Bioinspired calcium phosphate mineralization on Net-Shape-Nonwoven chitosan scaffolds stimulates human bone marrow stromal cell differentiation.

Chitosan fibers were processed using the Net-Shape-Nonwoven (NSN) technique in order to create porous scaffolds which were functionalized in two bioinspired ways: collagen type I coating and unique mineralization with organically modified hydroxyapatite (ormoHAP). While collagen is common to enhance cell attachment on surfaces, the electric-field assisted migration and deposition of ormoHAP on the surface of the NSN-scaffolds is a novel technique which enables sub-micrometer sized mineralization while maintaining the original pore structure. Microscopy revealed fast attachment and morphological adaptation of the cells on both, the pure and the functionalized NSN-scaffolds. Remarkably, the cell number of osteogenically induced hBMSC on ormoHAP-modified NSN-scaffolds increased 3.5-5 fold compared to pure NSN-scaffolds. Osteogenic differentiation of hBMSC/osteoblasts was highest on collagen-functionalized NSN-scaffolds. RT-PCR studies revealed gene expression of ALP, BSP II, and osteocalcin to be high for all NSN-scaffolds. Overall, the NSN-scaffold functionalization with collagen and ormoHAP improved attachment, proliferation, and differentiation of hBMSC and therefore revealed the remarkable potential of their application for the tissue engineering of bone.

Sorafenib paradoxically activates the RAS/RAF/ERK pathway in polyclonal human NK cells during expansion and thereby enhances effector functions in a dose- and time-dependent manner.

Natural killer (NK) cells play a major role in host immunity against leukaemia and lymphoma. However, clinical trials applying NK cells have not been as efficient as hoped for. Patients treated with rapidly accelerated fibrosarcoma (RAF) inhibitors exhibit increased tumour infiltration by immune cells, suggesting that a combination of RAF inhibitors with immunotherapy might be beneficial. As mitogen-activated protein kinases (MAPKs) such as raf-1 proto-oncogene, serine/threonine kinase (CRAF) regulate NK cell functions, we performed an in-vitro investigation on the potential of clinically relevant short-acting tyrosine kinase inhibitors (TKIs) as potential adjuvants for NK cell therapy: NK cells from healthy human blood donors were thus treated with sorafenib, sunitinib or the pan-RAF inhibitor ZM336372 during ex-vivo expansion. Functional outcomes assessed after washout of the drugs included cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction with/without target cell contact. Paradoxically, sorafenib enhanced NK cell effector functions in a time- and dose-dependent manner by raising the steady-state activation level. Of note, this did not lead to NK cell exhaustion, but enhanced activity against target cells such as K562 or Daudis mediated via the RAS/RAF/extracellular-regulated kinase (ERK) pathway, but not via protein kinase B (AKT). Our data will pave the path to develop a rationale for the considered use of RAF inhibitors such as sorafenib for pre-activation in NK cell-based adoptive immune therapy.

Efficiency of the sulfate pathway in comparison to the Δ4- and Δ5-pathway of steroidogenesis in the porcine testis.

Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis.

Effect of sulfonated steroids on steroidogenic cytochrome P450-dependent steroid hydroxylases.

In the last decades, sulfonated steroids evolved from inactive metabolites intended for excretion to highly relevant compounds involved in many physiological processes. Investigations of the impact of sulfonated steroids on the steroid hormone biosynthesis revealed that, on the one hand, these can serve as substrate for steroidogenic cytochromes P450 and, on the other hand, these are able to influence the catalytic properties of these enzymes. In this review the relevance of sulfonated steroids for the steroid hormone biosynthesis will be discussed.

The steroid metabolite 16(ß)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1.

The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(ß)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased Km and decreased kcat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites.

2ß- and 16ß-hydroxylase activity of CYP11A1 and direct stimulatory effect of estrogens on pregnenolone formation.

The biosynthesis of steroid hormones in vertebrates is initiated by the cytochrome P450 CYP11A1, which performs the side-chain cleavage of cholesterol thereby producing pregnenolone. In this study, we report a direct stimulatory effect of the estrogens estradiol and estrone onto the pregnenolone formation in a reconstituted in vitro system consisting of purified CYP11A1 and its natural redox partners. We demonstrated the formation of new products from 11-deoxycorticosterone (DOC), androstenedione, testosterone and dehydroepiandrosterone (DHEA) during the in vitro reaction catalyzed by CYP11A1. In addition, we also established an Escherichia coli-based whole-cell biocatalytic system consisting of CYP11A1 and its redox partners to obtain sufficient yields of products for NMR-characterization. Our results indicate that CYP11A1, in addition to the previously described 6ß-hydroxylase activity, possesses a 2ß-hydroxylase activity towards DOC and androstenedione as well as a 16ß-hydroxylase activity towards DHEA. We also showed that CYP11A1 is able to perform the 6ß-hydroxylation of testosterone, a reaction that has been predominantly attributed to CYP3A4. Our results are the first evidence that sex hormones positively regulate the overall production of steroid hormones suggesting the need to reassess the role of CYP11A1 in steroid hormone biosynthesis and its substrate-dependent mechanistic properties.

