Krisztina Fischer

Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication.

We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.

Focused ultrasound modulates region-specific brain activity

We demonstrated the in vivo feasibility of using focused ultrasound (FUS) to transiently modulate (through either stimulation or suppression) the function of regional brain tissue in rabbits. FUS was delivered in a train of pulses at low acoustic energy, far below the cavitation threshold, to the animals somatomotor and visual areas, as guided by anatomical and functional information from magnetic resonance imaging (MRI). The temporary alterations in the brain function affected by the sonication were characterized by both electrophysiological recordings and functional brain mapping achieved through the use of functional MRI (fMRI). The modulatory effects were bimodal, whereby the brain activity could either be stimulated or selectively suppressed. Histological analysis of the excised brain tissue after the sonication demonstrated that the FUS did not elicit any tissue damages. Unlike transcranial magnetic stimulation, FUS can be applied to deep structures in the brain with greater spatial precision. Transient modulation of brain function using image-guided and anatomically-targeted FUS would enable the investigation of functional connectivity between brain regions and will eventually lead to a better understanding of localized brain functions. It is anticipated that the use of this technology will have an impact on brain research and may offer novel therapeutic interventions in various neurological conditions and psychiatric disorders.

On-Demand Three-Dimensional Freeform Fabrication of Multi-Layered Hydrogel Scaffold With Fluidic Channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186.

Focused ultrasound-mediated suppression of chemically-induced acute epileptic EEG activity

BackgroundEpilepsy is a common neurological disorder, which is attributed to uncontrollable abnormal hyper-excitability of neurons. We investigated the feasibility of using low-intensity, pulsed radiation of focused ultrasound (FUS) to non-invasively suppress epileptic activity in an animal model (rat), which was induced by the intraperitonial injection of pentylenetetrazol (PTZ).ResultsAfter the onset of induced seizures, FUS was transcranially administered to the brain twice for three minutes each while undergoing electroencephalographic (EEG) monitoring. An air-backed, spherical segment ultrasound transducer (diameter: 6 cm; radius-of-curvature: 7 cm) operating at a fundamental frequency of 690 KHz was used to deliver a train of 0.5 msec-long pulses of sonication at a repetitive rate of 100 Hz to the thalamic areas of the brain. The acoustic intensity (130 mW/cm2) used in the experiment was sufficiently within the range of safety guidelines for the clinical ultrasound imaging. The occurrence of epileptic EEG bursts from epilepsy-induced rats significantly decreased after sonication when it was compared to the pre-sonication epileptic state. The PTZ-induced control group that did not receive any sonication showed a sustained number of epileptic EEG signal bursts. The animals that underwent sonication also showed less severe epileptic behavior, as assessed by the Racine score. Histological analysis confirmed that the sonication did not cause any damage to the brain tissue.ConclusionsThese results revealed that low-intensity, pulsed FUS sonication suppressed the number of epileptic signal bursts using acute epilepsy model in animal. Due to its non-invasiveness and spatial selectivity, FUS may offer new perspectives for a possible non-invasive treatment of epilepsy.

Three-dimensional bioprinting of rat embryonic neural cells

We present a direct cell printing technique to pattern neural cells in a three-dimensional (3D) multilayered collagen gel. A layer of collagen precursor was printed to provide a scaffold for the cells, and the rat embryonic neurons and astrocytes were subsequently printed on the layer. A solution of sodium bicarbonate was applied to the cell containing collagen layer as nebulized aerosols, which allowed the gelation of the collagen. This process was repeated layer-by-layer to construct the 3D cell–hydrogel composites. Upon characterizing the relationship between printing resolutions and the growth of printed neural cells, single/multiple layers of neural cell–hydrogel composites were constructed and cultured. The on-demand capability to print neural cells in a multilayered hydrogel scaffold offers flexibility in generating artificial 3D neural tissue composites.

Focused ultrasound as a local therapy for liver cancer.

Conventional surgical treatments of liver cancer are invasive (including minimally invasive) with a high incidence of new metastasis and poor success, even after multiple resections or ablations. These limitations motivated research into new, less invasive solutions for liver cancer treatment. Focused ultrasound surgery (FUS), or high-intensity focused ultrasound, has been recognized as a noninvasive technology for benign and malignant tumor treatment. Previously, FUS was guided with ultrasound that has limited target definition and monitoring capability of the ablation process. Combining magnetic resonance imaging (MRI) with multiple-element phased-array transducers to create MRI-guided focused ultrasound thermal therapy provides more accurate targeting and real-time temperature monitoring. This treatment is hindered by the ribcage that limits the acoustic windows to the liver and the respiratory motion of the liver. New advances in MRI and transducer design will likely resolve these limitations and make MRI-guided FUS a powerful tool in local liver cancer therapy. This article reviews this technology and advances that can expand its use for cancer treatment in general and liver cancer in particular.

Noninvasive transcranial stimulation of rat abducens nerve by focused ultrasound.

Nonpharmacologic and nonsurgical transcranial modulation of the nerve function may provide new opportunities in evaluation and treatment of cranial nerve diseases. This study investigates the possibility of using low-intensity transcranial focused ultrasound (FUS) to selectively stimulate the rat abducens nerve located above the base of the skull. FUS (frequencies of 350 kHz and 650 kHz) operating in a pulsed mode was applied to the abducens nerve of Sprague-Dawley rats under stereotactic guidance. The abductive eyeball movement ipsilateral to the side of sonication was observed at 350 kHz, using the 0.36-msec tone burst duration (TBD), 1.5-kHz pulse repetition frequency (PRF), and the overall sonication duration of 200 msec. Histologic and behavioral monitoring showed no signs of disruption in the blood brain barrier (BBB), as well as no damage to the nerves and adjacent brain tissue resulting from the sonication. As a novel functional neuro-modulatory modality, the pulsed application of FUS has potential for diagnostic and therapeutic applications in diseases of the peripheral nervous system.

