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Abstract Patient age is critical for mesenchymal stem cell quality and differentiation capacity. We demonstrate that proliferation and adipogenic capacity of subcutaneous adipose stem cells (ASCs) from female patients declined with advanced age, associated with reduction in cell nucleus size, increase in nuclear lamina protein lamin B1/B2, and lamin A, upregulation of senescence marker p16INK4a and senescence-associated β-galactosidase activity. Adipogenic induction resulted in differentiation of adipocytes and upregulation of adipogenic genes CCAAT enhancer binding protein alpha, fatty acid binding protein 4, lipoprotein lipase, and peroxisome proliferator-activated receptor-γ, which was not affected by the Sirt-1 activator YK-3-237 or the Sirt-1 inhibitor EX-527. Protein expression of the stem cell markers Oct4 and Sox2 was not significantly downregulated with advanced patient age. Mitochondrial reactive oxygen species were increased in ASCs from old-aged patients, whereas protein expression of NADPH oxidases NOX1 and NOX4 was downregulated, and dual oxidase isoforms remained unchanged. Generation of nitric oxide and iNOS expression was downregulated. Protein expression of Sirt-1 and Sirt-3 decreased with patient age, whereas Sirt-2 and Sirt-5 remained unchanged. Induction of adipogenesis stimulated protein expression of Sirt-1 and Sirt-3, which was not affected upon pre-incubation with the Sirt-1-activator YK-3-237 or the Sirt-1-inhibitor EX-527. The Sirt-1 inhibitor Sirtinol downregulated adiponectin protein expression and the number of adipocytes, whereas YK-3-237 exerted stimulatory effects. In summary, our data demonstrate increased oxidative stress in ASCs of aging patients, and decline of adipogenic capacity due to Sirt-1- mediated adiponectin downregulation in elderly patients.

Abstract One of the biggest challenges in tissue engineering and regenerative medicine is to ensure oxygen supply of cells in the (temporary) absence of vasculature. With the vision to exploit photosynthetic oxygen production by microalgae, co-cultivated in close vicinity to oxygen-consuming mammalian cells, we are searching for culture conditions that are compatible for both sides. Herein, we investigated the impact of long-term illumination on mammalian cells which is essential to enable photosynthesis by microalgae: four different cell types—primary human fibroblasts, dental pulp stem cells, and osteoblasts as well as the murine beta-cell line INS-1—were continuously exposed to warm white light, red or blue light over seven days. We observed that illumination with red light has no adverse effects on viability, metabolic activity and growth of the cells whereas exposure to white light has deleterious effects that can be attributed to its blue light portion. Quantification of intracellular glutathione did not reveal a clear correlation of this effect with an enhanced production of reactive oxygen species. Finally, our data indicate that the cytotoxic effect of short-wavelength light is predominantly a direct effect of cell illumination; photo-induced changes in the cell culture media play only a minor role.

Bone remodelling, important for homeostasis and regeneration involves the controlled action of osteoblasts, osteocytes and osteoclasts. The present study established a three-dimensional human in vitro bone model as triple culture with simultaneously differentiating osteocytes and osteoclasts, in the presence of osteoblasts. Since high sulfated hyaluronan (sHA3) was reported as a biomaterial to enhance osteogenesis as well as to dampen osteoclastogenesis, the triple culture was exposed to sHA3 to investigate cellular responses compared to the respective bone cell monocultures. Osteoclast formation and marker expression was stimulated by sHA3 only in triple culture. Osteoprotegerin (OPG) gene expression and protein secretion, but not receptor activator of NF-κB ligand (RANKL) or sclerostin (SOST), were strongly enhanced, suggesting an important role of sHA3 itself in osteoclastogenesis with other targets than indirect modulation of the RANKL/OPG ratio. Furthermore, sHA3 upregulated osteocalcin (BGLAP) in osteocytes and osteoblasts in triple culture, while alkaline phosphatase (ALP) was downregulated.