Taurine modulates catalytic activity of cytochrome P450 3A4.

The influence of the biologically active compound taurine on the stability and catalytic properties of the hemoprotein cytochrome P450 3A4 has been investigated. The catalytic properties were analyzed by electrochemical methods (cyclic and square-wave voltammetry) using cytochrome P450 3A4 immobilized on the electrode. Taurine at concentrations in the range 10-70 µM stimulated the electrochemical reduction of cytochrome P450 3A4, and the reduction was the highest (115 ± 3%) in the presence of 50 µM taurine. Taurine pronouncedly attenuated the itraconazol-caused inhibition of the P450 isoenzyme P450 3A4. Taurine protected cytochrome P450 3A4 due to stabilizing it during electrolysis at controlled voltage in the presence of erythromycin as a substrate. This protection was manifested by an increase in the amount of the "residual" reduced form of the hemoprotein (52 ± 5 and 71 ± 8%, respectively).

ECM inspired coating of embroidered 3D scaffolds enhances calvaria bone regeneration.

Resorbable polymeric implants and surface coatings are an emerging technology to treat bone defects and increase bone formation. This approach is of special interest in anatomical regions like the calvaria since adults lose the capacity to heal large calvarial defects. The present study assesses the potential of extracellular matrix inspired, embroidered polycaprolactone-co-lactide (PCL) scaffolds for the treatment of 13 mm full thickness calvarial bone defects in rabbits. Moreover the influence of a collagen/chondroitin sulfate (coll I/cs) coating of PCL scaffolds was evaluated. Defect areas filled with autologous bone and empty defects served as reference. The healing process was monitored over 6 months by combining a novel ultrasonographic method, radiographic imaging, biomechanical testing, and histology. The PCL coll I/cs treated group reached 68% new bone volume compared to the autologous group (100%) and the biomechanical stability of the defect area was similar to that of the gold standard. Histological investigations revealed a significantly more homogenous bone distribution over the whole defect area in the PCL coll I/cs group compared to the noncoated group. The bioactive, coll I/cs coated, highly porous, 3-dimensional PCL scaffold acted as a guide rail for new skull bone formation along and into the implant.

A steroidogenic pathway for sulfonated steroids: the metabolism of pregnenolone sulfate.

In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstituted in-vitro system. Difference spectroscopy revealed a Kd-value of 74.8±4.2µM for the CYP17A1-PregS complex, which is 2.5-fold higher compared to the CYP17A1-pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higher Km- and a lower kcat-value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome b5 (b5) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon-carbon bond occurs, when b5 is added to the reconstituted in-vitro system, while b5 promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids.

The bone architecture is enhanced with combined PTH and alendronate treatment compared to monotherapy while maintaining the state of surface mineralization in the OVX rat.

This study examined the effect of PTH and alendronate alone and in combination on the bone architecture, mineralization, and estimated mechanics in the OVX rat. Female Wistar rats aged 7-9months were assigned to one of five groups: (1) sham+vehicle, (2) OVX+vehicle, (3) OVX+PTH, (4) OVX+alendronate, and (5) OVX+PTH and alendronate. Surgery was performed at baseline (week 0), and biweekly treatment (15µg/kg of alendronate and/or daily (5days/week) 40µg/kg hPTH(1-34)) was administered from week 6 to week 14. Micro-CT scans of the right proximal tibial metaphysis were made in vivo at weeks 0, 6, 8, 10, 12 and 14 and measurements of bone microarchitecture and estimated mechanical parameters (finite element analysis) were made from the images. Synchrotron radiation micro-CT scans of the proximal tibia and fourth lumbar vertebrae were conducted ex vivo at the study endpoint to determine the degree and spatial distribution of the bone mineralization. Alendronate preserved the microarchitecture after OVX, and increased cortical (9%, p<0.05) and trabecular thickness (5%, p<0.05). PTH mono- and combined therapy induced increases in cortical (25-35%, p<0.05) and trabecular thicknesses (46-48%, p<0.05), resulting in a full restoration of bone volume in the PTH group, and an increase beyond baseline in the combined group. Improvements in estimated mechanical outcomes were observed in all treatment groups by the end of the study, with the combined group experiencing the greatest increase in predicted stiffness (63%, p<0.05). Alendronate treatment increased the peak mineral content above the other treatment groups at the trabecular (tibia: 6% above PTH, 6% above combined, L4: 4% above PTH, 4% above combined) and endocortical (tibia: 4% above PTH, 3% above combined, L4: 1% above PTH, 2% above combined) surfaces, while no differences in mineralization between the PTH and combined groups were observed. Combined treatment resulted in more pronounced improvements of the bone architecture than PTH monotherapy, while maintaining the state of mineralization observed with PTH treatment.

