Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering

Articular cartilage injuries and degeneration affect a large proportion of the population in developed countries world wide. Stem cells can be differentiated into chondrocytes by adding transforming growth factor-beta1 and dexamethasone to a pellet culture, which are unfeasible for tissue engineering purposes. We attempted to achieve stable chondrogenesis without any requirement for exogenous growth factors. Human mesenchymal stem cells were transduced with an adenoviral vector containing the SRY-related HMG-box gene 9 (SOX9), and were cultured in a three-dimensional (3D) hydrogel scaffold composite. As an additional treatment, mechanical stimulation was applied in a custom-made bioreactor. SOX9 increased the expression level of its known target genes, as well as its cofactors: the long form of SOX5 and SOX6. However, it was unable to increase the synthesis of sulfated glycosaminoglycans (GAGs). Mechanical stimulation slightly enhanced collagen type X and increased lubricin expression. The combination of SOX9 and mechanical load boosted GAG synthesis as shown by (35)S incorporation. GAG production rate corresponded well with the amount of (endogenous) transforming growth factor-beta1. Finally, cartilage oligomeric matrix protein expression was increased by both treatments. These findings provide insight into the mechanotransduction of mesenchymal stem cells and demonstrate the potential of a transcription factor in stem cell therapy.

Quantitative assessment of physiological and behavioural parameters in healthy dairy cows evoked by transcutaneous electrical nerve stimulation of the udder

The pain and distress associated with transcutaneous electrical nerve stimulation (TENS) of the udder was evaluated by treating 20 healthy dairy cows with an electrical udder stimulator. This generated a sequence of pulses (frequency: 160+/-10% impulses per second, duration 250 mus) and provided voltage ranges from 0 to 10 volts (+/-10%). Trials took place on three consecutive days, twice daily after morning and evening milking. Daily sessions were divided into two periods: (1) control (sham treatment) and (2) treatment (real treatment). Physiological (heart rate, respiratory rate, and plasma cortisol concentration) as well as ethological parameters (kicking, weight shifting, and looking backwards to udder) were defined as pain-indicating parameters and observed. Evaluation of data showed that only one parameter (kicking) was significantly increased during real treatment compared to sham treatment. It is concluded that the TENS therapy tested in this study can evoke changes in behaviour (increased kicking) consistent with an experience of pain in some cows.

Combined use of transcranial magnetic stimulation and metal electrode implants: a theoretical assessment of safety considerations

This paper provides a theoretical assessment of the safety considerations encountered in the simultaneous use of transcranial magnetic stimulation (TMS) and neurological interventions involving implanted metallic electrodes, such as electrocorticography. Metal implants are subject to magnetic forces due to fast alternating magnetic fields produced by the TMS coil. The question of whether the mechanical movement of the implants leads to irreversible damage of brain tissue is addressed by an electromagnetic simulation which quantifies the magnitude of imposed magnetic forces. The assessment is followed by a careful mechanical analysis determining the maximum tolerable force which does not cause irreversible tissue damage. Results of this investigation provide useful information on the range of TMS stimulator output powers which can be safely used in patients having metallic implants. It is shown that conventional TMS applications can be considered safe when applied on patients with typical electrode implants as the induced stress in the brain tissue remains well below the limit of tissue damage.

Repetitive transcranial magnetic stimulation of the dorsolateral prefrontal cortex affects divided attention immediately after cessation of stimulation

Transcranial magnetic stimulation has evolved into a powerful neuroscientific tool allowing to interfere transiently with specific brain functions. In addition, repetitive TMS (rTMS) has long-term effects (e.g. on mood), probably mediated by neurochemical alterations. While long-term safety of rTMS with regard to cognitive functioning is well established from trials exploring its therapeutic efficacy, little is known on whether rTMS can induce changes in cognitive functioning in a time window ranging from minutes to hours, a time in which neurochemical effects correlated with stimulation have been demonstrated. This study examined effects of rTMS on three measures of executive function in healthy subjects who received one single rTMS session (40 trains of 2 s duration 20 Hz stimuli) at the left dorsolateral prefrontal cortex (DLPFC). Compared to a sham condition one week apart, divided attention performance was significantly impaired about 30-60 min after rTMS, while Stroop-interference and performance in the Wisconsin Card Sorting Test was unaffected after rTMS. Repetitive TMS of the left DLPFC, at stimulation parameters used in therapeutic studies, does not lead to a clinically relevant impairment of executive function after stimulation. However, the significant effect on divided attention suggests that cognitive effects of rTMS are not limited to the of acute stimulation, and may possibly reflect known neurochemical alterations induced by rTMS. Sensitive cognitive measures may be useful to trace those short-term effects of rTMS non-invasively in humans.

