Sports-Related Motor Processing at Different Rates of Force Development.

How does our brain manage to process vast quantities of sensory information that define movement performance? By extracting the required movement parameters for which brain dynamics are, inter alia, assumed to be functionally related to, we used electroencephalography to investigate motor-related brain oscillations. Visually guided movement (i.e., motor) tasks at explosive, medium and slow rates of force development (RFD) revealed increased broad-band activity at explosive RFD, whereas decreasing activity could be observed during both intermediate and slow RFD. Moreover, a continuously decreasing activity pattern from faster to slower RFD and a return to baseline activity after full muscle relaxation was found. We suggest oscillatory activity to desynchronize in sensorimotor demanding tasks, whereas task-specific synchronization mirrors movement acceleration. The pre/post-stimulus activity steady state may indicate an inhibitory baseline that provides attentional focus and timing.

Effects of longer vs. shorter timed movement sequences on alpha motor inhibition when combining contractions and relaxations.

Alpha inhibitory processes reflect motor stimuli by either increasing or decreasing amplitude (i.e., power). However, the functional role and interplay of event-related alpha oscillations remains a regulatory domain that has not been sufficiently addressed, particularly with respect to different muscle activation types and durations in consecutive movement (i.e., motor) tasks. The aim of this study was to investigate alpha-band activity (7-13 Hz) in longer vs. shorter timed isometric muscle activations at distinct torques (20% and 40% of maximum voluntary contraction, MVC) when combined in one motor task sequence. In a randomized and controlled design, 18 healthy males volunteered to perform 40 longer (i.e., 6 s) and 40 shorter (i.e., 3 s) motor task sequences, each comprising isometric contractions (i.e., palmar flexion) from baseline to 20% and 40% MVC subsequent to relaxations from 40% and 20% MVC to baseline. Continuous, synchronized EEG, EMG and torque recordings served to determine alpha-band activity over task-relevant motor areas at distinct torques. Main findings revealed increases in alpha activity during subsequent progressive muscle relaxation (from 20% MVC in long and short: p < .001; from 40% MVC in short: p < .05), whereas modulations in relevant motor areas were not significant (p = .84). It may be suggested that an active task-relevant inhibitory process indicates motor task sequence-related relaxation mirrored by an increasing alpha activity.

Performance control in one consecutive motor task sequence - Αpproaching central neuronal motor behaviour preceding isometric contraction onsets and relaxation offsets at lower distinct torques.

OBJECTIVE: Motor-related cortical potentials (MRCP) often compared separated muscle activations; however, MRCP preceding combined contraction onsets and relaxation offsets of one consecutive motor task sequence remain to be elucidated. METHODS: Twelve healthy males (27.92±4.33 years, 181.83±7.15 cm, 84.58±7.15 kg) performed 40 submaximal isometric right-limb wrist flexions (i.e. motor task sequences). Each motor task sequence combined timed contractions to and relaxations from distinct torque levels, i.e. 20% and 40% of maximum voluntary contractions (MVC). Synchronized continuous EEG (32 Ag/AgCl-electrodes mounted over motor-related areas) and EMG (i.e. flexor carpi radialis, FCR) recordings served to detect torque level-on/offsets for MRCP analyses. RESULTS: Motor task sequences were accurately maintained with participants' mean values of FCR muscle activity revealing no signs of fatigue (p⟩0.05). Main findings (i.e. readiness potential) were larger amplitudes over frontal electrode sites (p⟨0.05) preceding contractions compared to relaxations, whereas amplitudes were larger (i.e. peak) over centro-parietal electrode sites (p⟨0.05) preceding 40% compared to 20% MVC. CONCLUSION: When performed in one consecutive motor task sequence, controlling the production as well as the releasing of force may require similar proprioceptive and visuo-motor processing preceding the same force level (i.e. 20% or 40%); however, this is irrespective of the muscle activation type (i.e. contraction or relaxation).

In Vitro Detection System to Evaluate the Immunogenic Potential of Xenografts.