Atomic Force Microscopy Study of Protein-Protein Interactions in the Cytochrome CYP11A1 (P450scc)-Containing Steroid Hydroxylase System.

Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) were used for monitoring of the procedure for cytochrome CYP11A1 monomerization in solution without phospholipids. It was shown that the incubation of 100 µM CYP11A1 with 12% Emulgen 913 in 50 mM KP, pH 7.4, for 10 min at T = 22°C leads to dissociation of hemoprotein aggregates to monomers with the monomerization degree of (82 ± 4)%. Following the monomerization procedure, CYP11A1 remained functionally active. AFM was employed to detect and visualize the isolated proteins as well as complexes formed between the components of the cytochrome CYP11A1-dependent steroid hydroxylase system. Both Ad and AdR were present in solution as monomers. The typical heights of the monomeric AdR, Ad and CYP11A1 images were measured by AFM and were found to correspond to the sizes 1.6 ± 0.2 nm, 1.0 ± 0.2 nm and 1.8 ± 0.2 nm, respectively. The binary Ad/AdR and AdR/CYP11A1mon complexes with the heights 2.2 ± 0.2 nm and 2.8 ± 0.2 nm, respectively, were registered by use of AFM. The Ad/CYP11A1mon complex formation reaction was kinetically characterized based on optical biosensor data. In addition, the ternary AdR/Ad/CYP11A1 complexes with a typical height of 4 ± 1 nm were AFM registered.

Discovery of a steroid 11α-hydroxylase from Rhizopus oryzae and its biotechnological application.

To overcome the chemically laborious stereo- and regioselective hydroxylation steps in the pharmaceutical production of corticosteroids and progestogens, certain fungal species, e.g. Rhizopus spp. and Aspergillus spp., are employed to perform the 11α-hydroxylation of the steroid skeleton, thereby significantly simplifying steroid drug production. Here we report for the first time the identification and expression of a fungal 11α-steroid hydroxylase, CYP509C12. The newly identified cytochrome P450, which is one of the 48 putative CYP genes in Rhizopus oryzae, was induced in the fungus by progesterone. By functionally expressing CYP509C12 in recombinant fission yeast, we were able to determine that its substrate spectrum includes progesterone as well as testosterone, 11-deoxycorticosterone, and 11-deoxycortisol, with the hydroxylations taking place predominantly at 11α and 6ß positions of the steroid ring system. To increase the 11α-hydroxylation activity of CYP509C12 in recombinant fission yeast, its natural redox partner, the R. oryzae NAD(P)H-dependent reductase, was coexpressed. The coexpression improved electron transfer to CYP509C12 and thus an increase in productivity from 246 to 300 µM hydroxyPg d(-1) was observed, as well as a 7-fold increase of rate of hydroxyprogesterone formation within the linear phase of transformation. This newly developed strain displayed total bioconversion of progesterone into 11α-hydroxyprogesterone and small amounts of 6ß-hydroxyprogesterone within the first 6h of incubation with progesterone as substrate, hence demonstrating its potential for biotechnological application.

Endogenous corticosteroid biosynthesis in subjects after bilateral adrenalectomy.