Marine litter is one of the most pervasive and fast-growing aspects of contamination in the global ocean, and has been observed in every environmental setting, including the deep seafloor where little is known about the magnitude and consequences of the problem. Submarine canyons, the main conduits for the transport of sediment, organic matter and water masses from shallow to abyssal depths, have been claimed to be preferential pathways for litter transport and accumulation in the deep sea. This is supported by ongoing evidence of large litter piles at great water depths, highlighting efficient transfer via canyons. The aim of this article is to present an overview of the current knowledge about marine litter in submarine canyons, taking a geological, process-based point of view. We evaluate sources, transport mechanisms and deposition of litter within canyons to assess the main factors responsible for its transport and accumulation in the deep sea. Few studies relate litter distribution to transport and depositional processes; nevertheless, results from available literature show that canyons represent accumulation areas for both land-based and maritime-based litter. Particularly, accumulation of fishing-related debris is mainly observed at the canyon heads and walls and is related to fishing activities carried out in and adjacent to canyons, while transport and accumulation of general waste and plastic along canyon axes can be related to different mechanisms, encompassing enhanced bottom currents, dense water cascading and turbidity currents, and is related to the proximity of canyons to shore. Global assessment of canyons exposure to riverine plastic inputs and fishing-related debris indicates varying susceptibility of canyons to litter, also highlighting that most of the canyons prone to receive large amounts of anthropogenic debris have not yet been surveyed. Considering that litter research in canyons is still in its infancy, several knowledge gaps need to be filled before the role of canyons as litter traps and the implication for benthic ecosystems can be fully understood.

In this study, the in vitro and in vivo bone formation behavior of mesoporous bioactive glass (MBG) particles incorporated in a pasty strontium-containing calcium phosphate bone cement (pS100G10) was studied in a metaphyseal fracture-defect model in ovariectomized rats and compared to a plain pasty strontium-containing calcium phosphate bone cement (pS100) and control (empty defect) group, respectively. In vitro testing showed good cytocompatibility on human preosteoblasts and ongoing dissolution of the MBG component. Neither the released strontium nor the BMG particles from the pS100G10 had a negative influence on cell viability. Forty-five female Sprague–Dawley rats were randomly assigned to three different treatment groups: (1) pS100 (n = 15), (2) pS100G10 (n = 15), and (3) empty defect (n = 15). Twelve weeks after bilateral ovariectomy and multi-deficient diet, a 4 mm wedge-shaped fracture-defect was created at the metaphyseal area of the left femur in all animals. The originated fracture-defect was substituted with pS100 or pS100G10 or left empty. After six weeks, histomorphometrical analysis revealed a statistically significant higher bone volume/tissue volume ratio in the pS100G10 group compared to the pS100 (p = 0.03) and empty defect groups (p = 0.0001), indicating enhanced osteoconductivity with the incorporation of MBG. Immunohistochemistry revealed a significant decrease in the RANKL/OPG ratio for pS100 (p = 0.004) and pS100G10 (p = 0.003) compared to the empty defect group. pS100G10 showed a statistically higher expression of BMP-2. In addition, a statistically significant higher gene expression of alkaline phosphatase, osteoprotegerin, collagen1a1, collagen10a1 with a simultaneous decrease in RANKL, and carbonic anhydrase was seen in the pS100 and pS100G10 groups compared to the empty defect group. Mass spectrometric imaging by time-of-flight secondary ion mass spectrometry (ToF-SIMS) showed the release of Sr2+ ions from both pS100 and pS100G10, with a gradient into the interface region. ToF-SIMS imaging also revealed that resorption of the MBG particles allowed for new bone formation in cement pores. In summary, the current work shows better bone formation of the injectable pasty strontium-containing calcium phosphate bone cement with incorporated mesoporous bioactive glass compared to the bioactive-free bone cement and empty defects and can be considered for clinical application for osteopenic fracture defects in the future.

