Analysis of the Differential Gene and Protein Expression Profiles of Corneal Epithelial Cells Stimulated with Alternating Current Electric Fields.

In cells, intrinsic endogenous direct current (DC) electric fields (EFs) serve as morphogenetic cues and are necessary for several important cellular responses including activation of multiple signaling pathways, cell migration, tissue regeneration and wound healing. Endogenous DC EFs, generated spontaneously following injury in physiological conditions, directly correlate with wound healing rate, and different cell types respond to these EFs via directional orientation and migration. Application of external DC EFs results in electrode polarity and is known to activate intracellular signaling events in specific direction. In contrast, alternating current (AC) EFs are known to induce continuous bidirectional flow of charged particles without electrode polarity and also minimize electrode corrosion. In this context, the present study is designed to study effects of AC EFs on corneal epithelial cell gene and protein expression profiles in vitro. We performed gene and antibody arrays, analyzed the data to study specific influence of AC EFs, and report that AC EFs has no deleterious effect on epithelial cell function. Gene Ontology results, following gene and protein array data analysis, showed that AC EFs influence similar biological processes that are predominantly responsive to organic substance, chemical, or external stimuli. Both arrays activate cytokine-cytokine receptor interaction, MAPK and IL-17 signaling pathways. Further, in comparison to the gene array data, the protein array data show enrichment of diverse activated signaling pathways through several interconnecting networks.

Alternating Electric Fields Modify the Function of Human Osteoblasts Growing on and in the Surroundings of Titanium Electrodes.

Endogenous electric fields created in bone tissue as a response to mechanical loading are known to influence the activity and differentiation of bone and precursor cells. Thus, electrical stimulation offers an adjunct therapy option for the promotion of bone regeneration. Understanding the influence of electric fields on bone cell function and the identification of suitable electrical stimulation parameters are crucial for the clinical success of stimulation therapy. Therefore, we investigated the impact of alternating electric fields on human osteoblasts that were seeded on titanium electrodes, which delivered the electrical stimulation. Moreover, osteoblasts were seeded on collagen-coated coverslips near the electrodes, representing the bone stock surrounding the implant. Next, 0.2 V, 1.4 V, or 2.8 V were applied to the in vitro system with 20 Hz frequency. After one, three, and seven days, the osteoblast morphology and expression of osteogenic genes were analysed. The actin organisation, as well as the proliferation, were not affected by the electrical stimulation. Changes in the gene expression and protein accumulation after electrical stimulation were voltage-dependent. After three days, the osteogenic gene expression and alkaline phosphatase activity were up to 2.35-fold higher following the electrical stimulation with 0.2 V and 1.4 V on electrodes and coverslips compared to controls. Furthermore, collagen type I mRNA, as well as the amount of the C-terminal propeptide of collagen type I were increased after the stimulation with 0.2 V and 1.4 V, while the higher electrical stimulation with 2.8 V led to decreased levels, especially on the electrodes.

Human Osteoblast Migration in DC Electrical Fields Depends on Store Operated Ca2+-Release and Is Correlated to Upregulation of Stretch-Activated TRPM7 Channels.

Fracture healing and bone regeneration, particularly in the elderly, remains a challenge. There is an ongoing search for methods to activate osteoblasts, and the application of electrical fields is an attractive approach in this context. Although it is known that such electromagnetic fields lead to osteoblast migration and foster mesenchymal osteogenic differentiation, so far the mechanisms of osteoblast activation remain unclear. Possible mechanisms could rely on changes in Ca2+-influx via ion channels, as these are known to modulate osteoblast activity, e.g., via voltage-sensitive, stretch-sensitive, transient-receptor-potential (TRP) channels, or store-operated release. In the present in vitro study, we explored whether electrical fields are able to modulate the expression of voltage-sensitive calcium channels as well as TRP channels in primary human osteoblast cell lines. We show migration speed is significantly increased in stimulated osteoblasts (6.4 ± 2.1 µm/h stimulated, 3.6 ± 1.1 µm/h control), and directed toward the anode. However, within a range of 154-445 V/m, field strength did not correlate with migration velocity. Neither was there a correlation between electric field and voltage-gated calcium channel (Cav3.2 and Cav1.4) expression. However, the expression of TRPM7 significantly correlated positively to electric field strength. TRPM7 channel blockade using NS8593, in turn, did not significantly alter migration speed, nor did blockade of Cav3.2 and Cav1.4 channels using Ni+ or verapamil, respectively, while a general Ca2+-influx block using Mg2+ accelerated migration. Stimulating store-operated Ca2+-release significantly reduced migration speed, while blocking IP3 had only a minor effect (at low and high concentrations of 2-APB, respectively). We conclude that (i) store operated channels negatively modulate migration speed and that (ii) the upregulation of TRPM7 might constitute a compensatory mechanism-which might explain how increasing expression levels at increasing field strengths result in constant migration speeds.

Daratumumab Interference in Pretransfusion Testing Is Overcome by Addition of Daratumumab Fab Fragments to Patients' Plasma.