A Novel Implantable Hearing System with Direct Acoustic Cochlear Stimulation

A new implantable hearing system, the direct acoustic cochlear stimulator (DACS) is presented. This system is based on the principle of a power-driven stapes prosthesis and intended for the treatment of severe mixed hearing loss due to advanced otosclerosis. It consists of an implantable electromagnetic transducer, which transfers acoustic energy directly to the inner ear, and an audio processor worn externally behind the implanted ear. The device is implanted using a specially developed retromeatal microsurgical approach. After removal of the stapes, a conventional stapes prosthesis is attached to the transducer and placed in the oval window to allow direct acoustical coupling to the perilymph of the inner ear. In order to restore the natural sound transmission of the ossicular chain, a second stapes prosthesis is placed in parallel to the first one into the oval window and attached to the patient's own incus, as in a conventional stapedectomy. Four patients were implanted with an investigational DACS device. The hearing threshold of the implanted ears before implantation ranged from 78 to 101 dB (air conduction, pure tone average, 0.5-4 kHz) with air-bone gaps of 33-44 dB in the same frequency range. Postoperatively, substantial improvements in sound field thresholds, speech intelligibility as well as in the subjective assessment of everyday situations were found in all patients. Two years after the implantations, monosyllabic word recognition scores in quiet at 75 dB improved by 45-100 percent points when using the DACS. Furthermore, hearing thresholds were already improved by the second stapes prosthesis alone by 14-28 dB (pure tone average 0.5-4 kHz, DACS switched off). No device-related serious medical complications occurred and all patients have continued to use their device on a daily basis for over 2 years. Copyright (c) 2008 S. Karger AG, Basel.

Non-invasive gene-expression-based detection of well-developed collateral function in individuals with and without coronary artery disease

BACKGROUND: In patients with coronary artery disease (CAD), a well grown collateral circulation has been shown to be important. The aim of this prospective study using peripheral blood monocytes was to identify marker genes for an extensively grown coronary collateral circulation. METHODS: Collateral flow index (CFI) was obtained invasively by angioplasty pressure sensor guidewire in 160 individuals (110 patients with CAD, and 50 individuals without CAD). RNA was extracted from monocytes followed by microarray-based gene-expression analysis. 76 selected genes were analysed by real-time polymerase chain reaction (PCR). A receiver operating characteristics analysis based on differential gene expression was then performed to separate individuals with poor (CFI<0.21) and well-developed collaterals (CFI>or=0.21) Thereafter, the influence of the chemokine MCP-1 on the expression of six selected genes was tested by PCR. RESULTS: The expression of 203 genes significantly correlated with CFI (p = 0.000002-0.00267) in patients with CAD and 56 genes in individuals without CAD (p = 00079-0.0430). Biological pathway analysis revealed 76 of those genes belonging to four different pathways: angiogenesis, integrin-, platelet-derived growth factor-, and transforming growth factor beta-signalling. Three genes in each subgroup differentiated with high specificity among individuals with low and high CFI (>or=0.21). Two out of these genes showed pronounced differential expression between the two groups after cell stimulation with MCP-1. CONCLUSIONS: Genetic factors play a role in the formation and the preformation of the coronary collateral circulation. Gene expression analysis in peripheral blood monocytes can be used for non-invasive differentiation between individuals with poorly and with well grown collaterals. MCP-1 can influence the arteriogenic potential of monocytes.

Thalamic stimulation for tremor. Subtle changes in episodic memory are related to stimulation per se and not to a microthalamotomy effect