Preventing hypoxic zones in 3D bioprinted mammalian cell-laden constructs using an internal oxygen supply could enable a more successful cultivation both in vitro and in vivo. In this study, the suitability of green microalgae as photosynthetic oxygen generators within bioprinted constructs was evaluated by defining and investigating important parameters for a successful co-culture. First, we assessed the impact of light–necessary for photosynthesis–on two non-light adapted mammalian cell types and defined red-light illumination and a temperature of 37°C as essential factors in a co-culture. The four thermotolerant microalgae strains Chlorella sorokiniana, Coelastrella oocystiformis, Coelastrella striolata, and Scenedesmus sp. were cultured both in suspension culture and 3D bioprinted constructs to assess viability and photosynthetic activity under these defined co-culture conditions. Scenedesmus sp. proved to be performing best under red light and 37°C as well as immobilized in a bioprinted hydrogel based on alginate. Moreover, the presence of the antibiotic ampicillin and the organic carbon-source glucose, both required for mammalian cell cultures, had no impact on bioprinted Scenedesmus sp. cultures regarding growth, viability, and photosynthetic activity. This study is the first to investigate the influence of mammalian cell requirements on the metabolism and photosynthetic ability of different microalgal strains. In a co-culture, the strain Scenedesmus sp. could provide a stable oxygenation that ensures the functionality of the mammalian cells.

Biopolymer hydrogels have become an important group of biomaterials in experimental and clinical use. However, unlike metallic or mineral materials, they are quite sensitive to sterilization. The aim of this study was to compare the effects of gamma irradiation and supercritical carbon dioxide (scCO2) treatment on the physicochemical properties of different hyaluronan (HA)- and/or gelatin (GEL)-based hydrogels and the cellular response of human bone marrow-derived mesenchymal stem cells (hBMSC). Hydrogels were photo-polymerized from methacrylated HA, methacrylated GEL, or a mixture of GEL/HA. The composition and sterilization methods altered the dissolution behavior of the biopolymeric hydrogels. There were no significant differences in methacrylated GEL release but increased methacrylated HA degradation of gamma-irradiated samples. Pore size/form remained unchanged, while gamma irradiation decreased the elastic modulus from about 29 kPa to 19 kPa compared to aseptic samples. HBMSC proliferated and increased alkaline phosphatase activity (ALP) particularly in aseptic and gamma-irradiated methacrylated GEL/HA hydrogels alike, while scCO2 treatment had a negative effect on both proliferation and osteogenic differentiation. Thus, gamma-irradiated methacrylated GEL/HA hydrogels are a promising base for multi-component bone substitute materials.

An in vitro bone triple culture involving human primary osteoblasts, osteocytes and osteoclasts enables the investigation of bone healing factors, drugs or biomaterials in a model system for native bone tissue. The present study analyses the impact of Sr2+ as well as hypoxic cultivation (5% O2 content or chemically induced by Co2+) on bone cells. The three cell types were cultivated together in the presence of 100 µM Sr2+, hypoxic conditions or in the presence of 75 µM Co2+. After cultivation the cell types were separated and analysed on mRNA and protein level individually. In response to Sr2+ osteoblasts showed a downregulation of IBSP expression and a stimulation of ALP activity. Osteocyte gene marker expression of PDPN, MEPE, RANKL, OPG, osteocalcin and likewise the amount of secreted osteocalcin was reduced in the presence of Sr2+. Activity of osteoclast-specific enzymes TRAP and CAII was enhanced compared to the Sr2+ free control. Hypoxic conditions induced by both 5% O2 or a Co2+ treatment led to decreased DNA content of all bone cells and downregulated expression of osteoblast markers ALPL and IBSP as well as osteocyte markers PDPN, RANKL and OPG. In addition, Co2+ induced hypoxia decreased gene and protein expression of osteocalcin in osteocytes. In response to the Co2+ treatment, the TRAP gene expression and activity was increased. This study is the first to analyse the effects of Sr2+ or hypoxia on triple cultures with primary human bone cells. The investigated in vitro bone model might be suitable to reduce animal experiments in early stages of biomaterial and drug development.

