Comparison of viable cell yield from excised versus aspirated adipose tissue

The correction of soft-tissue defects by adipose tissue transplantation often produces poor and unpredictable results. The implantation of isolated and cultured preadipocytes offers a solution to this problem since these cells differentiate into adipocytes when implanted in vivo. A field of major interest is to maximize the yield of preadipocytes isolated from adipose tissue showing only low contamination with other cell types. Aspiration and excision are two concurrent clinical ways of harvesting adipose tissue for the isolation of preadipocytes. This tissue is usually discarded after surgery. In this study, the yield of preadipocytes obtained from liposuction material was compared to that of excised adipose tissue. Furthermore, we determined the loss of precursor cells if isolation of preadipocytes was delayed for 24 h. Preadipocytes were isolated from the stromal cell fraction of human subcutaneous adipose tissue samples. Harvesting of adipose tissue by suction was performed according to the Coleman procedure (manually applied negative pressure using a 10-ml syringe with a blunt tip cannula). Isolation was either carried out within 60 min after extraction or after storage for 24 h in culture medium at 4 degrees C. Isolated preadipocytes were cultured for 24 h, trypsinized and counted in a Neubauer chamber. Our results show clearly that the yield of preadipocytes isolated from liposuction material (within 60 min after extraction and after 24 h of storage) is higher than the cell yield from excised adipose tissue. Overnight storage for 24 h leads to a significant loss of preadipocytes in excised tissue but not in liposuction material. The high yield of cells isolated from liposuction material proves that extraction by suction does not damage the stromal cell fraction in the adipose tissue. If cell isolation is not performed immediately after the operation, liposuction material is clearly the better alternative for storage.     

The “Mozart Effect”: An Electroencephalographic Analysis Employing the Methods of Induced Event-Related Desynchronization/Synchronization and Event-Related Coherence

The event-related responses of 18 individuals were recorded while they were listening to 3 music clips of 6 s duration which were repeated 30 times each. The music clips differed in the level of their complex structure, induced mood, musical tempo and prominent frequency. They were taken from Mozart's sonata (K. 448), and Brahms' Hungarian dance (no. 5). The third clip was a simplified version of the theme taken from Haydn's symphony (no. 94) played by a computer synthesizer. Significant differences in induced event-related desynchronization between the 3 music clips were only observed in the lower-1 alpha band which is related to attentional processes. A similar pattern was observed for the coherence measures. While respondents listened to the Mozart clip, coherence in the lower alpha bands increased more, whereas in the gamma band a less pronounced increase was observed as compared with the Brahms and Haydn clips. The clustering of the three clips based on EEG measures distinguished between the Mozart clip on the one hand, and the Haydn and Brahms clips on the other, even though the Haydn and Brahms clips were at the opposite extremes with regard to the mood they induced in listeners, musical tempo, and complexity of structure. This would suggest that Mozart's music--with no regard to the level of induced mood, musical tempo and complexity--influences the level of arousal. It seems that modulations in the frequency domain of Mozart's sonata have the greatest influence on the reported neurophysiological activity.     

Modulation of the responses of human osteoblast-like cells to physiologic mechanical strains by biomaterial surfaces

In a previous study we demonstrated that MG-63 cells cultured on Ti-6Al-4V discs covered by alumina ceramic and submitted to intermittent mechanical strain (IMS) presented morphological alteration associated with enhanced differentiation. Here we examine how the mechanical response of osteoblasts can be modulated by the nature of the substrate. MG-63 cells were cultured on four materials: polystyrene and Ti-6Al-4V (average roughness = 0.48 microm) as smooth substrates; Ti-6Al-4V (average roughness = 5.76 microm) and Ti-6Al-4V covered with alumina (average roughness = 5.21 microm) as rough substrates. Mechanical strains were applied for 15 min, three times a day for 1-5 days with a 600 microstrains magnitude and a 0.25 Hz frequency. IMS stimulated alkaline phosphatase activity by 25-35% on all substrates and had no effect on cell growth on either substrate. Fibronectin (FN) was chosen as representative of cell-matrix interaction. FN production was increased by 60% after 1 day of stretching only on alumina-coated discs. FN organization examined on smooth substrates was affected by 5 days of IMS, showing a thickening of the fibres. The same modifications induced by IMS were previously observed on alumina-covered discs. Vinculin expression was not affected by IMS whatever the substrate. Cell-cell interactions were determined by N-cadherin immunoblotting. N-cadherin expression was increased by IMS specifically on rough substrates. Our results suggest that the nature of the surface did not influence the up-regulation of alkaline phosphatase activity induced by IMS, but modulates specifically cell-substrate as well as cell-cell interactions in response to IMS.     