Renal Ultrafiltration Changes Induced by Focused US

PURPOSE To determine if focused ultrasonography (US) combined with a diagnostic microbubble-based US contrast agent can be used to modulate glomerular ultrafiltration and size selectivity. MATERIALS AND METHODS The experiments were approved by the animal care committee. The left kidney of 17 healthy rabbits was sonicated by using a 260-kHz focused US transducer in the presence of a microbubble-based US contrast agent. The right kidney served as the control. Three acoustic power levels were applied: 0.4 W (six rabbits), 0.9 W (six rabbits), and 1.7 W (five rabbits). Three rabbits were not treated with focused US and served as control animals. The authors evaluated changes in glomerular size selectivity by measuring the clearance rates of 3000- and 70,000-Da fluorescence-neutral dextrans. The creatinine clearance was calculated for estimation of the glomerular filtration rate. The urinary protein-creatinine ratio was monitored during the experiments. The authors assessed tubular function by evaluating the fractional sodium excretion, tubular reabsorption of phosphate, and gamma-glutamyltransferase-creatinine ratio. Whole-kidney histologic analysis was performed. For each measurement, the values obtained before and after sonication were compared by using the paired t test. RESULTS Significant (P < .05) increases in the relative (ratio of treated kidney value/nontreated kidney value) clearance of small- and large-molecule agents and the urine flow rates that resulted from the focused US treatments were observed. Overall, 1.23-, 1.23-, 1.61-, and 1.47-fold enhancement of creatinine clearance, 3000-Da dextran clearance, 70 000-Da dextran clearance, and urine flow rate, respectively, were observed. Focal tubular hemorrhage and transient functional tubular alterations were observed at only the highest (1.7-W) acoustic power level tested. CONCLUSION Glomerular ultrafiltration and size selectivity can be temporarily modified with simultaneous application of US and microbubbles. This method could offer new opportunities for treatment of renal disease.

PET/CT imaging evidence of FUS‐mediated (18)F‐FDG uptake changes in rat brain

PURPOSE Transcranial focused ultrasound (FUS) delivers highly focused acoustic energy to a small region of the brain in a noninvasive manner. Recent studies have revealed that FUS, which is administered either in pulsed or continuous waves, can elicit or suppress neural tissue excitability. This neuromodulatory property of FUS has been demonstrated via direct motion detection, electrophysiological recordings, functional magnetic resonance imaging (fMRI), confocal imaging, and microdialysis sampling of neurotransmitters. This study presents new evidence of local increase in glucose metabolism induced by FUS to the rat brain using FDG (18-fludeoxyglucose) positron emission tomography (PET). METHODS Sprague-Dawley rats underwent sonication to a unilateral hemispheric area of the brain prior to PET scan. The pulsed sonication (350 kHz, tone burst duration of 0.5 ms, pulse repetition frequency of 1 kHz, and duration of 300 ms) was applied in 2 s intervals for 40 min immediately after the FDG injection via tail vein. Subsequently, the PET was acquired in dynamic list-mode to image FDG activity for an hour, and reconstructed into a single volume representing standardized uptake value (SUV). The raw SUV as well as its asymmetry index (AI) were measured from five different volume-of-interests (VOIs) of the brain for both hemispheres, and compared between sonicated and unsonicated groups. RESULTS Statistically significant hemispheric changes in SUV were observed only at the center of sonication focus within the FUS group [paired t-test; t(7) = 3.57, p < 0.05]. There were no significant hemispheric differences in SUV within the control group in any of the VOIs. A statistically significant elevation in AI (t-test; t(7) = 3.40, p < 0.05) was observed at the center of sonication focus (7.9 ± 2.5%, the deviations are in standard error) among the FUS group when compared to the control group (-0.8 ± 1.2%). CONCLUSIONS Spatially distinct increases in the glucose metabolic activity in the rat brain is present only at the center of sonication focus, suggesting localized functional neuromodulation mediated by the sonication.

The results of the expression array studies correlate and enhance the known genetic basis of gastric and colorectal cancer

Gastric and colorectal cancers belong to the most frequent cancer types in the world today. This fact emphasizes the importance of identification of useful diagnostic and prognostic markers, in the earliest stage of the disease. The examination of gene expression profile in gastric and colorectal cancer may develop the bases of early diagnosis and of individual therapeutic strategies. In the microarray examinations done so far for these types of cancers, the expression of hundreds and thousands of genes were studied, however, both the sample collection and the results showed wide variations. The diversity of expression array methods and data analysis makes the comparison of microarray results difficult. Beside the exposition of the practical aspects of the chip technology, our aims are the systematization of data that are currently available in the international scientific literature and the description of the results in a comprehensive way. Microarray results show that the gene expression pattern, detected in gastric and colon cancers, highly depends on the histological type and heterogeneity of the sample, array type, and softwares, used for data analysis. Recent experiments point out not just the changes of the alterations of tumor suppression, apoptosis, cell‐cycle regulation, and signal transduction, but tumor cell metabolism and cell‐microenvironment interactions also. Results show connection to and make more complete the already known molecular background of gastric and colorectal cancers. Based on the accumulation of recent and further data, such kind of multifunctional diagnostic microarrays that can be suited for completing the conventional histological diagnostics and subtypization will certainly become available in the near future.