Human cells with osteogenic potential in bone tissue research.

Bone regeneration after injury or after surgical bone removal due to disease is a serious medical challenge. A variety of materials are being tested to replace a missing bone or tooth. Regeneration requires cells capable of proliferation and differentiation in bone tissue. Although there are many possible human cell types available for use as a model for each phase of this process, no cell type is ideal for each phase. Osteosarcoma cells are preferred for initial adhesion assays due to their easy cultivation and fast proliferation, but they are not suitable for subsequent differentiation testing due to their cancer origin and genetic differences from normal bone tissue. Mesenchymal stem cells are more suitable for biocompatibility testing, because they mimic natural conditions in healthy bone, but they proliferate more slowly, soon undergo senescence, and some subpopulations may exhibit weak osteodifferentiation. Primary human osteoblasts provide relevant results in evaluating the effect of biomaterials on cellular activity; however, their resources are limited for the same reasons, like for mesenchymal stem cells. This review article provides an overview of cell models for biocompatibility testing of materials used in bone tissue research.

Enzymatically Cross-linked Hydrogels Based on Synthetic Poly(α-amino acid)s Functionalized with RGD Peptide for 3D Mesenchymal Stem Cell Culture.

Injectable hydrogel scaffolds combined with stem cell therapy represent a promising approach for minimally invasive surgical tissue repair. In this study, we developed and characterized a fully synthetic, biodegradable poly(N5-(2-hydroxyethyl)-l-glutamine)-based injectable hydrogel modified with integrin-binding arginine-glycine-aspartic acid (RGD) peptide (PHEG-Tyr-RGD). The biodegradable hydroxyphenyl polymer precursor derivative of PHEG-Tyr was enzymatically cross-linked to obtain injectable hydrogels with different physicochemical properties. The gelation time, gel yield, swelling behavior, and storage modulus of the PHEG-Tyr hydrogels were tuned by varying the concentrations of the PHEG-Tyr precursors and horseradish peroxidase as well as the nH2O2/nTyr ratio. The mechanical properties and gelation time of the PHEG-Tyr hydrogel were optimized for the encapsulation of rat mesenchymal stem cells (rMSCs). We focused on the 2D and 3D spreading and viability of rMSCs within the PHEG-Tyr-RGD hydrogels with different physicochemical microenvironments in vitro. Encapsulation of rMSCs shows long-term survival and exhibits cell-matrix and cell-cell interactions reflective of both the RGD concentration and hydrogel stiffness. The presented biomaterial represents a suitable biological microenvironment to guide 3D spreading and may act as a promising 3D artificial extracellular matrix for stem cell therapy.

In Situ Hydroxyapatite Synthesis Enhances Biocompatibility of PVA/HA Hydrogels.

Bone tissue engineering tries to simulate natural behavior of hard tissues. This study aimed to produce scaffolds based on polyvinyl alcohol (PVA) and hyaluronic acid (HA) with hydroxyapatite (HAp) incorporated in two different ways, by in situ synthesis and physical mixing of pre-prepared HAp. In situ synthesis resulted in calcium deficient form of HAp with lower crystallinity. The proliferation of human osteoblast-like cells MG-63 proved to be better in the scaffolds with in situ synthesized HAp compared to those with physically mixed pre-prepared HAp. For scaffolds with PVA/HA/HAp ratio 3:1:2, there was significantly higher initial adhesion (p = 0.0440), as well as the proliferation in the following days (p < 0.001). It seemed to be advantageous improve the properties of the scaffold by in situ synthesizing of HAp directly in the organic matrix.

Biological Evaluation of Polyvinyl Alcohol Hydrogels Enriched by Hyaluronic Acid and Hydroxyapatite.