Fluoropyrimidine chemotherapy in a rat model: comparison of fluorouracil metabolite profiles determined by high-field 19F-NMR spectroscopy of tissues ex vivo with therapy response and toxicity for locoregional vs systemic infusion protocols

A Sprague-Dawley rat model with DS sarcoma transplanted in the thigh was used to compare transcatheter locoregional i.a. and systemic i.v. administration of 5-fluorouracil (FU) at 12 dose-rate schedules: 25, 50 and 100 mg/kg; bolus, 1, 5 and 24 h infusions. In experiment A tumor (62/67 animals) as well as liver and kidney (56/67 animals) were excised 1 h after a single bolus or 1 h infusion or at the end of 5 and 24 h infusions. (19)F-NMR spectroscopy at 11.7 T was used to quantitate FU and its metabolites in ca. 1 g of tissue at 4 degrees C. In experiment B analogous FU treatments were repeated for 5 days (rats 80+11 controls). Tumor volumes vs time, various blood parameters and survival times were recorded, and a log growth rate parameter log GR, a response index RI, and a toxicity index TI were calculated. The i.a. vs i.v. ratios for tumor concentrations of FU and total anabolites (F-Nucl) were >1 for nearly all treatments and increased with infusion time at the higher doses. F-Nucl in tumor correlated linearly with total fluorine concentration (Tot. F range 30-1100 nmol/g) over all treatments (r=0.92, slope=0.45, p<0.0001). For non-bolus i.v. treatments [FU+F-Nucl] decreased linearly with decreasing FU dose rate (r(2)=0.74, zero intercept), while i.a. treatments showed non-linear behavior. For non-bolus treatments the mean log GR per treatment group showed a negative correlation (r=-0.87) with log[F-Nucl]. The most effective non-toxic treatments were 25 mg/kg over 5 or 24 h; the i.a. route was superior to i.v. on the basis of [FU+F-Nucl], RI, the reduction in log GR, and Kaplan-Meier survival statistics. For liver and kidney Tot. F (>83% FU catabolites) reached ca. 3-4 and 6-7 micromol/g, respectively, at the highest dose rates for either route; F-Nucl were detected only for Tot. F>500 nmol/g and increased exponentially as Tot. F increased (toxic treatments). The concentrations of the main catabolite (alpha-fluoro-beta-alanine, FBAL) in tumor did not correlate with Tot. F but rather with FBAL levels in kidney (r=0.90, all treatments), indicating that uptake of liver-derived FBAL from the circulation is the major source of FBAL in tumor.     

Die Atemarbeit von Säuglingen bei Atmung durch Endotrachealkatheter

Zusammenfassung Wir bestimmten an sechs Säuglingen im Alter von 6 Monaten die zusätzliche Atemarbeit, die bei Atmung durch Loennecken-Katheter der Charrièrestufen 18, 16, 14 und durch einen neuen Katheter der Stufe 14 mit nur sehr kurzem stenotischem Anteil von den Säuglingen aufgewendet wird. Dabei stellten wir fest, daß im günstigsten Fall bei einem Katheter der Stufe 18 die Atemarbeit verdoppelt und im ungünstigsten Fall bei einem Katheter der Stufe 14 auf das 28fache erhöht wird. Der neue Katheter der Stufe 14 liegt in seiner Wirkung zwischen den Stufen 18 und 16 der Loennecken-Katheter.Ausgeführt mit Mitteln der Deutsehen Forschungsgemeinschaft und der Dr. Karl Wilder-Stiftung des Verbandes der Lebensversicherungsunternehmen e. V.Wir danken unserer technischen Assistentin Frau U.Loebell und Herrn cand. med. K. J.Grusz für ihre Mithilfe bei der Ausführung der Messungen und der Auswertung der Meßergebnisse.     

