Design and Dosimetric Characterization of a Broadband Exposure Facility for In Vitro Experiments in the Frequency Range 18-40.5 GHz.

A novel exposure facility for exposing cell monolayers to centimeter and millimeter waves (18-40.5 GHz) used by future 5G mobile communication technology and similar applications has been developed. A detailed dosimetric characterization of the apparatus for frequencies of 27 and 40.5 GHz and 60 mm petri dishes, used in a presently ongoing study on human dermal fibroblasts and keratinocytes, was carried out. The exposure facility enables a well-defined, randomized, and blinded application of sham exposure and exposure with selectable values of incident power flux density, and additionally provides the possibility of continuous monitoring of the sample temperature during exposure while it does not require significant deviations from routine in vitro handling procedures, i.e. petri dishes are not required to be placed inside waveguides or TEM cells. Mean specific absorption rate (SAR) values inside the cell monolayer of 115 W/kg (27 GHz) and 160 W/kg (40.5 GHz) per watt antenna input power and corresponding transmitted power density (St ) values at the bottom of the cell monolayer of 65 W/m2 (27 GHz) and 70 W/m2 (40.5 GHz) per watt antenna input power can be achieved, respectively. For reasonable amounts of harvested cells (80% of petri dish bottom area), the variation (max/min) of SAR and St over the cell monolayer remains below 3.7 dB (27 GHz) and 3.0 dB (40.5 GHz), respectively. © 2021 Bioelectromagnetics Society.

Effects of Long-Term Exposure of Intermediate Frequency Magnetic Fields (20 kHz, 360 µT) on the Development, Pathological Findings, and Behavior of Female Mice.

The use of magnetic fields in the intermediate-frequency (IF) range to wirelessly charge electric cars with power transfer in the kilowatt range has become increasingly widespread, leading to unavoidable stray fields in the microtesla range. Only a handful of studies have assessed the potential biological risks associated with exposure to such fields. We exposed female mice (n = 80 per group) to either 20 kHz, 360 µT (rms), or sham in Helmholtz coils to conduct a blind design study. Exposure started at 3 months of age (24 h/day). Body mass was recorded every 1-2 weeks. At 10 months of age, three behavioral tests were performed on 24 animals per group. Three months later, the mice were sacrificed and organs (brain, liver, kidney, spleen, and lung) were removed and prepared for microscopic analysis. Our findings demonstrate no differences in the development of body mass and survival rates (96% and 89%, respectively). Similarly, no significant differences were observed in tumor incidence rates. When it comes to behavioral tests, the 8-arm maze results revealed no significant differences. In contrast, the Rotarod data were significantly (P < 0.001) different with longer retention times seen in the exposed mice. In the open field, the number of supported rears was significantly lower (P < 0.01), whereas the other endpoints did not show any differences. Overall, our data reveal no adverse effects of exposure to 20 kHz, 360 µT on the development and tumor incidences, while the significant differences in the behavioral tests may indicate higher levels of alertness in mice.

Forced exercise-induced osteoarthritis is attenuated in mice lacking the small leucine-rich proteoglycan decorin.

OBJECTIVE: Interterritorial regions of articular cartilage matrix are rich in decorin, a small leucine-rich proteoglycan and important structural protein, also involved in many signalling events. Decorin sequesters transforming growth factor ß (TGFß), thereby regulating its activity. Here, we analysed whether increased bioavailability of TGFß in decorin-deficient (Dcn-/-) cartilage leads to changes in biomechanical properties and resistance to osteoarthritis (OA). METHODS: Unchallenged knee cartilage was analysed by atomic force microscopy (AFM) and immunohistochemistry. Active transforming growth factor ß-1 (TGFß1) content within cultured chondrocyte supernatants was measured by ELISA. Quantitative real-time (RT)-PCR was used to analyse mRNA expression of glycosaminoglycan (GAG)-modifying enzymes in C28/I2 cells following TGFß1 treatment. In addition, OA was induced in Dcn-/- and wild-type (WT) mice via forced exercise on a treadmill. RESULTS: AFM analysis revealed a strikingly higher compressive stiffness in Dcn-/- than in WT cartilage. This was accompanied by increased negative charge and enhanced sulfation of GAG chains, but not by alterations in the levels of collagens or proteoglycan core proteins. In addition, decorin-deficient chondrocytes were shown to release more active TGFß1. Increased TGFß signalling led to enhanced Chst11 sulfotransferase expression inducing an increased negative charge density of cartilage matrix. These negative charges might attract more water resulting in augmented compressive stiffness of the tissue. Therefore, decorin-deficient mice developed significantly less OA after forced exercise than WT mice. CONCLUSIONS: Our study demonstrates that the disruption of decorin-restricted TGFß signalling leads to higher stiffness of articular cartilage matrix, rendering joints more resistant to OA. Therefore, the loss of an important structural component can improve cartilage homeostasis.

4-Phenylbutyric Acid Reduces Endoplasmic Reticulum Stress in Chondrocytes That Is Caused by Loss of the Protein Disulfide Isomerase ERp57.

OBJECTIVE: The integrity of cartilage depends on the correct synthesis of extracellular matrix (ECM) components. In case of insufficient folding of proteins in the endoplasmic reticulum (ER) of chondrocytes, ECM proteins aggregate, ER stress evolves, and the unfolded protein response (UPR) is initiated. By this mechanism, chondrocytes relieve the stress condition or initiate cell death by apoptosis. Especially persistent ER stress has emerged as a pathogenic mechanism in cartilage diseases, such as chondrodysplasias and osteoarthritis. As pharmacological intervention is not available yet, it is of great interest to understand cartilage ER stress in detail and to develop therapeutics to intervene. METHODS: ERp57-deficient chondrocytes were generated by CRISPR/Cas9-induced KO. ER stress and autophagy were studied on mRNA and protein level as well as by transmission electron microscopy (TEM) in chondrocyte micromass or cartilage explant cultures of ERp57 KO mice. Thapsigargin (Tg), an inhibitor of the ER-residing Ca2+-ATPase, and 4-Phenylbutyric acid (4-PBA), a small molecular chemical chaperone, were applied to induce or inhibit ER stress. RESULTS: Our data reveal that the loss of the protein disulfide isomerase ERp57 is sufficient to induce ER stress in chondrocytes. 4-PBA efficiently diffuses into cartilage explant cultures and diminishes excessive ER stress in chondrocytes dose dependently, no matter if it is induced by ERp57 KO or stimulation with Tg. CONCLUSION: ER-stress-related diseases have different sources; therefore, various targets for therapeutic treatment exist. In the future, 4-PBA may be used alone or in combination with other drugs for the treatment of ER-stress-related skeletal disorders in patients.