Alterations in Protein Expression of Arabidopsis thaliana Cell Cultures During Hyper- and Simulated Micro-Gravity

Callus cell cultures of Arabidopsis thaliana exposed to hypergravity (8×g), 2D clinorotation and random positioning exhibit changes in gene expression (Martzivanou et al., Protoplasma 229:155–162, 2006). In a recent investigation we could show that after 2 h of exposure also the protein complement shows treatment-related changes which are indicative for reactive oxygen species being involved in the perception of/response to changes in the gravitational field. In the present study we have extended these investigations for a period of up to 16 h of exposure. We report on changes in abundance of 28 proteins which have been identified by nano HPLC-ESI-MS/MS, and which were altered in amount after 2 h of treatment. According to changes between 2 and 16 h we could distinguish four groups of proteins which either declined, increased from down-regulated to control levels, showed a transient decline or a transient increase. With regard to function, our data indicate stress relief or adaptation to a new gravitational steady state under prolonged exposure. The latter assumption is supported by the appearance of a new set of 19 proteins which is changed in abundance after 8 h of hypergravity. A comparative analysis of the different treatments showed some similarities in response between 8×g centrifugation and 2D clinorotation, while random positioning showed the least responses.     

Actin Microfilament Organization in the Transition Zone of Arabidopsis-ABD2-GFP Roots Under Clinorotation

Seedlings of Arabidopsis thaliana-ABD2-GFP were grown under slow clinorotation (2?rpm) and treated with actin and tubulin disrupting drugs in order to characterize the role of actin microfilaments in cell growth and gravisensing. Changes in microfilament organization and cell parameters have shown that the transition root zone (TZ) is rather sensitive to microfilament disruption in control plants. It is assumed that under clinorotation, organization of actin cytoskeleton in the TZ is coordinated in a different way than in the control. Organization of microfilaments depends upon organization of microtubules and clinorotation does not influence this interrelation significantly.     

Spectroscopic study of poly(3-alkylthiophenes) electrochemically synthesized in different conditions

In this work, poly(3-alkylthiophenes) films were synthesized electrochemically in acetonitrile with LiClO₄ or Et₄NBF₄ at 5 or 18 °C. The films were deposited on platinum plates, and the polymer as-prepared was referred to as-oxidized. The as-reduced polymer was obtained by subjecting the as-oxidized product to ?1.70 V for 1 min. These various products were characterized by cyclic voltammetry, reflectance UV–Vis and Raman spectroscopy, and the results were used to estimate the ionization potential (I _P), electron affinity (E.A.) and energy gap (E _g) parameters. This study was possible because the Raman spectra demonstrated three structures (aromatic, radical cation and dication segments) which formed the P3MT and P3OT polymer chains.     

Determining early adhesion of cells on polysaccharides/PCL surfaces by a quartz crystal microbalance

The early adhesions of cells to various biopolymers are important to their growths and proliferations. Here, the adhesion of cells (e.g., fibroblasts) on the electrode of a quartz crystal microbalance (QCM) that was coated by PCL or PEG/PCL and further adsorbed by chitosan (CS) or CS/hyaluronic acid (HA) layers, was examined by cell-counting technique, QCM method and MTS assay under a serum-free condition for 3 h. The surfaces on electrodes of the QCM were confirmed to have been modified by measuring their contact angles, FT-IR spectra and the weights of biopolymers affected the frequency shifts of the QCM. Among tested surfaces on electrodes, the adhesion of fibroblasts on a HA/CS/PCL surface was the most (e.g., 3.08 × 10⁵ cells/cm²) while that on a PEG/PCL surface was the least (e.g., 0.7 × 10⁵ cells/cm²), as determined by cell-counting technique. The frequency shift and the mass of adhering fibroblasts on HA/CS/PCL electrodes were ?3,537 ± 770 Hz and 3.78 ± 0.22 μg (n = 3), respectively, that were significantly exceeded those on other electrodes (?393 ± 58 Hz and 0.32 ± 0.06 μg, n = 3, respectively, for PEG/PCL electrodes). These results were consistent with cell-counting technique. Although MTS assay yielded similar results, it was less sensitive than the two aforementioned methods. In conclusion, modified electrodes of a QCM provide a convenient and sensitive method for examining the early adhesion of cells (e.g., 3 h) to biopolymer surfaces.     

