Katja Myllymaa

Fabrication and testing of polyimide-based microelectrode arrays for cortical mapping of evoked potentials.

Modern microfabrication techniques make it possible to develop microelectrode arrays that may be utilized not only in neurophysiological research but also in the clinic, e.g. in neurosurgery and as elements of neural prostheses. The aim of this study was to test whether a flexible microelectrode array is suitable for recording cortical surface field potentials in rats. Polyimide-based microelectrode arrays were fabricated by utilizing microfabrication techniques e.g. photolithography and magnetron sputter deposition. The present microelectrode array consists of eight platinum microelectrodes (round-shaped, Ø: 200 microm), transmission lines and connector pads sandwiched between two thin layers of biocompatible polyimide. The microelectrode arrays were electrochemically characterized by impedance spectroscopy in physiological saline solution and successfully tested in vivo by conducting acute and chronic measurements of evoked potentials on the surface of rat cortex. The arrays proved excellent flexibility and mechanical strength during handling and implantation onto the surface of cortex. The excellent electrochemical characteristics and stable in vivo recordings with high spatiotemporal resolution highlight the potential of these arrays. The fabrication protocol described here allows implementation of several other neural interfaces with different layouts, material selections or target areas either for recording or stimulation purposes.

Formation and retention of staphylococcal biofilms on DLC and its hybrids compared to metals used as biomaterials

Staphylococcus epidermidis and Staphylococcus aureus cause most of the implant-related infections. Antibiotic treatment often fails and cure requires surgical intervention. It was hypothesized that biomaterial coatings resistant to biofilms offer a preventive option. Physical vapour deposited diamond-like carbon (DLC) and its polytetrafluoroethylene (DLC-PTFE-h) and polydimethylsiloxane (DLC-PDMS-h) hybrids were compared to titanium (Ti), tantalum (Ta) and chromium (Cr) thin films on silicon wafers for their resistance against formation and/or retention of biofilms produced by S. epidermidis and S. aureus in vitro. Sample surfaces were characterized for surface topography, contact angle and zeta-potential, because such properties might affect the biofilm. Biofilm was stained using calcofluor white and analysed in fluorescence microscopy using morphometry. Sixteen hour incubation was selected in pilot tests; at this checkpoint Ti, Ta, Cr and DLC-PDMS-h were almost fully covered by biofilm, but DLC and DLC-PTFE-h were only partially biofilm coated by S. epidermidis (88±26%, p<0.001 and 56±39%, p<0.001, respectively) or S. aureus (81±24%, p<0.001 and 51±26%, p<0.001, respectively). DLC and its PTFE hybrid offer a potential biofilm hostile surface coating for implants and medical devices. This ability to resist biofilm formation and attachment could not be explained by only one factor, but it seems to be related to a combination of various properties, with electrokinetic streaming potential and protein coating being particularly important for its outcome.

Osteogenesis of human mesenchymal stem cells on micro-patterned surfaces:

Osteogenic responses of human mesenchymal stromal cells (hMSCs) were compared on square-patterned, inverse square-patterned, and planar titanium, chromium, diamond-like carbon (DLC), and tantalum; hypothesis was that both the materials and patterns affect osteogenesis. Samples were produced using photolithography and physical vapor deposition. Early-marker alkaline phosphatase (ALP) and mid-markers, small body size and mothers against decapentaplegic-related protein-1 (SMAD1), runt-related transcription factor-2 (RUNX2), and osteopontin were studied using quantitative real-time polymerase chain reaction. ALP and hydroxyapatite, were colorimetrically studied. ALP reached highest values on both patterned titanium samples, but mid-markers disclosed that it was already lagging behind planar and inverse patterned tantalum. Hydroxyapatite formation disclosed that osteo-induced hMSCs passed all the differentiation stages (except on planar chromium). Presence of hydroxyapatite disclosed that both types of patterning promoted (p < 0.001) osteogenesis compared to planar samples. Results suggest that the osseocompatibility/integration of implants could be improved by changing the monotonous and featureless implant–host interface into micro-patterned interface to provide physical differentiation cues.