This study aimed to develop polyvinyl alcohol (PVA) -based scaffold enriched with hyaluronic acid (HA) and hydroxyapatite (HAp) using physical crosslinking by freezing-thawing method. We accomplished biological evaluation of scaffolds, swelling degree, bioactivity assessment, and hemolytic test. The results showed that all types of scaffolds should be safe for use in the human body. The culturing of human osteoblast-like cells MG-63 and their proliferation showed better adhesion of cells due to the presence of HA and confirmed better proliferation depending on the amount of HAp. This paper gives the optimal composition of the scaffold and the optimal amount of the particular components of the scaffold. Based on our results we concluded that the best PVA/HA/HAp combination is in the ratio 3:1:2.

Effect of n-3 fatty acids supplementation during life style modification in women with overweight.

OBJECTIVE: The marine n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert numerous beneficial effects on health, but their potency to defend against development of peripheral insulin resistance of healthy person with overweight remains poorly characterized. We aimed to evaluate the effect of a combination intervention using EPA + DHA and the lifestyle modification (LSM) in women with overweight. METHOD: In a parallel-group, three-arm, randomized trial (UMIN Clinical Trials Registry - R000031131), 34 women were assigned to a 12-week-intervention using corn oil (1.5 g/day; placebo); LSM and corn oil (1.5 g/day; LSM); or LSM and EPA + DHA concentrate (1.5 g/day, containing ~ 0.6 g EPA + DHA; LSM & n-3). At baseline and after intervention, anthropometric measurements including bioelectrical impedance analysis, spiroergometry, 24-hours dietary recall, and various metabolic markers, adiponectin and cytokines were evaluated in serum using standard procedures. Data from 29 women were used for the final evaluation. Wilcoxon two-sided rank-sum test was used to inspect the differences between LSM and LSM & n-3, and placebo groups, with a p-value of ≤ 0.05. All computations were performed with MATLAB Statistics Toolbox. RESULTS: In comparison with placebo, LSM and LSM & n-3 decreased body weight, waist circumference, and body fat, and increased VO2max/kg. LSM & n-3 increased adiponectin levels in comparison to LSM. Fasting insulin, IL8, and cholesterol were decreased by LSM, but were unchanged by LSM & n-3. IL6 was not affected in LSM & n-3, while it was increased in LSM. Other inflammatory markers, as well as leptin, LIF, follistatin, BDNF, and fasting triacylglycerol were not significantly affected by any of the interventions. CONCLUSION: Besides preventing a modest negative effect of LSM on IL6 and adiponectin level, the combination of LSM and EPA + DHA supplementation could be probably used to improve the functional capacity of adipose tissue in women with overweight.

The origin and development of fat droplet and its role in health and illness.

Lipid droplets are found in most cells, where they are needed for lipid metabolism and energy homeostasis. Their biogenesis is still not entirely described process, in which an organelle with monomolecular layer on the surface originates from two-layer membrane of the smooth endoplasmic reticulum. Large lipid droplets can be formed either by growth or by fusion of smaller lipid droplets. Their basic functions are controlled lipid deposition and lipid metabolism and the protection of the intracellular environment from lipotoxicity. Currently, dysfunction of lipid droplet is associated with a number of diseases such as obesity, cardiometabolic syndrome, tumor and neurodegenerative diseases, lipodystrophy and cachexia. Keywords: lipid droplet, adipocyte, endoplasmic reticulum, perilipin, obesity, type 2 diabetes mellitus, lipodystroph.

PERSISTENT ORGANOCHLORINE POLLUTANTS IN OBESE WOMEN AFTER DIET INDUCED WEIGHT LOSS: FIVE YEARS FOLLOW UP STUDY.

AIM: Persistent organochlorine pollutants (POPs) from the environment are still bioaccumulating in human tissues. The aim of our study was to analyze the development of plasma POPs levels in obese women in relationship with their weight loss success in five year follow-up study. METHODS: 20 obese women aged 25-73 years were studied just before and after having completed a 3 month controlled low calorie diet (LCD) intervention (5 MJ daily), and again after 6 and 60 months since the beginning of the study. Body weight and plasma levels of 7 POPs were measured: polychlorinated biphenyls (PCB) 153, 138, 180; 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethylene (p,p'-DDE); 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (p,p'-DDT), hexachlorocyclobenzene (HCB), hexachlorocyclohexane ß (HCH ß). RESULTS: Data shows that after 3 months of a completely controlled restrictive diet regimen, the weight loss was associated with an increase in POP plasma levels. However, after a five year follow-up, there were no differences in POPs plasma levels between those who kept losing weight or maintained the initial weight (WL/M) and the group of weight gainers (WG), except for HCB where the WG had a significantly higher level (p < 0.05). CONCLUSIONS: These results suggest that contrary to the long-term weight gain, the long-term weight loss or weight maintenance caused by diet restriction is associated with lower plasma levels of HCB.