Comparison Study of Gravity-Dependent Displacement of Amyloplasts in Statocytes of Cress Roots and Hypocotyls

In this study, the kinetics of gravity-dependent movement of amyloplasts (statoliths) along root statocytes and hypocotyls (endodermis cells) has been analyzed and compared in order to testify cytoskeleton involvement in the displacement of statoliths in cress (Lepidium sativum L.) seedling statocytes. After 32 h of growth at 1 g or under a fast clinorotation (50 rpm), the seedlings were treated for 24 min as follows: exposition to clinorotation or 180° inversion and the action of gravitational force in root tip or hypocotyl tip direction. Statolith displacement was studied by light microscopy on semi-thin longitudinal sections of hypocotyls and root caps, measuring the distance between the centre of plastids and morphological cell bottom. Considerable temporal differences have been determined between the kinetics of the longitudinal motion of amyloplasts in root and hypocotyl statocytes of 1-g seedlings upon exposition to fast clinorotation and inversion. In statocytes of both organs of seedlings grown under fast clinorotation, the gravity provoked displacement of statoliths in the direction of its action; however, the displacement was much faster in hypocotyl than in root statocytes. It has been assumed that the gravity-determined longitudinal transport of amyloplasts, both in hypocotyl endodermic cells and root statocytes of cress seedlings, is modulated by the cytoskeleton.     

Physiological Responses of Synechocystis sp. PCC 6803 under Clinorotation

Photosystem efficiency and the characteristic on oxidative stress were examined to elucidate the metabolic responses of Synechocystis sp. PCC 6803 to short-term clinorotation. Results compiled when using clinostat to simulate microgravity for 60?h, showed that clinorotation clearly prohibited the photochemical quantum yield, but promoted the synthesis of chlorophyll and total protein. This may be a compensatory mechanism for the algal cell to maintain its normal metabolism. An increased malondialdehyde (MDA) content of algal cell upon clinorotation, together with an enhanced catalase (CAT) activity was observed during the whole period of clinorotation. One conclusion is that short-term clinorotation acts as a kind of stress, and that these physiological responses may be a special way for an algal cell to adapt itself to a different environment other than earth gravity.     

Clinorotation Affects the Ultrastructure of Pea Root Mitochondria

Effects of clinorotation on the mitochondrial ultrastructure in cells of meristem, distal and central elongation zones of 3- and 5-day-old etiolated roots of pea seedling roots were studied. It was shown that mitochondria in cells of examined root growth zones revealed a different sensitivity to clinorotation. The ultrastructure of mitochondria in the meristem and central elongation zone cells did not substantially change in comparison with stationary control. At the same time, changes in the mitochondrial ultrastructure in cells of the distal elongation zone under clinorotation were observed, namely: decrease in the size of mitochondria, as well as increase in both matrix electron density and crista volume. Such changes in the mitochondrial ultrastructure under clinorotation are supposed to display the rearrangements of energy metabolism in cells of the distal elongation zone in these conditions.     

Physicochemical properties and osteogenic activity of radiopaque calcium silicate–gelatin cements

The purpose of this study is to evaluate the physicochemical properties and in vitro osteogenic activity of radiopaque calcium silicate–gelatin cements. The radiopacity, setting time, working time, flow, diametral tensile strength, pH value, washout resistance and morphology of the cements with gelatin (0, 5 and 10 % by weight) were measured, which compared to a popular endodontic material, ProRoot white-colored mineral trioxide aggregate (WMTA). The cell morphology, cell attachment and proliferation, alkaline phosphatase and osteocalcin levels on the cements were measured by culturing the specimens with dental pulp cells. The results indicated that the presence of gelatin significantly (P < 0.05) reduced radiopacity and diametral tensile strength and prolonged setting time. Nevertheless, the 5 wt% gelatin cement had a radiopacity (5.1 mm of Al thickness) higher than ISO 6876:2001 standards (3 mm of Al thickness). The setting time (33 min), working time (9 min) and flow value (17.4 mm) of the 5 wt% gelatin cement were significantly (P < 0.05) better than those of WMTA (corresponding 165, 6 min and 14.2 mm). The fresh WMTA completely degraded after soaking in a physiological solution for 1 h, while the gelatin cements resisted washout, showing no noticeable breakdown even after 1 day of soaking. The gelatin cement enhanced the higher expression of cell attachment, proliferation and differentiation as compared to WMTA. It was concluded that the 5 wt% gelatin–calcium silicate hybrid cement appears to be promising as a radiopaque biomaterial for medical applications such as endodontics and vertebroplasty.     

