₂O₂ were not responsible for the virucidal effect of plasma. The oxidation of viral capsid proteins by plasma-produced reactive oxygen and nitrogen species in the solution was thought to be responsible for the virucidal effect. In conclusion, CGP exhibits virucidal activity in vitro and has the potential to combat viral contamination in foods and on food preparation surfaces.