The aim of this study was to investigate the impact of unilateral deep brain stimulation (DBS) of the ventrointermediate (Vim) thalamic nucleus on neuropsychological functioning comparing stimulation-on with stimulation-off conditions. Nine patients [five patients with Parkinson's Disease (PD), two patients with essential tremor (ET) and 2 patients with multiple sclerosis (MS)] underwent comprehensive neuropsychological testing for cognitive functions, including general mental impairment, aphasia, agnosia, executive and constructional abilities, learning, memory, cognitive processing speed and attention as well as depression. The neuropsychological assessments were performed at least 6 months postoperatively (mean 9 months). Testing in the stimulation-on and stimulation-off condition was obtained within a period of 3 to 4 weeks. Unilateral DBS resulted in improvement of tremor in all patients. There were no significant differences between the stimulation-on and the stimulation-off condition with the exception of a decrement of word-recall in the short delay free-recall subtest of the Rey Auditory-Verbal Learning Test (RAVLT). Subgroup analysis indicated that the impairment in word-recall was related to left-sided thalamic stimulation. Our study confirms that chronic unilateral DBS is a safe method with regard to cognitive function. The subtle changes in episodic memory are related to stimulation per se and not to a microthalamotomy effect.

Sympathetic stimulation using the cold pressor test increases coronary collateral flow

BACKGROUND Little is known about the vasomotor function of human coronary collateral vessels. The purpose of this study was to examine collateral flow under a strong sympathetic stimulus (cold pressor test, CPT). METHODS In 30 patients (62 +/- 12 years) with coronary artery disease, two subsequent coronary artery occlusions were performed with random CPT during one of them. Two minutes before and during the 1 minute-occlusion, the patient's hand was immerged in ice water. For the calculation of a perfusion pressure-independent collateral flow index (CFI), the aortic (Pao), the central venous (CVP) and the coronary wedge pressure (Poccl) were measured: CFI = (Poccl - CVP)/(Pao - CVP). RESULTS CPT lead to an increase in Pao from 98 +/- 14 to 105 +/- 15 mm Hg (p = 0.002). Without and with CPT, CFI increased during occlusion from 14% +/- 10% to 16% +/- 10% (p = 0.03) and from 17% +/- 9% to 19% +/- 9% (p = 0.006), respectively, relative to normal flow. During CPT, CFI was significantly higher at the beginning as well as at the end of the occlusion compared to identical instants without CPT. CFI at the end of the control occlusion did not differ significantly from the CFI at the beginning of occlusion with CPT. CONCLUSIONS During balloon occlusion, collateral flow increased due to collateral recruitment independent of external sympathetic stimulation. Sympathetic stimulation using CPT additionally augmented collateral flow. The collateral-flow-increasing effect of CPT is comparable to the recruitment effect of the occlusion itself. This may reflect a coronary collateral vasodilation mediated by the sympathetic nervous system.

High frequency repetitive transcranial magnetic stimulation (rTMS) of the left dorsolateral cortex: EEG topography during waking and subsequent sleep.

Repetitive transcranial magnetic stimulation (rTMS) is a novel research tool in neurology and psychiatry. It is currently being evaluated as a conceivable alternative to electroconvulsive therapy for the treatment of mood disorders. Eight healthy young (age range 21-25 years) right-handed men without sleep complaints participated in the study. Two sessions at a 1-week interval, each consisting of an adaptation night (sham stimulation) and an experimental night (rTMS in the left dorsolateral prefrontal cortex or sham stimulation; crossover design), were scheduled. In each subject, 40 trains of 2-s duration of rTMS (inter-train interval 28 s) were applied at a frequency of 20 Hz (i.e. 1600 pulses per session) and at an intensity of 90% of the motor threshold. Stimulations were scheduled 80 min before lights off. The waking EEG was recorded for 10-min intervals approximately 30 min prior to and after the 20-min stimulations, and polysomnographic recordings were obtained during the subsequent sleep episode (23.00-07.00 h). The power spectra of two referential derivations, as well as of bipolar derivations along the antero-posterior axis over the left and right hemispheres, were analyzed. rTMS induced a small reduction of sleep stage 1 (in min and percentage of total sleep time) over the whole night and a small enhancement of sleep stage 4 during the first non-REM sleep episode. Other sleep variables were not affected. rTMS of the left dorsolateral cortex did not alter the topography of EEG power spectra in waking following stimulation, in the all-night sleep EEG, or during the first non-REM sleep episode. Our results indicate that a single session of rTMS using parameters like those used in depression treatment protocols has no detectable side effects with respect to sleep in young healthy males.