Analysis of Statoliths Displacement in <Emphasis Type="Italic">Chara</Emphasis> Rhizoids for Validating the Microgravity-Simulation Quality of Clinorotation Modes

In single-celled rhizoids of the green algae Chara, positively gravitropic growth is governed by statoliths kept in a dynamically stable position 10–25 μ m above the cell tip by a complex interaction of gravity and actomyosin forces. Any deviation of the tube-like cells from the tip-downward orientation causes statoliths to sediment onto the gravisensitive subapical cell flank which initiates a gravitropic curvature response. Microgravity experiments have shown that abolishing the net tip-directed gravity force results in an actomyosin-mediated axial displacement of statoliths away from the cell tip. The present study was performed to critically assess the quality of microgravity simulation provided by different operational modes of a Random Positioning Machine (RPM) running with one axis (2D mode) or two axes (3D mode) and different rotational speeds (2D), speed ranges and directions (3D). The effects of 2D and 3D rotation were compared with data from experiments in real microgravity conditions (MAXUS sounding rocket missions). Rotational speeds in the range of 60–85 rpm in 2D and 3D modes resulted in a similar kinetics of statolith displacement as compared to real microgravity data, while slower clinorotation (2–11 rpm) caused a reduced axial displacement and a more dispersed arrangement of statoliths closer to the cell tip. Increasing the complexity of rotation by adding a second rotation axis in case of 3D clinorotation did not increase the quality of microgravity simulation, however, increased side effects such as the level of vibrations resulting in a more dispersed arrangement of statoliths. In conclusion, fast 2D clinorotation provides the most appropriate microgravity simulation for investigating the graviperception mechanism in Chara rhizoids, whereas slower clinorotation speeds and rotating samples around two axes do not improve the quality of microgravity simulation.     

Effects of two low-shrinkage composites on dental stem cells (viability, cell damaged or apoptosis and mesenchymal markers expression)

To investigate the effects of new two low-shrinkage composites SDR^® and Venus^®Bulk Fill on the cell viability, cellular damage and expression of mesenchymal markers on dental stem cells. Specimens from two low-shrinkage composites were eluted with culture medium for 24 h. After 24 h of incubation, cytotoxicity of elutes were evaluated by MTT assay; apoptosis was determined using the DNA-specific fluorochrome Hoechst 33342 and the mesenchymal stem cells markers expression was analyzed by immunofluorescence staining. After 24 h of cell exposure to each extract media, dental stem cells expressed MSCs markers. The interaction among the material and cell line was not significantly correlated [F(1,60) = 2.251, P = 0.39], whereas statistically significant differences among cells lines were observed [F(1,60) = 9.157, P = 0.004], being dental pulp stem cells more resistant that periodontal ligament stem cells. Also, we did not find any significant effect between the tested materials [F(1,60) = 0.090, P = 0.765]. Furthermore, a very low proportion of exposed cells showed condensed or fragmented nuclei, typical of apoptotic cells at 24 h. The results suggest that SDR^® and Venus^® Bulk fill and should be considered when selecting an appropriate resin-based dental restorative material.     

Spheroid model study comparing the biocompatibility of Biodentine and MTA

The primary objective of this study was to assess the biological effects of a new dentine substitute based on Ca₃SiO₅ (Biodentine?) for use in pulp-capping treatment, on pseudo-odontoblastic (MDPC-23) and pulp (Od-21) cells. The secondary objective was to evaluate the effects of Biodentine and mineral trioxide aggregate (MTA) on gene expression in cultured spheroids. We used the acid phosphatase assay to compare the biocompatibility of Biodentine and MTA. Cell differentiation was investigated by RT-qPCR. We investigated the expression of genes involved in odontogenic differentiation (Runx2), matrix secretion (Col1a1, Spp1) and mineralisation (Alp). ANOVA and PLSD tests were used for data analysis. MDPC-23 cells cultured in the presence of MTA had higher levels of viability than those cultured in the presence of Biodentine and control cells on day 7 (P = 0.0065 and P = 0.0126, respectively). For Od-21 cells, proliferation rates on day 7 were significantly lower in the presence of Biodentine or MTA than for control (P < 0.0001). Col1a1 expression levels were slightly lower in cells cultured in the presence of MTA than in those cultured in the presence of Biodentine and in control cells. Biodentine and MTA may modify the proliferation of pulp cell lines. Their effects may fluctuate over time, depending on the cell line considered. The observed similarity between Biodentine and MTA validates the indication for direct pulp-capping claimed by the manufacturers.     