BACKGROUND: Daratumumab (DARA), an IgG1κ human monoclonal anti-CD38 antibody, is used for the treatment of refractory myeloma for example. Binding of DARA to CD38 on red blood cells (RBCs), however, leads to panagglutination in indirect antiglobulin testing and possibly masks clinically relevant alloantibodies. Dithiothreitol eliminates panreactivity by destroying CD38 but has the drawback of modifying certain blood group antigens and, thereby, impairs the detection of alloantibodies. METHODS: DARA was digested for 16 h at 37°C using immobilized papain in a spin column, centrifuged, and washed, and the DARA-Fab fragments in pooled flow-throughs were stored at -20°C. DARA-Fab and test cells (ID-DiaCell I-II-III or ID-DiaPanel; BioRad) were incubated with human plasma spiked with DARA (plasma concentration up to 1,000 mg/L) or plasma from patients under DARA therapy at 37°C for 15 min. Thereafter, ID-Cards LISS/Coombs were used. RESULTS: Immunofixation electrophoresis showed complete fragmentation of DARA into Fc and Fab fragments by papain proteolysis. DARA-Fab efficiently prevented RBC agglutination by patients' plasma and by plasma spiked with DARA. Moreover, DARA-Fab did not interfere with the detection of alloantibodies. CONCLUSION: We present a quite easy, reproducible, and cost-effective method for DARA-Fab fragment preparation. Blocking CD38 epitopes with DARA-Fab easily overcomes DARA interference in pretransfusion testing without affecting alloantibody detection.

Effect of electric stimulation on human chondrocytes and mesenchymal stem cells under normoxia and hypoxia.

During joint movement and mechanical loading, electric potentials occur within cartilage tissue guiding cell development and regeneration. Exposure of cartilage exogenous electric stimulation (ES) may imitate these endogenous electric fields and promote healing processes. Therefore, the present study investigated the influence of electric fields on human chondrocytes, mesenchymal stem cells and the co­culture of the two. Human chondrocytes isolated from articular cartilage obtained post­mortally and human mesenchymal stem cells derived from bone marrow (BM­MSCs) were seeded onto a collagen­based scaffold separately or as co­culture. Following incubation with the growth factors over 3 days, ES was performed using titanium electrodes applying an alternating electric field (700 mV, 1 kHz). Cells were exposed to an electric field over 7 days under either hypoxic or normoxic culture conditions. Following this, metabolic activity was investigated and synthesis rates of extracellular matrix proteins were analyzed. ES did not influence metabolic activity of chondrocytes or BM­MSCs. Gene expression analyses demonstrated that ES increased the expression of collagen type II mRNA and aggrecan mRNA in human chondrocytes under hypoxic culture conditions. Likewise, collagen type II synthesis was significantly increased following exposure to electric fields under hypoxia. BM­MSCs and the co­culture of chondrocytes and BM­MSCs revealed a similar though weaker response regarding the expression of cartilage matrix proteins. The electrode setup may be a valuable tool to investigate the influence of ES on human chondrocytes and BM­MSCs contributing to fundamental knowledge including future applications of ES in cartilage repair.

Magnetically induced electrostimulation of human osteoblasts results in enhanced cell viability and osteogenic differentiation.

The application of electromagnetic fields to support the bone-healing processes is a therapeutic approach for patients with musculoskeletal disorders. The ASNIS-III s-series screw is a bone stimulation system providing electromagnetic stimulation; however, its influence on human osteoblasts (hOBs) has not been extensively investigated. Therefore, in the present study, the impact of this system on the viability and differentiation of hOBs was examined. We used the ASNIS-III s screw system in terms of a specific experimental test set-up. The ASNIS-III s screw system was used for the application of electromagnetic fields (EMF, 3 mT, 20 Hz) and electromagnetic fields combined with an additional alternating electric field (EMF + EF) (3 mT, 20 Hz, 700 mV). The stimulation of primary hOBs was conducted 3 times per day for 45 min over a period of 72 h. Unstimulated cells served as the controls. Subsequently, the viability, the gene expression of differentiation markers and pro-collagen type 1 synthesis of the stimulated osteoblasts and corresponding controls were investigated. The application of both EMF and EMF + EF using the ASNIS-III s screw system revealed a positive influence on bone cell viability and moderately increased the synthesis of pro-collagen type 1 compared to the unstimulated controls. Stimulation with EMF resulted in a slightly enhanced gene expression of type 1 collagen and osteocalcin; however, stimulation with EMF + EF resulted in a significant increase in alkaline phosphatase (1.4-fold) and osteocalcin (1.6-fold) levels, and a notable increase in the levels of runt-related transcription factor 2 (RUNX-2; 1.54-fold). Our findings demonstrate that stimulation with electromagnetic fields and an additional alternating electric field has a positive influence on hOBs as regards cell viability and the expression of osteoblastic differentiation markers.

A Novel In Vitro System for Comparative Analyses of Bone Cells and Bacteria under Electrical Stimulation.

Electrical stimulation is a promising approach to enhance bone regeneration while having potential to inhibit bacterial growth. To investigate effects of alternating electric field stimulation on both human osteoblasts and bacteria, a novel in vitro system was designed. Electric field distribution was simulated numerically and proved by experimental validation. Cells were stimulated on Ti6Al4V electrodes and in short distance to electrodes. Bacterial growth was enumerated in supernatant and on the electrode surface and biofilm formation was quantified. Electrical stimulation modulated gene expression of osteoblastic differentiation markers in a voltage-dependent manner, resulting in significantly enhanced osteocalcin mRNA synthesis rate on electrodes after stimulation with 1.4VRMS. While collagen type I synthesis increased when stimulated with 0.2VRMS, it decreased after stimulation with 1.4VRMS. Only slight and infrequent influence on bacterial growth was observed following stimulations with 0.2VRMS and 1.4VRMS after 48 and 72 h, respectively. In summary this novel test system is applicable for extended in vitro studies concerning definition of appropriate stimulation parameters for bone cell growth and differentiation, bacterial growth suppression, and investigation of general effects of electrical stimulation.