Dramatic decrease in muscular fitness in the Czech schoolchildren over the Last 20 years.

AIM: Obesity and physical inactivity had already reached epidemic, becoming one of the leading causes of death and disability worldwide. The objective of this study was to investigate the current level and a nearly three decades' trend of muscular fitness (MF) and the nutritional state of Czech children. METHODS: In 2013, cross-sectional epidemiological survey was conducted in schoolchildren with examination of the anthropometric characteristics and measured MF using a UNIFIT test, which normative for MF categories was given as results of national monitoring data collected in 1987. In total 896 current schoolchildren (472 aged 8-9 years and 424 aged 12-13 years) were examined from selected children through stratified sampling from the Czech Republic. RESULTS: Against 31% of children from 1986 there were 74% of current children classified as "poor" or "below normal" in the MF category (p<0.001). MF was inversely associated with BMI. Poor posture was diagnosed in 24% of children, more frequently in overweight (OW) and obese (OB) children in comparison to normal body weight children (p<0.001). In comparison to 10% of OW and OB children in 1991, using these cut-off points for body mass index references, the current prevalence of OW and OB doubled (19.8%). CONCLUSIONS: Current Czech schoolchildren showed a doubled prevalence of OW and OB during the last two decades and simultaneously during nearly three decades there were more than doubled prevalence of "poor" or "under normal" MF of children, with overall dramatic decrease of MF in current schoolchildren.

Fully synthetic, tunable poly(α-amino acids) as the base of bioinks curable by visible light.

Bioinks play a crucial role in tissue engineering, influencing mechanical and chemical properties of the printed scaffold as well as the behavior of encapsulated cells. Recently, there has been a shift from animal origin materials to their synthetic alternatives. In this context, we present here bioinks based on fully synthetic and biodegradable poly(α,L-amino acids) (PolyAA) as an alternative to animal-based gelatin methacrylate (Gel-Ma) bioinks. Additionally, we first reported the possibility of the visible light photoinitiated incorporation of the bifunctional cell adhesive RGD peptide into the PolyAA hydrogel matrix. The obtained hydrogels are shown to be cytocompatible, and their mechanical properties closely resemble those of gelatin methacrylate-based scaffolds. Moreover, combining the unique properties of PolyAA-based bioinks, the photocrosslinking strategy, and the use of droplet-based printing allows the printing of constructs with high shape fidelity and structural integrity from low-viscosity bioinks without using any sacrificial components. Overall, presented PolyAA-based materials are a promising and versatile toolbox that extends the range of bioinks for droplet bioprinting.

Soft Hydrogels with Double Porosity Modified with RGDS for Tissue Engineering.

This study develops and characterizes novel biodegradable soft hydrogels with dual porosity based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers cross-linked by hydrolytically degradable linkers. The structure and properties of the hydrogels are designed as scaffolds for tissue engineering and they are tested in vitro with model mesenchymal stem cells (rMSCs). Detailed morphological characterization confirms dual porosity suitable for cell growth and nutrient transport. The dual porosity of hydrogels slightly improves rMSCs proliferation compared to the hydrogel with uniform pores. In addition, the laminin coating supports the adhesion of rMSCs to the hydrogel surface. However, hydrogels modified by heptapeptide RGDSGGY significantly stimulate cell adhesion and growth. Moreover, the RGDS-modified hydrogels also affect the topology of proliferating rMSCs, ranging from single-cell to multicellular clusters. The 3D reconstruction of the hydrogels with cells obtained by laser scanning confocal microscopy (LSCM) confirms cell penetration into the inner structure of the hydrogel and its corresponding microstructure. The prepared biodegradable oligopeptide-modified hydrogels with dual porosity are suitable candidates for further in vivo evaluation in soft tissue regeneration.