Surface characterization and in vitro biocompatibility assessment of photosensitive polyimide films

Polyimide (PI) is a commonly used polymer in microelectronics. Recently, numerous PI-based flexible neural interfaces have been developed for reducing mechanical mismatch between rigid implant and soft neural tissue. Most approaches employ non-photosensitive PI, which has been proven earlier to be biocompatible. However, photosensitive polyimide (PSPI) would simplify device fabrication remarkably, but its biocompatibility has been only sparsely reported. In this study, cytotoxicity of spin-coated PSPI (HD Microsystems PI-2771) and conventional PI (HD Microsystems PI-2525) films were evaluated in vitro using BHK-21 fibroblasts according to the ISO-10993-5 standard. PSPIs were tested as cured at a temperature of 200 degrees C (PI-2771-200) and 350 degrees C (PI-2771-350). The PI film surfaces were characterized in terms of their roughness, energy and zeta potential which are hypothesized to affect cell-material interactions. The values of the total surface free energy (SFE), and its polar and dispersive component, were significantly (p<0.001) greater for the PI-2525 film (SFE: 47.3 mJ/m2) than for the PI-2771-200 (25.6 mJ/m2) or PI-2771-350 films (26.2 mJ/m2). The curing temperature of the PI-2771 had a significant effect on the zeta potential values (p<0.001), but not on surface energy (p=0.091) or roughness (p=0.717). The results from the MTS proliferation assays and live/dead staining revealed that PSPI is almost as non-cytotoxic as conventional PI and polyethylene (negative control). The morphology and spreading of BHK-21 cells were similar on all the PI materials tested. In conclusion, PSPI seems to be a promising biocompatible material, while further studies in vitro and in vivo are needed to clarify the long-term effects.

Improved adherence and spreading of Saos-2 cells on polypropylene surfaces achieved by surface texturing and carbon nitride coating

The adhesion and contact guidance of human primary osteogenic sarcoma cells (Saos-2) were characterized on smooth, microstructured (MST) and micro- and nano-structured (MNST) polypropylene (PP) and on the same samples with a silicon-doped carbon nitride (C3N4-Si) coating. Injection molding was used to pattern the PP surfaces and the coating was obtained by using ultra-short pulsed laser deposition (USPLD). Surfaces were characterized using atomic force microscopy and surface energy components were calculated according to the Owens-Wendt model. The results showed C3N4-Si coated surfaces to be significantly more hydrophilic than uncoated ones. In addition, there were 86% more cells in the smooth C3N4-Si coated PP compared to smooth uncoated PP and 551%/476% more cells with MST/MNST C3N4-Si coated PP than could be obtained with MST/MNST uncoated PP. Thus the adhesion, spreading and contact guidance of osteoblast-like cells was effectively improved by combining surface texturing and deposition of osteocompatible C3N4-Si coating.

Improving electrochemical performance of flexible thin film electrodes with micropillar array structures

For reliable function, bioelectrodes require a stable, low-impedance contact with the target tissue. In biosignal monitoring applications, in which low ion current densities are recorded, it is important to minimize electrode contact impedances. Recently, several flexible electrode concepts have been introduced for single-patient use. These electrodes conform well on the patient skin enabling an artifact-free, low-noise recording. In this study, polydimethylsiloxane (PDMS) elastomer was used as an electrode substrate material. One half of the substrates were surface-patterned with micropillars produced by using micro-working robot-made mold inserts and a replica molding technique. The substrates were subsequently coated with thin films of titanium (Ti), copper (Cu), silver (Ag) or silver?silver chloride (Ag/AgCl). Electrical impedance spectroscopy studies revealed that the micropillar structure caused statistically significant reductions in impedance modulus and phase for each coating candidate. The relative effect was strongest for pure Ag, for which the values of the real part (Z?) and the imaginary part (Z?) decreased to less than one tenth of the original (smooth) values. However, Ag/AgCl, as expected, proved to be a superior electrode material. Coating with chloride drastically reduced the interfacial impedance compared to pure Ag. Further significant reduction was achieved by the micropillars, since the phase angle declined from 10?13? (for smooth samples, f < 50?Hz) to a value as low as 5?. Equivalent circuit modeling was used to obtain a better understanding of phenomena occurring at various electrode?electrolyte interfaces. The knowledge obtained in this study will be exploited in the further development of flexible electrodes and miniaturized biointerfaces with improved electrochemical characteristics.

Patterned macroarray plates in comparison of bacterial adhesion inhibition of tantalum, titanium, and chromium compared with diamond‐like carbon

Staphylococcus aureus device-related infection is a common complication in implantology. Bacterial adhesion on implant surfaces is the initial step in the infective process. The aim was to develop a method suitable for quantitative bacterial adherence studies and to test a new diamond-like carbon (DLC) coating against commonly used metallic biomaterials with regards to Staphylococcus aureus adhesion. Patterned silicon chips with spots of tantalum, titanium, chromium, and DLC were produced using ultraviolet lithography and physical vapor deposition. These patterned chips were used as such or glued to array plates, pretreated with serum and exposed to S. aureus (S-15981) for 90 min, followed by acridine orange staining and fluorescence microscopy. An adhesion index showed that the ranking order of the biomaterials was titanium, tantalum, chromium, and DLC, with the DLC being clearly most resistant against colonization with S. aureus. Micropatterned surfaces are useful for quantitative comparison of bacterial adherence on different biomaterials. In the presence of serum, DLC is superior in its ability to resist adhesion and colonization by S. aureus compared with the commonly used biomaterial metals tantalum, titanium, and chromium.