Electrode location and clinical outcome in hippocampal electrical stimulation for mesial temporal lobe epilepsy

PURPOSE To study the clinical outcome in hippocampal deep brain stimulation (DBS) for the treatment of patients with refractory mesial temporal lobe epilepsy (MTLE) according to the electrode location. METHODS Eight MTLE patients implanted in the hippocampus and stimulated with high-frequency DBS were included in this study. Five underwent invasive recordings with depth electrodes to localize ictal onset zone prior to chronic DBS. Position of the active contacts of the electrode was calculated on postoperative imaging. The distances to the ictal onset zone were measured as well as atlas-based hippocampus structures impacted by stimulation were identified. Both were correlated with seizure frequency reduction. RESULTS The distances between active electrode location and estimated ictal onset zone were 11±4.3 or 9.1±2.3mm for patients with a >50% or <50% reduction in seizure frequency. In patients (N=6) showing a >50% seizure frequency reduction, 100% had the active contacts located <3mm from the subiculum (p<0.05). The 2 non-responders patients were stimulated on contacts located >3mm to the subiculum. CONCLUSION Decrease of epileptogenic activity induced by hippocampal DBS in refractory MTLE: (1) seems not directly associated with the vicinity of active electrode to the ictal focus determined by invasive recordings; (2) might be obtained through the neuromodulation of the subiculum.

Prediction of water–rock interaction and porosity evolution in a granitoid-hosted enhanced geothermal system, using constraints from the 5 km Basel-1 well

Numerical simulations based on plans for a deep geothermal system in Basel, Switzerland are used here to understand chemical processes that occur in an initially dry granitoid reservoir during hydraulic stimulation and long-term water circulation to extract heat. An important question regarding the sustainability of such enhanced geothermal systems (EGS), is whether water–rock reactions will eventually lead to clogging of flow paths in the reservoir and thereby reduce or even completely block fluid throughput. A reactive transport model allows the main chemical reactions to be predicted and the resulting evolution of porosity to be tracked over the expected 30-year operational lifetime of the system. The simulations show that injection of surface water to stimulate fracture permeability in the monzogranite reservoir at 190 °C and 5000 m depth induces redox reactions between the oxidised surface water and the reduced wall rock. Although new calcite, chlorite, hematite and other minerals precipitate near the injection well, their volumes are low and more than compensated by those of the dissolving wall-rock minerals. Thus, during stimulation, reduction of injectivity by mineral precipitation is unlikely. During the simulated long-term operation of the system, the main mineral reactions are the hydration and albitization of plagioclase, the alteration of hornblende to an assemblage of smectites and chlorites and of primary K-feldspar to muscovite and microcline. Within a closed-system doublet, the composition of the circulated fluid changes only slightly during its repeated passage through the reservoir, as the wall rock essentially undergoes isochemical recrystallization. Even after 30 years of circulation, the calculations show that porosity is reduced by only ∼0.2%, well below the expected fracture porosity induced by stimulation. This result suggests that permeability reduction owing to water–rock interaction is unlikely to jeopardize the long-term operation of deep, granitoid-hosted EGS systems. A peculiarity at Basel is the presence of anhydrite as fracture coatings at ∼5000 m depth. Simulated exposure of the circulating fluid to anhydrite induces a stronger redox disequilibrium in the reservoir, driving dissolution of ferrous minerals and precipitation of ferric smectites, hematite and pyrite. However, even in this scenario the porosity reduction is at most 0.5%, a value which is unproblematic for sustainable fluid circulation through the reservoir.

Directional deep brain stimulation: an intraoperative double-blind pilot study.

Deep brain stimulation of different targets has been shown to drastically improve symptoms of a variety of neurological conditions. However, the occurrence of disabling side effects may limit the ability to deliver adequate amounts of current necessary to reach the maximal benefit. Computed models have suggested that reduction in electrode size and the ability to provide directional stimulation could increase the efficacy of such therapies. This has never been demonstrated in humans. In the present study, we assess the effect of directional stimulation compared to omnidirectional stimulation. Three different directions of stimulation as well as omnidirectional stimulation were tested intraoperatively in the subthalamic nucleus of 11 patients with Parkinson's disease and in the nucleus ventralis intermedius of two other subjects with essential tremor. At the trajectory chosen for implantation of the definitive electrode, we assessed the current threshold window between positive and side effects, defined as the therapeutic window. A computed finite element model was used to compare the volume of tissue activated when one directional electrode was stimulated, or in case of omnidirectional stimulation. All but one patient showed a benefit of directional stimulation compared to omnidirectional. A best direction of stimulation was observed in all the patients. The therapeutic window in the best direction was wider than the second best direction (P = 0.003) and wider than the third best direction (P = 0.002). Compared to omnidirectional direction, the therapeutic window in the best direction was 41.3% wider (P = 0.037). The current threshold producing meaningful therapeutic effect in the best direction was 0.67 mA (0.3-1.0 mA) and was 43% lower than in omnidirectional stimulation (P = 0.002). No complication as a result of insertion of the directional electrode or during testing was encountered. The computed model revealed a volume of tissue activated of 10.5 mm(3) in omnidirectional mode, compared with 4.2 mm(3) when only one electrode was used. Directional deep brain stimulation with a reduced electrode size applied intraoperatively in the subthalamic nucleus as well as in the nucleus ventralis intermedius of the thalamus significantly widened the therapeutic window and lowered the current needed for beneficial effects, compared to omnidirectional stimulation. The observed side effects related to direction of stimulation were consistent with the anatomical location of surrounding structures. This new approach opens the door to an improved deep brain stimulation therapy. Chronic implantation is further needed to confirm these findings.