Optimizing PCL/PLGA Scaffold Biocompatibility Using Gelatin from Bovine, Porcine, and Fish Origin.

This research introduces a novel approach by incorporating various types of gelatins, including bovine, porcine, and fish skin, into polycaprolactone and poly (lactic-co-glycolic acid) using a solvent casting method. The films are evaluated for morphology, mechanical properties, thermal stability, biodegradability, hemocompatibility, cell adhesion, proliferation, and cytotoxicity. The results show that the incorporation of gelatins into the films alters their mechanical properties, with a decrease in tensile strength but an increase in elongation at break. This indicates that the films become more flexible with the addition of gelatin. Gelatin incorporation has a limited effect on the thermal stability of the films. The composites with the gelatin show higher biodegradability with the highest weight loss in the case of fish gelatin. The films exhibit high hemocompatibility with minimal hemolysis observed. The gelatin has a dynamic effect on cell behavior and promotes long-term cell proliferation. In addition, all composite films reveal exceptionally low levels of cytotoxicity. The combination of the evaluated parameters shows the appropriate level of biocompatibility for gelatin-based samples. These findings provide valuable insights for future studies involving gelatin incorporation in tissue engineering applications.

Discrepant results of adrenal venous sampling in seven patients with primary aldosteronism.

BACKGROUND: Adrenal venous sampling serves as a discrimination between uni- and bilateral forms of primary aldosteronism (PA). Even correctly performed adrenal venous sampling may lead to non-diagnostic results in some cases. RESULTS: We describe 7 subjects with PA in whom correct cannulation of adrenal veins (high selectivity index defined as cortisol((adrenal))/cortisol((periphery)) ratio) was associated with aldosterone (ALDO) suppression (ALDO/cortisol((adrenal))/ALDO/cortisol((periphery)) ratio <1) in the left adrenal gland and in whom all subjects underwent a successful adrenalectomy on the right side. In 3 subjects, samples from the right side with lower selectivity indexes (1.11-1.7) compared to those samples with a higher index of selectivity (10.4-44.9) pointed to lateralization. Next, 2 subjects were operated because of relatively large adrenal masses in the right adrenal gland on CT despite ALDO suppression on this side. One subject presented with high selectivity indexes from the right side (19.5 and 37.6), but only one sample showed ALDO secretion. Patient 7 was treated with right-sided adrenalectomy despite a low lateralization index (ALDO/cortisol((right))/ALDO/cortisol((left)) 1.78). CONCLUSIONS: Our results document some uncertainties in interpreting results of adrenal venous sampling in subjects with PA which may result from deep catheter insertion, anomalous venous drainage, or fluctuations in ALDO secretion.

Stimuli-responsive polypeptide nanogels for trypsin inhibition.