Cryostat for Fixed-Point Calibration of Capsule-Type SPRTs

A cryostat for fixed-point calibration of capsule-type SPRTs (standard platinum resistance thermometers) was developed. Using this system, cryogenic fixed points defined on the International Temperature Scale of 1990 (ITS-90) were realized. The cryogenic cells were argon, oxygen, neon, and two equilibrium-hydrogen (e-H₂) cells, made by INRiM, Italy. The uncertainty of the realization of each fixed point was estimated to range from 0.53 mK to 0.43 mK (k = 2). The realizations of the triple point of e-H₂ using two sealed cells coincided within 0.1 mK. Therefore, we are able to calibrate capsule-type SPRTs down to 24.5561 K within an uncertainty of 1 mK (k = 2) by this system. A closed-cycle helium gas refrigerator was used for the cryostat. Each sealed cell was designed so that it could accommodate three sealed cells in the thermometer wells made within the cell. Therefore, the cryostat was designed to accommodate only one sealed cell at a time. The base temperature of this liquid-free cryostat, when one sealed cell and three capsule-type SPRTs were attached for calibration, was ~17 K. For the realization of the triple point of e-H₂, we used liquid helium for additional cooling. Adiabatic melting of the triple point was realized by controlling the inner-most radiation shield at a temperature very close to that of the triple point, and by applying a heat pulse by a heater directly wound to the cell. The amount of the heater power and the waiting time for the thermal equilibrium after each heat pulse were chosen in a way that the adiabatic melting could be finished within ~6 h for each cell. The triple point of each cryogenic fixed point was deduced from the equilibrium temperatures between the heat pulses and subsequent extrapolation to the liquidus point. For the oxygen cell, temperatures of two solid?Csolid transitions (???C?? and ???C?? transitions) were also measured, and the results were consistent with values reported in the literature within the designated uncertainty.     

Axially aligned electrically conducting biodegradable nanofibers for neural regeneration

In this study, electrically conducting axially aligned nanofibers have developed to provide both electrical and structural cues. Poly(lactide-co-glycolide) (PLGA) with poly(3-hexylthiophene) (PHT) was electrospun into 2D random (196 ± 98 nm) and 3D axially aligned nanofibers (200 ± 80 nm). Electrospun random and aligned PLGA–PHT fibers were characterized for surface morphology, mechanical property, porosity, degradability, and electrical conductivity. The pore size of random PLGA–PHT nanofibers (6.0 ± 3.3 μm) were significantly higher than the aligned (1.9 ± 0.4 μm) (P < 0.05) and the Young’s modulus of aligned scaffold was significantly lower than the random. Aligned nanofibers showed significantly lesser degradation rate and higher electrical conductivity (0.1 × 10^?5 S/cm) than random nanofibers (P < 0.05). Results of in vitro cell studies indicate that aligned PLGA–PHT nanofibers have a significant influence on the adhesion and proliferation of Schwann cells and could be potentially used as scaffold for neural regeneration.     

An exploratory study on the efficacy of rat dedifferentiated fat cells (rDFATs) with a poly lactic-co-glycolic acid/hydroxylapatite (PLGA/HA) composite for bone formation in a rat calvarial defect model

In the last two decades, tissue-engineering approaches using scaffolds, growth factors, and cells, or their combination, have been developed for the regeneration of periodontal tissue and bone. The aim of this study was to examine the effects of rat dedifferentiated fat cells (rDFATs) with a poly lactic-co-glycolic acid/hydroxylapatite (PLGA/HA) composite on bone formation in rat calvarial defects. Twenty animals surgically received two calvarial defects (diameter, 5 mm) bilaterally in each parietal bone. The defects were treated by one of the following procedures: PLGA/HA+osteo-differentiated rDFATs implantation (PLGA/HA+rDFATs (OD)); PLGA/HA+rDFATs implantation (PLGA/HA+rDFATs); PLGA/HA implantation (PLGA/HA); no implantation as a control. The animals were euthanized at 8 weeks after the surgery for histological evaluation. The PLGA/HA composite was remarkably resorbed and the amounts of residual PLGA/HA were very slight at 8 weeks after the surgery. The PLGA/HA-implanted groups (PLGA/HA+rDFATs (OD), PLGA/HA+rDFATs and PLGA/HA) showed recovery of the original volume and contour of the defects. The newly formed bone area was significantly larger in the PLGA/HA group (42.10 ± 9.16 %) compared with the PLGA/HA+rDFATs (21.35 ± 13.49 %) and control (22.17 ± 13.08 %) groups (P < 0.05). The percentage of defect closure (DC) by new bone in the PLGA/HA+rDFATs (OD) group (83.16 ± 13.87 %) was significantly greater than that in the control group (40.61 ± 29.62 %) (P < 0.05). Furthermore, the PLGA/HA+rDFATs (OD) group showed the highest level of DC among all the groups. The present results suggest that the PLGA/HA composite is a promising scaffold and that PLGA/HA+DFATs (OD) may be effective for bone formation.     