Disruption of right prefrontal cortex by low-frequency repetitive transcranial magnetic stimulation induces risk-taking behavior

Decisions require careful weighing of the risks and benefits associated with a choice. Some people need to be offered large rewards to balance even minimal risks, whereas others take great risks in the hope for an only minimal benefit. We show here that risk-taking is a modifiable behavior that depends on right hemisphere prefrontal activity. We used low-frequency, repetitive transcranial magnetic stimulation to transiently disrupt left or right dorsolateral prefrontal cortex (DLPFC) function before applying a well known gambling paradigm that provides a measure of decision-making under risk. Individuals displayed significantly riskier decision-making after disruption of the right, but not the left, DLPFC. Our findings suggest that the right DLPFC plays a crucial role in the suppression of superficially seductive options. This confirms the asymmetric role of the prefrontal cortex in decision-making and reveals that this fundamental human capacity can be manipulated in normal subjects through cortical stimulation. The ability to modify risk-taking behavior may be translated into therapeutic interventions for disorders such as drug abuse or pathological gambling.

Suppressing versus releasing a habit: frequency-dependent effects of prefrontal transcranial magnetic stimulation.

When subjects are required to generate a random sequence of numbers they typically produce too many forward and backward 'counts' (e.g. 5-6, 4-3). This counting bias is interpreted as the consequence of an interference by overlearned tendencies to arrange numbers according to their natural order. Inhibition of such well-learned routines is known to rely on frontal lobe functioning. We examined differential effects of slow (1 Hz) and fast (10 Hz) repetitive transcranial magnetic stimulation (rTMS) over the left or right dorsolateral prefrontal cortex (DLPFC) on random number generation (RNG) performance. Eighteen healthy men performed an RNG task. Those subjects stimulated over the left DLPFC showed a frequency-dependent rTMS effect: counting bias was significantly reduced after the 1 Hz stimulation compared with baseline, but significantly exaggerated after the 10 Hz stimulation compared with 1 Hz stimulation. In contrast, the sequences of the subjects stimulated over the right DLPFC showed the well-known excess of counting in all conditions (i.e. baseline, 1 Hz and 10 Hz). These findings confirm the functional importance of specifically the left DLPFC in sequential response production and show, for the first time, that rTMS affects cognitive processing in a frequency-dependent manner.

Bladder function in patients with dystonia undergoing deep brain stimulation

INTRODUCTION Neurogenic bladder dysfunction is well described in Parkinson's disease and has a major impact on quality of live. In contrast, little is known about the extent of urinary symptoms in other movement disorders such as dystonia and about the role of the basal ganglia in bladder control.. PATIENTS AND METHODS A consecutive series of 11 patients with severe dystonia undergoing deep brain stimulation (DBS) of the globus pallidus internus was prospectively enrolled. Bladder function was assessed by the International Prostate Symptom Score and urodynamic investigation (UDI) before DBS surgery and afterwards in the conditions with and without DBS. RESULTS In UDI before DBS surgery, detrusor overactivity was found in 36% (4/11) of dystonia patients. With pallidal DBS ON, maximum flow rate significantly decreased, post-void residual significantly increased and detrusor overactivity disappeared.. CONCLUSIONS Pathological urodynamic changes can be found in a relevant percentage of dystonia patients. Pallidal DBS has a relaxing effect on detrusor function indicating a role of the basal ganglia in lower urinary tract control. Thus, a better understanding on how subcortical networks influence lower urinary tract function might open new therapeutic perspectives..