A new type of hydrophilic, biocompatible, and biodegradable polypeptide nanogel depots loaded with the natural serine protease inhibitor α1-antitrypsin (AAT) was applied for the inhibition of the inflammatory mediator trypsin. Two types of nanogels were prepared from linear synthetic polypeptides based on biocompatible and biodegradable poly[N 5-(2-hydroxyethyl)-ʟ-glutamine-ran-N 5-propargyl-ʟ-glutamine-ran-N 5-(6-aminohexyl)-ʟ-glutamine]-ran-N 5-[2-(4-hydroxyphenyl)ethyl)-ʟ-glutamine] (PHEG-Tyr) or biocompatible N α-ʟ-lysine-grafted α,ß-poly[(2-propyne)-ᴅ,ʟ-aspartamide-ran-(2-hydroxyethyl)-ᴅʟ-aspartamide-ran-(2-(4-hydroxyphenyl)ethyl)-ᴅʟ-aspartamide] (N α-Lys-NG). Both nanogels were prepared by HRP/H2O2-mediated crosslinking in inverse miniemulsions with pH and temperature-stimuli responsive behavior confirmed by dynamic light scattering and zeta potential measurements. The loading capacity of PHEG-Tyr and N α-Lys-NG nanogels and their release profiles were first optimized with bovine serum albumin. The nanogels were then used for loading and release of AAT. PHEG-Tyr and N α-Lys-NG nanogels showed different loading capacities for AAT with the maximum (20%) achieved with N α-Lys-NG nanogel. In both cases, the nanogel depots demonstrated a burst release of AAT during the first 6 h, which could be favorable for quick inhibition of trypsin. A consequent pilot in vitro inhibition study revealed that both PHEG-Tyr and N α-Lys-NG nanogels loaded with AAT successfully inhibited the enzymatic activity of trypsin. Furthermore, the inhibitory efficiency of the AAT-loaded nanogels was higher than that of only AAT. Interestingly, also non-loaded PHEG-Tyr and N α-Lys-NG nanogels were shown to effectively inhibit trypsin because they contain suitable amino acids in their structures that effectively block the active site of trypsin.

Influence of 3D Printing Topology by DMLS Method on Crack Propagation.

The presented text deals with research into the influence of the printing layers' orientation on crack propagation in an AlSi10Mg material specimen, produced by additive technology, using the Direct Metal Laser Sintering (DMLS) method. It is a method based on sintering and melting layers of powder material using a laser beam. The material specimen is presented as a Compact Tension test specimen and is printed in four different defined orientations (topology) of the printing layers-0°, 45°, 90°, and twice 90°. The normalized specimen is loaded cyclically, where the crack length is measured and recorded, and at the same time, the crack growth rate is determined. The evaluation of the experiment shows an apparent influence of the topology, which is essential especially for possible use in the design and technical preparation of the production of real machine parts in industrial practice. Simultaneously with the measurement results, other influencing factors are listed, especially product postprocessing and the measurement method used. The hypothesis of crack propagation using Computer Aided Engineering/Finite Element Method (CAE/FEM) simulation is also stated here based on the achieved results.

Biocompatible polypeptide nanogel: Effect of surfactants on nanogelation in inverse miniemulsion, in vivo biodistribution and blood clearance evaluation.

Horseradish peroxidase (HRP)/H2O2-mediated crosslinking of polypeptides in inverse miniemulsion is a promising approach for the development of next-generation biocompatible and biodegradable nanogels. Herein, we present a fundamental investigation of the effects of three surfactants and their different concentrations on the (HRP)/H2O2-mediated nanogelation of poly[N5-(2-hydroxyethyl)-l-glutamine-ran-N5-propargyl-l-glutamine-ran-N5-(6-aminohexyl)-l-glutamine]-ran-N5-[2-(4-hydroxyphenyl)ethyl)-l-glutamine] (PHEG-Tyr) in inverse miniemulsion. The surfactants sorbitan monooleate (SPAN 80), polyoxyethylenesorbitan trioleate (TWEEN 85), and dioctyl sulfosuccinate sodium salt (AOT) were selected and their influence on the nanogel size, size distribution, and morphology was evaluated. The most effective nanogelation stabilization was achieved with 20 wt% nonionic surfactant SPAN 80. The diameter of the hydrogel nanoparticles was 230 nm (dynamic light scattering, DLS) and was confirmed also by nanoparticle tracking analysis (NTA) which showed the diameters ranging from 200 to 300 nm. Microscopy and image analyses showed that the nanogel in the dry state was spherical in shape and had number-average diameter Dn = 26 nm and dispersity Р= 1.91. In the frozen-hydrated state, the nanogel appeared porous and was larger in size with Dn = 182 nm and Р= 1.52. Our results indicated that the nanogelation of the polymer precursor required a higher concentration of surfactant than classical inverse miniemulsion polymerization to ensure effective stabilization. The developed polypeptide nanogel was radiolabeled with 125I, and in vivo biodistribution and blood clearance evaluations were performed. We found that the 125I-labeled nanogel was well-biodistributed in the bloodstream, cleared from mouse blood during 48 h by renal and hepatic pathways and did not provoke any sign of toxic effects.