Tissue antigenicity represents the main limitation for the use of xenografts in clinical practice. To eliminate xenoantigens and avoid graft rejection in human, decellularization is often used to remove all immunoreactive components from the extracellular matrix (ECM). After decellularization, acellular scaffolds are required to be investigated regarding the presence of antigens, but commonly used detection methods solely focus on known xenoantigens such as alpha Gal (Galα1,3-Galß1-4GlcNAc-R) or major histocompatibility complex-I (MHC-I). However, there are unknown xenoantigens that escape the standard methods. To evaluate the immunological potential of xenogenic tissues, new in vitro methods need to be developed. Therefore, we established a novel human serum-based approach, including dot blot, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). With these methods, we analyzed protein extracts and tissue samples of native and decellularized bovine carotid arteries. All methods verified an effective removal of potential immunogens from the ECM through decellularization, and relative quantification with ELISA showed that 99.9% (p < 0.01) of antigenic components were successfully eliminated. We compared our human serum-based methods with commonly used assays for the detection of alpha Gal and MHC-I. Our results showed highly increased sensitivity for xenoantigens using the human serum antibody pool. This novel in vitro detection system allows the direct determination of the immunogenic potential of xenografts and is a vast improvement in comparison to the methods used so far. That way, it is possible to optimize the decellularization process to prevent hyperacute graft rejection in patients.

A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments.

Detergent-Based Decellularization of Bovine Carotid Arteries for Vascular Tissue Engineering.

Vascular diseases are an increasing health issue, and common alloplastic, allogenic or autologous vascular grafts show frequent complications. The aim of this study is to develop an acellular, xenogenic bypass-graft from a bovine carotid artery (BAC) using detergent-based protocols. We compared decellularization with sodium desoxycholate (DOA), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), sodium dodecyl sulfate (SDS), and Triton X100 and improved suitable methods by variation of concentration, buffer system, incubation time, temperature, rinsing, and flow rate. All processes were evaluated systematically based on cellular residues, biocompatibility, structural and mechanical integrity. Decellularization with SDS and Triton X100 was not sufficient for the removal of cellular components. We optimized protocols using 1% DOA and Chaps by a buffered system at 37 °C with extended decellularization and rinsing. Decellularization with DOA depleted DNA to 0.5 ± 0.1% and soluble proteins to 0.6 ± 0.2%. Using Chaps, DNA was reduced to 0.2 ± 0.2% and proteins to 0.6 ± 0.3%. The improved protocols eliminated RNA completely from the matrix, and no cytotoxic effects were detected. Mechanical and structural integrity of decellularized tissues was comparable to non-decellularized controls. Our method effectively removed cellular components from the extracellular matrix while preserving the structural and mechanical integrity of the tissue. Decellularized BACs could be a promising alternative for vascular replacement therapy.

EDC Cross-linking of Decellularized Tissue: A Promising Approach?

BACKGROUND: Decellularization of xenogenous cardiovascular structures is a promising approach to create scaffolds for tissue engineering. Unfortunately, handling and pliability of the unfixed tissue is challenging. N-(3-dimethylaminopropyl)-N9-ethylcarbodiimide (EDC) is an alternative cross-linking agent to glutaraldehyde (GA). Applied in native tissue, it provides biocompatibility and shows no potential for calcification. In addition, EDC can be used to link growth factors (GFs) to tissue scaffolds after decellularization. EDC cross-linking could thereby help to improve decellularized tissue without the toxicity of GA. MATERIAL AND METHODS: Porcine aortic wall tissue specimens (TS) were decellularized, treated with EDC, and coated with fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF). Afterward, TS were subcutaneously implanted in 36 Lewis rats along with one decellularized TS without EDC treatment. After 2, 4, and 6 weeks TS were explanted from 12 rats, respectively. Vital cells were evaluated by RNA quantification, general cellular infiltration by hematoxylin and eosin staining (H&E), macrophage infiltration by CD68 staining, calcification by Von-Kossa staining, and tissue degradation by measurement of TS thickness. RESULTS: Quantification of vital cells showed reduced reseeding of EDC-treated TS compared to noncross-linked TS after 2 (p < 0.05) and 4 weeks (p < 0.05), while after 6 weeks only EDC+VEGF showed fewer viable cells (p < 0.01). Histological evaluation confirmed a reduced infiltration of EDC-treated TS. Macrophage infiltration decreased in all groups from 2 to 6 weeks, with the smallest population in EDC+VEGF-treated TS (p > 0.05). In EDC+FGF-treated TS, macrophages were reduced after 2 weeks compared to noncross-linked TS (p < 0.05), while after 4 and 6 weeks no significant difference was found (p > 0.05). Von-Kossa staining revealed no calcification in any of the specimens. Thickness of noncross-linked and EDC+FGF-treated TS was not different at the respective times of explantation, but decreased in both groups toward 6 weeks. CONCLUSION: EDC cross-linking combined with GF coating of decellularized aortic wall tissue showed encouraging results. The treatment did not impair the advantages of decellularized tissue such as long-term recellularization, absence of calcification, and tissue integrity. Based on the low macrophage infiltration and minimal tissue degradation, treatment with EDC and VEGF could be useful after decellularization. However, further research is necessary to verify these findings in models, including mechanical stress.