Formation of viscoelastic protein layers on polymeric surfaces relevant to platelet adhesion

The hemocompatibility of biomaterials is highly dependent on the adhesion and activation of platelets. Surface-adsorbed fibrinogen has a dominant role in promoting platelet adhesion to artificial surfaces by binding glycoprotein IIb-IIIa (GPIIb-IIIa), the major platelet membrane receptor. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we have investigated the material-dependent binding kinetics of purified GPIIb-IIIa to polymer-adsorbed fibrinogen. The following ranking of polymer-adsorbed mass (fibrinogen and GPIIb-IIIa) to test polymers could be established: poly[desaminotyrosyl-tyrosine ethyl (DTE) carbonate]/poly(lactide-co-glycolide)>poly[DTE co-5% poly(ethylene glycol) carbonate]. The QCM-D fibrinogen adsorption data were confirmed using an immunofluorescence assay. A synthetic RGD-containing peptide, but not a control peptide, inhibited GPIIb-IIIa binding to polymer-adsorbed fibrinogen, demonstrating the specificity of binding. Importantly, the binding efficiency of purified GPIIb-IIIa to polymer-adsorbed fibrinogen correlated with increased platelet adhesion in an in vitro model. Theoretical simulations using a Voight-based model provided quantitative data on the thickness and viscoelastic properties of the polymer-adsorbed protein layers. The precision of the modeling technique was limited with respect to the shear moduli values, leading to large variations. However, the other modeling parameters showed reproducible results. The thickness of both protein layers was polymer-dependent and ranged from 5 to 35 nm and the viscosity from 0.001 to 0.005 kg/ms, whereas the protein layer densities showed little differences between the test polymers. These results suggest that material-dependent changes in the thickness and viscoelastic properties of adsorbed fibrinogen-GPIIb-IIIa layers are crucial factors in the binding behavior of platelets to biomaterials.     

13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy

Chronic allograft nephropathy is characterized by chronic inflammation and fibrosis. Because retinoids exhibit anti-proliferative, anti-inflammatory, and anti-fibrotic functions, the effects of low and high doses of 13-cis-retinoic acid (13cRA) were studied in a chronic Fisher344-->Lewis transplantation model. In 13cRA animals, independent of dose (2 or 20 mg/kg body weight/day) and start (0 or 14 days after transplantation) of 13cRA administration, serum creatinine was significantly lower and chronic rejection damage was dramatically reduced, including subendothelial fibrosis of preglomerular vessels and chronic tubulointerstitial damage. The number of infiltrating mononuclear cells and their proliferative activity were significantly diminished. The mRNA expression of chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, IP-10/CXCL10, RANTES/CCL5) and proteins associated with fibrosis (plasminogen activator inhibitor-1, transforming growth factor-beta1, and collagens I and III) were strikingly lower in treated allografts. In vitro, activated peritoneal macrophages of 13cRA-treated rats showed a pronounced decrease in protein secretion of inflammatory cytokines (eg, tumor necrosis factor-alpha, interleukin-6). The suppression of the proinflammatory chemokine RANTES/CCL5 x 13cRA in fibroblasts could be mapped to a promoter module comprising IRF-1 and nuclear factor-kappaB binding elements, but direct binding of retinoid receptors to promoter elements could be excluded. In summary, 13cRA acted as a potent immunosuppressive and anti-fibrotic agent able to prevent and inhibit progression of chronic allograft nephropathy.     

Dipole Localization and Test-Retest Reliability of Frequency and Duration Mismatch Negativity Generator Processes

The mismatch negativity (MMN) is an event related potential component elicited by changes in duration, frequency or intensity of the stimuli during repetitive series of equal standard stimuli. In the present study we compared duration and frequency MMN using dipole source analysis concerning both the test-retest reliability of MMN-amplitudes and the locations of the potential sources. Furthermore, the influence of attention for test-retest-reliability was studied. Therefore, two groups of healthy subjects were investigated with different attentional manipulations. Twenty-one healthy subjects had to perform a visual attention task during the recording and 21 healthy subjects had no additional task to perform. All subjects were studied twice with a time interval of 3 weeks. Test-retest reliability was sufficiently high for the frequency but slightly lower for the duration MMN. The locations of the frequency and duration MMN-dipoles were in the auditory cortex with a more anterior and caudal location for the frequency MMN-dipoles. The latter finding supports the hypothesis that the frequency and duration MMNs have separate neuronal generators.