Efficacy of repetitive transcranial magnetic stimulation for the treatment of refractory chronic tinnitus: a randomized, placebo controlled study.

OBJECTIVE: The pathophysiologic mechanisms of idiopathic tinnitus remain unclear. Low frequency rTMS applied over the auditory cortex has been proposed as a new and causally oriented treatment approach for pathological conditions with abnormal, increased cortical activity including tinnitus with increased activity in the auditory cortex. However available studies are characterized by a positive reports on the therapeutic effects of repetitive transcranial magnetic stimulation (rTMS) for treatment of tinnitus, there are few details about the duration of specific treatment effects. DESIGN: The design of the study was randomized, prospective, placebo-controlled. Right-handed patients were treated with either real or sham 1 Hz frequency rTMS over a period of two weeks. Fifty-two patients with chronic, treatment resistant tinnitus and stable medication were enrolled in the study after giving written informed consent and forty-two patients completed the study and were included in data analysis. RESULTS: The ability to reduce the symptoms of tinnitus appeared in both randomized groups immediately after the 1 Hz rTMS and sham stimulation phase. There was a significant reduction in both groups of the tinnitus total score on the Tinnitus Handicap Inventory (THI) (real rTMS p=0.005; sham rTMS p=0.049) and Tinnitus Questionnaire (TQ) total score (real rTMS p=0.003; sham rTMS p=0.049). On the THI evaluation scale, in the real rTMS a mild worsening was noted during week 6 in comparison with the state attained in week 2. During the subsequent course of the study a significant reduction of the total score persisted in the case of THI (real rTMS week 14 p=0.033 and borderline week 26 p=0.058). The reduction of symptoms as evaluated using the TQ was significant compared to baseline in the real rTMS group at week 2, 6 and 14 (p=0.003; p=0.024; p=0.022). The group treated with sham stimulation reached significant reduction of symptoms only at week 2 (p=0.049). A comparison of the difference in the recorded values of the total score during follow-up in relation to baseline expressed as a percentage demonstrates the difference in the effect of rTMS and sham stimulation as evaluated by both the basic scales. Graphical analysis of mean patterns of treatment response according to stimulation type shows a similarity between treatment response patterns evaluated by reduction of the total scores using THI and TQ. CONCLUSIONS: The principal finding of this study is that real 1 Hz rTMS treatment was capable of significantly reducing the total baseline score of basic scales that measure tinnitus severity. This result is important as it proves that significant reduction of symptoms can be achieved even in a group of patients with long-term symptoms resistant to pharmacological treatment.

Experimental Measurements of Mechanical Properties of PUR Foam Used for Testing Medical Devices and Instruments Depending on Temperature, Density and Strain Rate.

Rigid polyurethane (PUR) foam is products used as a biomedical material for medical device testing. Thermal stability is a very important parameter for evaluating the feasibility of use for testing surgical instrument load during drilling. This work aimed to perform experimental measurements to determine the dependence of the mechanical properties of a certified PUR on temperature, strain rate and density. Experimental measurements were realised for three types of the PUR samples with different density 10, 25 and 40 pounds per cubic foot. The samples were characterised in terms of their mechanical properties evaluated from tensile and compression tests at temperatures of 25 °C, 90 °C and 155 °C. Furthermore, the structures of the samples were characterised using optical microscope, their thermal properties were characterised by thermogravimetric analysis, and their density and stiffness with the effect of temperature was monitored. The results show that it is optimal not only for mechanical testing but also for testing surgical instruments that generate heat during machining. On the basis of experimental measurements and evaluations of the obtained values, the tested materials are suitable for mechanical testing of medical devices. At the same time, this material is also suitable for testing surgical instruments that generate heat during machining.

Repeated Exposure of Nanostructured Titanium to Osteoblasts with Respect to Peri-Implantitis.

