M. De Mattei

Effects of pulsed electromagnetic fields on human chondrocytes: an in vitro study.

Abstract.3H-thymidine incorporation was studied in cultured human nasal and articular chondrocytes exposed to low-energy, low-frequency pulsed electromagnetic fields (PEMFs) (75 Hz, 2.3 mT). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows that human secondary chondrocytes derived from both nasal and articular cartilage express collagen type II mRNA, which is a specific marker of the chondrocyte phenotype. In a preliminary series of experiments, cells were exposed to PEMF for different time periods ranging from 6 to 30 hours (time-course), in medium supplemented with 10% or 0.5% fetal calf serum (FCS) and in serum-free medium. The ratios between the 3H-thymidine incorporation in PEMFs and control cultures show an increase of the cell proliferation in cultures exposed to PEMFs when serum is present in the culture medium, whereas no effect was observed in serum-free conditions. The increase in DNA synthesis, induced by PEMFs, was then evaluated only at the times of maximum induction and the results were analyzed by the three-factor analysis of variance (ANOVA).The data presented in this study show that even if 3H-thymidine incorporation is higher in nasal than in articular chondrocytes, PEMF induce an increase in the proliferation of both cell types. Moreover, the concentration of FCS in the culture medium greatly influences the proliferative response of human chondrocytes to the PEMF exposure. Though normal human osteoblast cells increase their proliferation when exposed to PEMFs if only 10% FCS is present in the medium, human chondrocytes are able to increase their cell proliferation when exposed to PEMFs in the presence of both 0.5% and 10% of FCS in the medium. The results obtained may help to explain the basic mechanisms of PEMF stimulation of fracture healing.

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts.

Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro‐inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin‐1β (IL‐1β), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A2A and A3 ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF‐exposed cells. Functional data obtained in the presence of selective A2A and A3 adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E2 (PGE2) and the proinflammatory cytokines interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8), while stimulating the release of interleukin‐10 (IL‐10), an antinflammatory cytokine. These effects seem to be mediated by the EMF‐induced upregulation of A2A and A3 ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti‐inflammatory effects in human OASFs, and that these EMF‐induced effects are in part mediated by the adenosine pathway, specifically by the A2A and A3 AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases. J. Cell. Physiol. 227: 2461–2469, 2012.

Large T antigen coding sequences of two DNA tumor viruses, BK and SV40, and nonrandom chromosome changes in two glioblastoma cell lines

The T antigen (TAg) coding sequences of two DNA tumor viruses, BKV and SV40, were detected by Polymerase Chain Reaction (PCR) amplification followed by Southern-blot hybridization in two human glioblastoma multiforme derived cell lines. RT-PCR analysis indicated that these two TAg coding sequences were expressed in both tumor cell lines carrying the viral early region DNAs. Moreover, analytical polyacrylamide gel electrophoresis (PAGE) and DNA sequence analyses showed that the amplified PCR products are indistinguishable from the TAg coding sequences of BKV and SV40 wildtype strains. Cytogenetic study performed in the two cell lines showed unbalanced changes, mainly gains of chromosomes 3p, 5, 6, 7, and 19 and losses of chromosomes 3, 3q, 16, 9p22-->pter, 18, and 20. Excess of chromosomes 6 and 7 are common to the two cell lines. The putative role of the TAg of the two DNA tumor viruses in transformation and karyotype changes is discussed.

Effects of electrical physical stimuli on articular cartilage.

Articular cartilage is a hypocellular, avascular, alymphatic tissue with a dense collagen and proteoglycan matrix that provides a low-friction and highly durable wear-resistant surface1 to both shear and compressive stress. Normal maintenance of articular cartilage results from the balance between anabolic and catabolic activity. Resident chondrocytes control the extracellular matrix turnover—collagen and proteoglycans synthesis and degradation—from the tidemark to the tangential zone of the cartilage. However, little is known about the physiological processes regulating cellular turnover and cartilage homeostasis, mainly because of the large number of factors involved (mechanical load, cell density, matrix composition, growth factors, cytokines, injury, and aging) and the complexity of their interactions. Insufficient knowledge of the physiology and homeostasis of articular cartilage greatly impairs the ability to stop or slow disease progression. It is commonly accepted that articular cartilage is a tissue with little or no regenerative potential and thus undergoes degradation over time. The notion that it is impossible to prevent or reverse degeneration of articular cartilage has been challenged recently by the growing body of evidence in the literature based on basic-research findings concerning the physiology and pathophysiology of articular cartilage, so that new strategies for its maintenance and repair are emerging2. In two recent studies by Glasson et al.3 and Stanton et al.4, a knockout mouse model of osteoarthritis, a degenerative disease that eventually leads to destruction of articular cartilage, demonstrated how a single protein, ADAMTS 5, is the main aggrecanase responsible for cartilage degradation and the principal mediator of the catabolic effects of pro-inflammatory cytokines, such as interleukin-1 (IL-1). Their findings identify a rational target for therapeutic intervention to limit cartilage degradation in osteoarthritis and demonstrate that interference with a single pathway can dramatically alter the natural history of joint disease. Chondral injury rapidly results in …