Extracting information about past tectonic or climatic environmental changes from sedimentary records is a key objective of provenance research. Interpreting the imprint of such changes remains challenging as signals might be altered in the sediment-routing system. We investigate the sedimentary provenance of the Oligocene/Miocene Upper Austrian Northern Alpine Foreland Basin and its response to the tectonically driven exhumation of the Tauern Window metamorphic dome (28 ± 1 Ma) in the Eastern European Alps by using the unprecedented combination of Nd isotopic composition of bulk-rock clay-sized samples and partly previously published multi-proxy (Nd isotopic composition, trace-element geochemistry, U-Pb dating) sand-sized apatite single-grain analysis. The basin offers an excellent opportunity to investigate environmental signal propagation into the sedimentary record because comprehensive stratigraphic and seismic datasets can be combined with present research results. The bulk-rock clay-sized fraction εNd values of well-cutting samples from one well on the northern basin slope remained stable at ∼−9.7 from 27 to 19 Ma but increased after 19 Ma to ∼−9.1. In contrast, apatite single-grain distributions, which were extracted from 22 drill-core samples, changed significantly around 23.3 Ma from apatites dominantly from low-grade (

Besides osteoconductivity and a high degradation rate, mesoporous bioactive glasses (MBGs) are specific for their highly ordered channel structure and high specific surface area, making them suitable as drug and/or growth factor delivery systems. On the other hand, the mesoporous channel structure and MBG composition can have an effect on common cell evaluation assays, leading to inconclusive results. This effect is especially important when MBG is mixed in composite bioinks, together with cells. Additionally, the hydrogel component of the ink can influence the degradation of MBG, leading to different ion releases, which can additionally affect the analyses. Hence, our aim here was to show how the MBG structure and composition influence common cell viability and differentiation assays when calcium (Ca)- or magnesium (Mg)-containing glass is part of an alginate-based composite bioink. We suggested pre-labeling of cells with DiI prior to bioprinting and staining with calcein-AM to allow identification of metabolically active cells expressing signals in both green and red channels, allowing the use of fluorescence imaging for cell viability evaluations in the presence of high amounts (7 wt %) of MBGs. The release and uptake of ions during degradation of CaMBG and MgMBG were significantly changed by alginate in the composite bioinks, as confirmed by higher release and uptake from bulk glasses. Additionally, we detected a burst release of Mg2+ from composites only after 24 h of incubation. Furthermore, we demonstrated that released ions and the mesoporous channel structure affect the measurement of lactate dehydrogenase (LDH) and alkaline phosphatase activity (ALP) in bioprinted composite scaffolds. Measured LDH activity was significantly decreased in the presence of CaMBG. On the other hand, the presence of MgMBG induced increased signal measured for the ALP. Taken together, our findings show how composite bioinks containing MBGs can interfere with common analyses, obtaining misleading results.

Sediment archives in the terrestrial and marine realm are regularly analyzed to infer changes in climate, tectonic, or anthropogenic boundary conditions of the past. However, contradictory observations have been made regarding whether short period events are faithfully preserved in stratigraphic archives; for instance, in marine sediments offshore large river systems. On the one hand, short period events are hypothesized to be non-detectable in the signature of terrestrially derived sediments due to buffering during sediment transport along large river systems. On the other hand, several studies have detected signals of short period events in marine records offshore large river systems. We propose that this apparent discrepancy is related to the lack of a differentiation between different types of signals and the lack of distinction between river response times and signal propagation times. In this review, we (1) expand the definition of the term ‘signal’ and group signals in sub-categories related to hydraulic grain size characteristics, (2) clarify the different types of ‘times’ and suggest a precise and consistent terminology for future use, and (3) compile and discuss factors influencing the times of signal transfer along sediment routing systems and how those times vary with hydraulic grain size characteristics. Unraveling different types of signals and distinctive time periods related to signal propagation addresses the discrepancies mentioned above and allows a more comprehensive exploration of event preservation in stratigraphy – a prerequisite for reliable environmental reconstructions from terrestrially derived sedimentary records.