OBJECTIVE: Corticosteroids can be synthesized in extra-adrenal tissues but the contribution of this to circulating levels in humans is not known. Previous in vitro studies suggest that the 'hybrid' corticosteroid 18-oxocortisol (18-oxoF) is produced from cortisol by aldosterone synthase. We looked for evidence of extra-adrenal production of this and other corticosteroids in 10 subjects stable on long-term glucocorticoid replacement following bilateral adrenalectomy. METHODS: In phase 1, patients were maintained on cortisol alone (30 mg/day), in phase 2 dexamethasone (2 mg/day), and in phase 3, both cortisol and dexamethasone. Each phase lasted 3 days. MEASUREMENTS: On the last day of each phase, 24-h urine collection was performed for analysis of steroid metabolite excretion [using gas chromatography-mass spectrometry (GCMS)] and plasma aldosterone and renin were measured (by radioimmunoassay). RESULTS: Cortisol metabolite excretion rate [tetrahydrocortisone (THE) + tetrahydrocortisol (THF) + allotetrahydrocortisol (aTHF)] fell from 9169 nmol/24 h in phase 1 to 22 nmol/24 h in phase 2, rising to 6843 nmol/24 h in phase 3. Tetrahydroaldosterone (THAldo) excretion was readily detectable and did not alter significantly between phases (26.5, 23.5 and 28.5 nmol/24 h, respectively; P = 0.474). 18-Hydroxycortisol (18-OHF) excretion was easily detectable in phases 1 and 3 (252.5 and 212 nmol/24 h), falling in phase 2 (12 nmol/24 h). 18-oxoF excretion rates were lower but followed a similar pattern (1.62, 0.085 and 1.785 nmol/24 h in phases 1, 2 and 3, respectively). CONCLUSIONS: Significant levels of adrenal steroids are found in adrenalectomized subjects. We speculate that this occurs at extra-adrenal sites or in residual adrenal cortex tissue in an ACTH-independent manner. Our data suggest that aldosterone synthase, acting on cortisol, is the source of 18-oxoF and 18-OHF in these subjects. Further studies of corticosteroid production within adrenalectomized subjects, looking for evidence of adrenal regrowth or residual adrenal tissue, are justified.

Function and engineering of the 15beta-hydroxylase CYP106A2.

CYP106A2 from Bacillus megaterium ATCC 13368 is a bacterial cytochrome P450 that is capable of transforming steroid hormones. It can be easily expressed in Escherichia coli with a high yield. Its activity in vitro can be achieved by using the adrenal redox proteins adrenodoxin and adrenodoxin reductase. So far, it was not possible to crystallize CYP106A2 because of degradation during the crystallization process. Nevertheless, CYP106A2 is an interesting enzyme for biotechnological use. It hydroxylates pharmaceutically important steroids such as progesterone and 11-deoxycortisol. However, it will be necessary for efficient application of CYP106A2 in biotechnology to improve the hydroxylation activity and manipulate the regiospecificity. The present paper gives an overview of recent developments in protein engineering of CYP106A2.

Immunosensor with a controlled orientation of antibodies by using NeutrAvidin-protein A complex at immunoaffinity layer.

The orientation of antibody was controlled by using NeutrAvidin-protein A complex on the gold surface of SPR biosensor. The surface density of receptor antibody (anti-hIgG) was compared by treatment of receptor antibody to the layer of avidin, NeutrAvidin, protein A, NeutrAvidin-protein A complex and bare gold surface of SPR biosensor. The ligand antibody (hIgG) was injected to each IA layer and the binding ratio of ligand antibody per unit receptor was estimated as a parameter of orientation control. The NeutrAvidin-protein A complex on gold surface of SPR biosensor showed the highest surface density of receptor antibody as well as the binding ratio of ligand antibody per receptor antibody. The NeutrAvidin-protein A complex was also prepared on biotin-labelled SAM, and the binding ratio of ligand per receptor was found to be significantly improved in comparison to the IA layer prepared by chemical coupling of receptor antibody to the SAM layer. The NeutrAvidin-protein A complex which showed the highest efficiency for the binding of ligand antibodies, was applied for the detection of a cancer marker called CEA. By using NeutrAvidin-protein A complex and sandwich assay for signal amplification, sensitivity was improved to be 1.5-fold higher than bare gold surface and the detection of CEA with the detection limit of 30 ng/ml was achieved.

Sequential analysis of multiple analytes using a surface plasmon resonance (SPR) biosensor.

A sequential analysis method for the analysis of two analytes was developed using a surface plasmon resonance (SPR) biosensor. A sample with both analytes was introduced into the single sensing region and then each analyte was analyzed sequentially. Two detection models were devised for the samples with the following composition: (1) one target analyte resulting in a sensor response without any label and the other analyte with only additional label, (2) both target analytes requiring additional labels for detection. A standard curve for each model was prepared and applied for sequential analysis of anti-bovine serum albumin (anti-BSA) antibodies and horseradish peroxidase (HRP). The errors of the sequential analysis of Models 1 and 2 were found to be less than 6%, and this method was therefore acceptable for application. No cross-reaction arising from non-specific binding among the participating antigens and antibodies was shown to occur in Models 1 and 2. For optimization of the analyte binding capacity of immunoaffinity (IA), the concentration ratio of the molecular recognition element at the immobilization step was adjusted. Subsequently, from the measurement of the maximum sensor response (R(max)), optimization of the analyte binding capacity could be made. Using Model 2, the feasibility of sequential analysis was demonstrated by detecting levels of human chorionic gonadotropin (hCG) and human albumin (hA) in healthy human urine, since both proteins are known to be related to abortion and preterm delivery during early pregnancy.