Titanium offers excellent biocompatibility and extraordinary mechanical properties. As a result, it is used as a material for dental implants. Implants infected by peri-implantitis can be cleaned for successful re-osseointegration. Optimal surface properties, such as roughness and wettability, have a significant impact on cell adhesion. The aim of this study was to evaluate the adhesion and proliferation of osteoblasts on the surface of repeatedly cleaned nanostructured titanium samples. Human osteoblast-like cells MG-63 were seeded on nanostructured titanium specimens manufactured from rods produced by the equal channel angular pressing. For surface characterization, roughness and wettability were measured. Cell adhesion after 2 h as well as cell proliferation after 48 h from plating was assessed. We have found that this repeated cleaning of titanium surface reduced cell adhesion as well as proliferation. These events depend on interplay of surface properties, such as wettability, roughness and topography. It is difficult to distinguish which factors are responsible for these events and further investigations will be required. However, even after the several rounds of repeated cleaning, there was a certain rate of adhesion and proliferation recorded. Therefore the attempts to save failing implants by using in situ cleaning are promising.

The comparison of impedance-based method of cell proliferation monitoring with commonly used metabolic-based techniques.

OBJECTIVES: Determination of cell numbers is a crucial step in studies focused on cytokinetics and cell toxicity. The impedance-based analysis employing electronic sensor array system xCELLigence System allowing label-free dynamic monitoring of relative viable adherent cell amounts was compared with the most utilized methods for relative quantification of viable cell numbers based on a determination of cellular metabolism. DESIGN: Colorimetric assay based on reduction of tetrazolium salt (MTT) by mitochondrial enzymes and chemiluminiscent assay based on intracellular adenosine triphosphate (ATP) determination were compared with the impedance-based system. Cell morphology was compared by microscopic evaluation. Normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF), together with 3T3 mouse fibroblast and HaCaT keratinocyte cell lines were employed. RESULTS: The progress of cell growth curves obtained by different methods during 72 hours reflected cell type and cell seeding densities. The impedance-based method was found to be applicable for the determination of the cell proliferation of 3T3 fibroblasts, HaCaT and NHDF, since the comparison of this method with ATP and MTT determinations showed a comparable results. In contrast, the proliferation of NHEK measured by the impedance-based method did not correlate with other methodological approaches. This could be accounted to the specific morphological appearance of these cells. CONCLUSION: The study shows the impedance-based detection of viable adherent cells is a valuable approach for cytokinetics and pharmacological studies. However, the specific morphological characteristics of cell lines have to be considered employing this method for determination of cell proliferation without using other reference methods.

High-frequency priming stimulation does not enhance the effect of low-frequency rTMS in the treatment of tinnitus.

Based on its ability to reduce the excitability of the cortex locally, low-frequency repetitive transcranial magnetic stimulation (rTMS) has been investigated for the treatment of hyperexcitability disorders such as auditory hallucinations and tinnitus. Results are promising, but characterized by only moderate improvement and a high inter-individual variability. Experimental data from motor cortex stimulation in healthy subjects indicates that the depressant effect of low-frequency rTMS can be enhanced by high-frequency priming stimulation. Here we will investigate whether high-frequency priming also improves the therapeutic efficacy of low-frequency rTMS in a clinical application. 32 patients with chronic tinnitus were randomly assigned to either a standard protocol of low-frequency rTMS (110% motor threshold, 1 Hz, 2000 stimuli/day) or a stimulation protocol in which priming stimulation with 6 Hz (90% motor threshold, 960 stimuli) preceded low-frequency rTMS (110% motor threshold, 1 Hz, 1040 stimuli/day). Stimulation was applied over the left auditory cortex by using MRI-guided coil positioning. The treatment outcome was assessed with a standardized tinnitus questionnaire. There was no significant difference between the standard protocol and the protocol involving priming stimulation. Both stimulation protocols resulted in significant clinical improvement after 10 days of stimulation, as compared to baseline. Our data does not support an enhancing effect of higher frequency priming on low-frequency rTMS in the treatment of tinnitus.