New disposable forehead electrode set with excellent signal quality and imaging compatibility

The use of emergency electroencephalography (EEG) in clinical practice is limited in part due to the lack of commercially available EEG monitoring sets that are suitable for rapid and simple use. The aim of this study was to develop a rapid and simple-to-use disposable forehead EEG electrode set for routine use that is also suitable for long-term monitoring. The EEG set we developed consists of 12 hydrogel-coated electrodes (10 recording electrodes, plus a reference and ground electrode) attached to a solid polymer film. The developed EEG set was compared to the full conventional 10-20 electrode setup in terms of the ability to detect epileptiform abnormalities in two critically ill patients. The technical quality of the EEG signal from the newly developed electrode set was excellent, and status epilepticus was reliably detected with this EEG set. Electric performance testing showed that the impedance spectra of the developed EEG electrodes were comparable to those of three commercially available, disposable electrodes, and the noise level was lower than that of the commercial electrodes. The developed EEG set is also MRI and CT compatible and lacks any signs of imaging artefacts or heat induction. These promising results provide a reason to expect that the developed EEG set may be applicable to situations in which the full, conventional 10-20 electrode setup is not available.

Assessment of the suitability of using a forehead EEG electrode set and chin EMG electrodes for sleep staging in polysomnography.

Recently, a number of portable devices designed for full polysomnography at home have appeared. However, current scalp electrodes used for electroencephalograms are not practical for patient self‐application. The aim of this study was to evaluate the suitability of recently introduced forehead electroencephalogram electrode set and supplementary chin electromyogram electrodes for sleep staging. From 31 subjects (10 male, 21 female; age 31.3 ± 11.8 years), sleep was recorded simultaneously with a forehead electroencephalogram electrode set and with a standard polysomnography setup consisting of six recommended electroencephalogram channels, two electrooculogram channels and chin electromyogram. Thereafter, two experienced specialists scored each recording twice, based on either standard polysomnography or forehead recordings. Sleep variables recorded with the forehead electroencephalogram electrode set and separate chin electromyogram electrodes were highly consistent with those obtained with the standard polysomnography. There were no statistically significant differences in total sleep time, sleep efficiency or sleep latencies. However, compared with the standard polysomnography, there was a significant increase in the amount of stage N1 and N2, and a significant reduction in stage N3 and rapid eye movement sleep. Overall, epoch‐by‐epoch agreement between the methods was 79.5%. Inter‐scorer agreement for the forehead electroencephalogram was only slightly lower than that for standard polysomnography (76.1% versus 83.2%). Forehead electroencephalogram electrode set as supplemented with chin electromyogram electrodes may serve as a reliable and simple solution for recording total sleep time, and may be adequate for measuring sleep architecture. Because this electrode concept is well suited for patients self‐application, it may offer a significant advancement in home polysomnography.

Shielded Design of Screen-Printed EEG Electrode Set Reduces Interference Pick-Up

Thick-film technologies, such as screen printing, are becoming more popular in the manufacturing of bioelectrodes due to their simplicity, low cost, high reproducibility, and efficiency in large-scale production. Since electroencephalography (EEG) is a method for measuring potential differences in the microvolt scale, it is important to minimize all electromagnetic interferences. However, protecting screen-printed electrodes and their conduction traces from electromagnetic interferences has not been adequately investigated. We hypothesized that interference pick-up could be effectively reduced by an optimized silver or graphite shielded construction. The interference pick-up of the electrodes was technically evaluated in an electromagnetic compatibility laboratory and with EEG recordings of healthy volunteers. The grounding layer significantly (p<; 0.001) reduced radio frequency (RF) interference in the standardized laboratory tests and the shielded electrodes exhibited significantly better power-line interference immunity in the EEG recordings (p<; 0.05). The silver layer achieved better shielding in the RF tests than the graphite layer (p= 0.029). Since the silver layer is straightforward and cheap to produce with the screen printing technique, it could be advantageously used to shield, e.g., in emergency rooms where many interfering electronic devices are close to patient.