Influence of calcium and phosphorus release from bioactive glasses on viability and differentiation of dental pulp stem cells

The release of ions that can significantly contribute toward cellular response is an important characteristic of bioactive glasses (BG). Here, ionic extracts of three different compositions of BG powders in 60 mol% SiO₂, x mol% CaO (x = 28, 32 and 36), x mol% P₂O₅ (x = 12, 8 and 4) compositional system were utilized to study their effect on the viability, differentiation and mineralization of dental pulp stem cells (DPSCs) in vitro. ICP was applied to detect the exact ionic concentrations released from different composition of BG. DPSCs treated with conditioned media from the glass with 4 mol% P₂O₅ (BGCM1, media containing 44.01 ± 0.6 mg/L Si, 61.72 ± 0.1 mg/L Ca and 7.57 ± 0.01 mg/L P) were more metabolically active compared to conditioned media from the glass with 8 mol% P₂O₅ (BGCM2, media with 47.36 ± 0.7 mg/L Si, 57.4 ± 0.1 mg/L Ca and 14.54 ± 0.2 mg/L P), at all times tested, but in all cases the process was slower than the control. Cells exposed to media conditioned by the glass with 12 mol% P₂O₅ (BGCM3, 40.46 ± 0.5 mg/L Si, 61. 85 ± 0.3 mg/L Ca and 28.43 ± 0.3 mg/L P) responded differently, such that cells showed to be more metabolically active than control at day 3, but then similar to or lower than control at higher time points. Differentiation of DPSCs toward osteogenic lineage in the presence of BGCM was assessed by Alizarin red staining. Cells treated with high phosphate BGCM3 displayed a higher density of red mineralized nodules than cells treated with BGCM1 and BGCM2 after 21 days of culture in non-osteogenic medium. BGCM3 was therefore chosen for gene expression studies. Osteogenic differentiation of DPSCs in the presence and/or absence of BGCM3 or osteogenic supplements were studied by RT-PCR. Overall, the results demonstrated that, in the absence of osteogenic supplements, BGCM3 group showed a significantly higher mRNA expression levels for alkaline phosphatase at day 7, osteopontin and osteonectin at days 7 and 14, and a high level of collagen I at day 14, compared to negative control group (BM?). Overall, the results obtained from BGCM3 group are beneficial for the design and manufacture of scaffolds or particulates with tailored ion release for a range of bone repair applications.     

Immobilized-OPG-Fc on a titanium surface inhibits RANKL-dependent osteoclast differentiation in vitro

The purpose of the present study was to examine the effect of osteoprotegerin (OPG)-Fc fusion protein immobilized on a titanium surface on the initial differentiation of osteoclast precursor RAW264.7 cells. These cells were cultured on titanium specimens over which OPG-Fc was immobilized. The enhancement of tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expression in RAW264.7 cells exposed to receptor activator of NF-κB ligand (RANKL) stimulation on OPG-Fc-coated titanium was significantly lower than that in RAW264.7 cells exposed to RANKL on titanium specimens without immobilized OPG-Fc (ANOVA, P < 0.01). Preincubation of OPG-Fc-coated titanium, in a medium supplemented with 10% fetal bovine serum at 37°C for two days before the cells were seeded, had no significant effect on the decrease in mRNA expression (ANOVA, P < 0.01). Taken together, these results indicate that OPG-Fc immobilized on a titanium surface blocks the differentiation of RAW264.7 cells induced by RANKL stimulation.     

Electrical properties of LiNi3/5Fe1/5Cu1/5VO4 ceramics

In this work, LiNi_3/5Fe_1/5Cu_1/5VO₄ ceramic was prepared by solution-based chemical route whose X-ray diffraction results reveal an orthorhombic unit cell structure with lattice parameters of a = 9.6329 (23) Å, b = 6.8527 (23) Å, c = 5.4667 (23) Å. Complex electrical impedance and electrical conductivity were studied using complex impedance spectroscopy technique. The analysis of complex electrical impedance shows: (i) grain interior, grain boundary and electrode-material interface contributions to electrical response and (ii) the presence of temperature dependent electrical relaxation phenomena in the material. A detailed study of electrical conductivity indicates that electrical conduction in the material is a thermally activated process.