Detailed characterization of variations in sediment architecture, flux, and transport processes in peri-orogenic basins offers insights into external climatic or tectonic forcings. We tested how four well-known tectonic/erosional events in the Oligocene/Miocene Alpine source area are recorded in the sediment-accumulation rates (SARs) of the deep marine sink in the Northern Alpine Foreland Basin (NAFB): exhumation of the Lepontine Dome (starting at 30 Ma) and the Tauern Window (23-21 Ma), erosion of the Augenstein Formation (∼21 Ma), and the visco-elastic relaxation of the European Plate. The Upper Austrian NAFB offers a unique opportunity to investigate external forcings on sedimentary infill due to the large amount of data on the Alpine hinterland and foreland. Deep-marine sedimentation, forming the Puchkirchen Group and the basal Hall Formation, was controlled by a basin-axial submarine channel (3–5 km wide, >100 km length). Two basin-wide unconformities were recognized in seismic-reflection data: the Northern Slope Unconformity (NSU) and the Base Hall Unconformity (BHU). We combine biostratigraphic and chemostratigraphic analyses of 316 drill-cutting samples from three wells with a large 3D-seismic-reflection data set (3300 km2, >5 km depth) to determine age and duration of the unconformities and to calculate spatially averaged SARs for the submarine channel and its overbanks, separately. Deepening of the basin, recorded by the NSU, occurred between 28.1 and 26.9 Ma. The Puchkirchen Group (26.9–19.6 Ma) is characterized by constant SARs (within standard deviation) in the channel [432–623 (t/m2/Ma)] and on the overbanks [240–340 (t/m2/Ma)]. The visco-elastic relaxation of the European Plate results in low SARs on the overbanks [186 (t/m2/Ma)], a decrease in sediment grain size in channel deposits and a decrease in sea level at the BHU (19.6–19.0 Ma). In the upper Hall Formation (19.0–18.1 Ma), clinoforms prograding from the south filled up the basin [1497 (t/m2/Ma)] within 1 Myrs. We conclude that only two of the tectonic signals are recorded in this part of the deep-marine sink, erosion of Augenstein Formation and visco-elastic relaxation of the European Plate; the exhumation of the Tauern Window and Lepontine Dome remain unrecorded.

Biologically active metal ions in low doses have the potential to accelerate bone defect healing. For successful remodelling the interaction of bone graft materials with both bone-forming osteoblasts and bone resorbing osteoclasts is crucial. In the present study brushite forming calcium phosphate cements (CPC) were doped with Co2+, Cu2+ and Cr3+ and the influence of these materials on osteoclast differentiation and activity was examined. Human osteoclasts were differentiated from human peripheral blood mononuclear cells (PBMC) both on the surface and in indirect contact to the materials on dentin discs. Release of calcium, phosphate and bioactive metal ions was determined using ICP-MS both in the presence and absence of the cells. While Co2+ and Cu2+ showed a burst release, Cr3+ was released steadily at very low concentrations (below 1 μM) and both calcium and phosphate release of the cements was considerably changed in the Cr3+ modified samples. Direct cultivation of PBMC/osteoclasts on Co2+ cements showed lower attached cell number compared to the reference but high activity of osteoclast specific enzymes tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (CAII) and cathepsin K (CTSK) and significantly increased gene expression of vitronectin receptor. Indirect cultivation with diluted Co2+ cement extracts revealed highest resorbed area compared to all other modifications and the reference. Cu2+ cements had cytotoxic effect on PBMC/osteoclasts during direct cultivation, while indirect cultivation with diluted extracts from Cu2+ cements did not provoke cytotoxic effects but a strictly inhibited resorption. Cr3+ doped cements did not show cytotoxic effects at all. Gene expression and enzyme activity of CTSK was significantly increased in direct culture. Indirect cultivation with Cr3+ doped cements revealed significantly higher resorbed area compared to the reference. In conclusion Cr3+ doped calcium phosphate cements are an innovative cement modification because of their high cytocompatibility and support of active resorption by osteoclasts.

Background: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Methods: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Results: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Conclusions: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