Successful implantation of a decellularized equine pericardial patch into the systemic circulation.

BACKGROUND: In the past, successful use of decellularized xenogenic tissue was shown in the pulmonary circulation. This study, however, evaluates a newly developed decellularized equine pericardial patch under high pressure circumstances. MATERIAL AND METHODS: Seven decellularized equine pericardial scaffolds were implanted into the descending aorta of the juvenile sheep. The implanted patches were oversized to evaluate the durability of the decellularized tissue under high surface tension (Law of Laplace). After 4 months of implantation, all decellularized patches were inspected by gross examination, light microscopy (H&E, Serius red, Gomori, Weigert, and von Kossa straining), and immunohistochemical staining. RESULTS: The juvenile sheep showed fast recovery after surgery. There was no mortality during follow-up. At explantation, only limited adhesion was seen at the surgical site. Gross examination showed a smooth and pliable surface without degeneration, as well as absence of aneurysmatic dilatation. Light microscopy showed a well preserved extracellular scaffold with a monolayer of endothelial cells covering the luminal side of the patch. On the outside part of the patch, a well developed neo-vascularization was seen. Host fibroblasts were seen in all layers of the scaffolds. There was no evidence for structural deterioration or calcification of the decellularized equine pericardial scaffolds. CONCLUSIONS: In the juvenile sheep, decellularized equine tissue showed no structural deterioration, but regeneration and remodeling processes at systemic circulation.

Extracellular matrix in deoxycholic acid decellularized aortic heart valves.

BACKGROUND: Only limited information is available regarding the influence of decellularization on the extracellular matrix in heart valves. Within the extracellular matrix proteoglycans (PG) play a central role in the structural organization and physical functioning of valves and in their capability of settling with endothelial and interstitial cells partially myofibroblasts. We have therefore estimated the effects of decellularization using deoxycholic acid on the structure of the extracellular matrix and PG´s in porcine aortic valves. MATERIAL/METHODS: Cupromeronic blue was used, alone or in combination with OsO4/thio-carbo-hydrazide/OsO4 for electron microscopic visualization. For PG and glycosaminoglycan (GAG) investigation a papain digestion was employed in combination with photometric determination using dimethylmethylene blue. RESULTS: The results indicate that deoxycholic acid affects the compartmentation of the PG-associated interstitial network not significantly. Compared to controls the PG-rich network was preserved even after deoxycholic acid treatment for 48 h. In parallel to electron microscopy immune assays (ELISA) showed smooth muscle cell -actin to be reduced to 0.96% ± 0.71 and total soluble protein to 6.68% ± 2.0 (n=3) of untreated controls. Protein loss corresponded well with the observations in electron micrographs of rupture and efflux of cell content. Further signs of lysis were irregular cell contours and loss of the basement membrane. CONCLUSIONS: Efficient cell-lysis without disintegration or loss of integrity of the interstitial PG network can be achieved by treatment of aortic valves with deoxycholic acid for 48h. This protocol might also be suitable for clinical use to optimize conditions for growth and autologous remodelling of valves.

Immune response in patients receiving a bioprosthetic heart valve: lack of response with decellularized valves.

Conventional biological heart valves treated with glutaraldehyde (GA) reveal a limited lifespan due to calcification. This is assumed to be an immune response initiated process, which is not seen with decellularized valves. However, their immunological potential is still a matter of debate. Therefore, serum samples from patients undergoing heart valve surgery were obtained before (Pre), after (Post), and 9-12 months after operation (Follow Up). Immunoglobulin G (IgG) and M (IgM) antibodies against porcine collagen I and α-Gal (Gal-alpha1,3-Gal-beta1,4-GlcNac-R) were determined for decellularized and GA treated valves. Antibody titers for collagen type I revealed no significant alteration for both types of valves. However, a considerable anti-α-Gal antibody response was observed in patients with GA-treated porcine valves. In detail, IgM antibodies were increased during follow up (p<0.05), whereas decellularized valves revealed a minor decrease in the IgM response (p<0.001). IgG antibodies were considerably increased with GA-treated porcine (p<0.05) and bovine (p<0.01) xenografts, whereas there was lack of response with decellularized valves. This indicates that GA treatment is not sufficient to eliminate immune response to the α-Gal epitope completely. Future investigations will have to verify whether immune response to α-Gal can be linked to the limited durability of conventional valves.