Comparison of microfocus- and synchrotron X-ray tomography for the analysis of osteointegration around Ti6Al4V implants.

Micro-computed tomography (microCT) provides quantitative three-dimensional information of bone around titanium implants similar to classical histology. The study, based on an animal model, containing cuboid-shaped biofunctionalised Ti6Al4V implants with surrounding bone after 4 weeks, is performed using 3 microCT-systems with X-ray tubes, one synchrotron-radiation-based microCT-system (SRmicroCT), and classical histology. Although the spatial resolution of the microCT-systems is comparable, only the results of SRmicroCT agree with results of classical histology. The X-ray tube sources give rise to huge artefacts in the tomograms (interface scattering, beam hardening), which impaired the quantitative analysis of bone up to about 200microm from the implant surface. Due to the non-destructive character of microCT the specimens can be subsequently examined by classical histology without restriction. The quantitative comparison of bone formation uncovers the strong dependence of the newly formed bone from the selected slice. This implies the necessity of 3D analysis. SRmicroCT and classical histology prove that surface modifications of the titanium implant significantly influence the bone formation. Using SRmicroCT, the preparation artefacts due to cutting and polishing are excluded.

Analysis of the interaction of a hybrid system consisting of bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119.

The mitochondrial steroid-hydroxylating system in vertebrates and the NADPH producing electron transfer chain in photosynthetic organisms contain structurally and functionally similar components. Examination of a potential hybrid reconstitution of the electron transfer chain between different components of both systems could help to improve our knowledge on protein-protein interaction and subsequent electron transfer. Here we analyzed the interaction between bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119. Optical biosensor as well as steady state and fast kinetic experiments showed their ability to form distinct productive complexes. Compared with the corresponding physiological systems the electron transfer is rather slow, probably due to the lack of specificity at the interaction surface.

Safety concept for robotic gait trainers.

The work presents a newly developed safety concept for application of robotic walking simulators based on the principle of programmable footplates in gait rehabilitation. Unlike robotic hand devices or exoskeleton robots for gait training on treadmills, which can be built relatively lightweight and require only small drives which can hardly do harm to the patient, a programmable footplate walking simulator with permanent foot fixture essentially needs to have powerful drives in order to carry and move the full body weight of the patient. The developed safety concept comprises several redundant algorithms and devices in the real-time robot control software, electrical emergency stop circuitry and machine mechanics. The mechanical core is a machine design offering maximum passive security by covering all moving parts (i.e. robot drives and linkages) and a newly developed foot safety release binding, which is mounted on each footplate. The release binding allows a safe release from the footplate in all directions in any degree of freedom in the sagittal plane. It is combined with an ankle goniometer which is equipped with adjustable emergency stop limit switches, thus ensuring that the allowed ankle range-of-motion is not exceeded.

[Development of a robotic walking simulator for gait rehabilitation]. / Entwicklung eines robotergestützten Laufsimulators zur Gangrehabilitation.

Restoration of gait is a major concern of rehabilitation after stroke or spinal cord injury. Modern concepts of motor learning favour a task-specific repetitive approach, i.e. "whoever wants to learn to walk again must walk." However, the physical demands this places on the therapist, is a limiting factor in the clinical routine setting. This article describes a robotic walking simulator for gait training that enables wheelchair-bound subjects to freely carry out repetitive practicing of an individually adapted gait pattern under simulation of the manual guidance of an experienced therapist. The technical principle applied makes use of programmable footplates with permanent foot/machine contact in combination with compliance control. The solution chosen comprises a planar parallel-serial hybrid kinematic system with three degrees of freedom that moves the feet in the sagittal plane. Gait analysis while floor walking and stair climbing, clinical practicability and safety aspects were the basis for the design. A variable compliance control enables man-machine interaction, ranging from purely position controlled movement to full compliance during swing phase above a virtual ground profile. In full compliance mode the robotic walking simulator behaves like a haptic device. The concept presented offers new prospects for individualized gait rehabilitation.