The development of bio-resorbable implant materials is rapidly going on. Sterilization of those materials is inevitable to assure the hygienic requirements for critical medical devices according to the medical device directive (MDD, 93/42/EG). Biopolymer-containing biomaterials are often highly sensitive towards classical sterilization procedures like steam, ethylene oxide treatment or gamma irradiation. Supercritical CO₂ (scCO₂) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO₂ treatment effectively inactivates microorganisms including bacterial spores. We established a scCO₂ sterilization procedure under addition of 0.25% water, 0.15% hydrogen peroxide and 0.5% acetic anhydride. The procedure was successfully tested for the inactivation of a wide panel of microorganisms including endospores of different bacterial species, vegetative cells of gram positive and negative bacteria including mycobacteria, fungi including yeast, and bacteriophages. For robust testing of the sterilization effect with regard to later application of implant materials sterilization all microorganisms were embedded in alginate/agarose cylinders that were used as Process Challenge Devices (PCD). These PCD served as surrogate models for bioresorbable 3D scaffolds. Furthermore, the impact of scCO₂ sterilization on mechanical properties of polysaccharide-based hydrogels and collagen-based scaffolds was analyzed. The procedure was shown to be less compromising on mechanical and rheological properties compared to established low-temperature sterilization methods like gamma irradiation and ethylene oxide exposure as well as conventional steam sterilization. Cytocompatibility of alginate gels and scaffolds from mineralized collagen was compared after sterilization with ethylene oxide, gamma irradiation, steam sterilization and scCO₂ treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO₂ treatment of these materials and scaffolds. We conclude that scCO₂ sterilization under addition of water, hydrogen peroxide and acetic anhydride is a very effective, gentle, non-cytotoxic and thus a promising alternative sterilization method especially for biomaterials.

Abstract Dry electroencephalography (EEG) electrodes provide rapid, gel-free, and easy EEG preparation, but with limited wearing comfort. We propose a novel dry electrode comprising multiple tilted pins in a flower-like arrangement. The novel Flower electrode increases wearing comfort and contact area while maintaining ease of use. In a study with 20 volunteers, we compare the performance of a novel 64-channel dry Flower electrode cap to a commercial dry Multipin electrode cap in sitting and supine positions. The wearing comfort of the Flower cap was rated as significantly improved both in sitting and supine positions. The channel reliability and average impedances of both electrode systems were comparable. Averaged VEP components showed no considerable differences in global field power amplitude and latency, as well as in signal-to-noise ratio and topography. No considerable differences were found in the power spectral density of the resting state EEGs between 1 and 40 Hz. Overall, our findings provide evidence for equivalent channel reliability and signal characteristics of the compared cap systems in the sitting and supine positions. The reliability, signal quality, and significantly improved wearing comfort of the Flower electrode allow new fields of applications for dry EEG in long-term monitoring, sensitive populations, and recording in supine position.

Warning of workers in dangerous situations is crucial. With the aim of deriving practical parameters for an electrocutaneous warning stimulation, we explore the design dimensions of pulse intervals, amplitudes, and locations of electrocutaneous stimulation in a study on healthy volunteers. Using biphasic rectangular current pulses on the upper right arm of 81 healthy participants, they evaluated temporal perception with varying intervals, ranging from 200 ms down to 0.5 ms, categorizing it as 'Individual pulses', 'Pulsating', 'Vibrating', or 'Continuous'. Next, we tested nine amplitude levels. Participants rated the perceived amplitude on a scale from 1 to 9 after a training phase. Finally, we presented five consecutive stimulation pulses in a pseudo-random order at eight electrode pair positions, asking participants to report the stimulated electrode pair. Participants perceived electrocutaneous pulses as 'Individual pulses' for median intervals above 74 ms, as 'Pulsating' between 44 ms and 74 ms, as 'Vibrating' between 12 ms and 44 ms, and as 'Continuous' below 12 ms. Pulse intervals below about 1 ms were perceived as weak and at about 5 ms as inconvenient, rendering these intervals less suitable for the design of a warning pattern. The median reported amplitudes [25%-75%-percentile] for presented amplitudes 1 to 9 are: 1[1-1], 2[2-3], 3[2-4], 3[3-4], 4[3-5], 5[4-6], 6[4-7], 7[5-8] and 7.5 [6-8] indicating a linear relationship between presented and perceived amplitude. These results suggest that the stimulation amplitude may be incorporated into a structured stimulation pattern. The majority of the electrode pair locations were reported correctly (64.3%-86.6%) or within the two neighboring electrode pairs (98%-99.7%). We conclude that the determined pulse intervals combined with the differentiability of locations offer the basis for designing a warning signal. Our research lays the groundwork for developing suitable signals for wearable electric warning devices.