Evaluation of the Electroporation Efficiency of a Grid Electrode for Electrochemotherapy: From Numerical Model to In Vitro Tests.

Electrochemotherapy (ECT) is a local anticancer treatment based on the combination of chemotherapy and short, tumor-permeabilizing, voltage pulses delivered using needle electrodes or plate electrodes. The application of ECT to large skin surface tumors is time consuming due to technical limitations of currently available voltage applicators. The availability of large pulse applicators with few and more spaced needle electrodes could be useful in the clinic, since they could allow managing large and spread tumors while limiting the duration and the invasiveness of the procedure. In this article, a grid electrode with 2-cm spaced needles has been studied by means of numerical models. The electroporation efficiency has been assessed on human osteosarcoma cell line MG63 cultured in monolayer. The computational results show the distribution of the electric field in a model of the treated tissue. These results are helpful to evaluate the effect of the needle distance on the electric field distribution. Furthermore, the in vitro tests showed that the grid electrode proposed is suitable to electropore, by a single application, a cell culture covering an area of 55 cm2. In conclusion, our data might represent substantial improvement in ECT in order to achieve a more homogeneous and time-saving treatment, with benefits for patients with cancer.

Identification of in vitro electropermeabilization equivalent pulse protocols.

Exposure of cells to an external sufficiently strong electric field results in the formation of pores across the membrane. This phenomenon, termed electropermeabilization, permits the transport of poorly permeant molecules into cytosol. In clinical practice, cell membrane permeabilization for drug electrotransfer is achieved using the ESOPE pulse protocol (1000 V/cm, 8 pulses, 100 μs, 5 kHz). The aim of this study was to investigate several combinations of electric field amplitude and pulse number able to induce electropermeabilization as the one observed when the ESOPE protocol was applied. Decreasing electric field amplitudes (1000 to 300 V/cm) in combination with increasing number of pulses (8 to 320) were applied to in vitro MG63 cells. Propidium iodide and Calcein blue AM uptake were used to evaluate cell electropermeabilization and viability. Results showed that the threshold of local electric field needed to obtain electropermeabilization decreased exponentially with increasing the number of pulses delivered (r2 50.92, p < 0.0001). The absorbed dose threshold was dependent on the number of pulses for each voltage applied (r2 50.96, p < 0.0001). In conclusion, the possibility of applying an increased number of pulses rather than increasing the electric field amplitude to perform electropermeabilization, may become an important tool for electropermeabilization - related clinical applications.

Effects of Low-Frequency Pulsed Electromagnetic Fields on Human Osteoblast-Like Cells In Wtro

We have studied the effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on human osteoblast-like cells in vitroin order to investigate the biochemical mechanism(s) by which electrical stimulation can effect changes in skeletal metabolism. Bony nature of the cells was assayed by immunolabeling with antiosteonectin antibody. Multidish plates were placed between a pair of Helmoltz coils powered by a pulse generator (1.3 ms, 75 Hz) in a tissue culture incubator for 24 h. 3H-thymidine was added to the culture medium to evaluate cell proliferation. The acid-precipitable 3H-thymidine incorporation significantly increases in PEMF-exposed cells as compared to the control. PEMF exposure induces a two- to fivefold increase of DNA synthesis. Osteoblasts cultured for 24 h in a serum-free medium show a very low proliferation rate, and the exposure to PEMF does not stimulate DNA synthesis.

Glycosaminoglycan analysis in amniotic fluid and in cultured fibroblasts from normal and holoprosencephalic human embryonic organs

Glycosaminoglycans are polysaccharides involved in epithelial–mesenchymal interaction and cell differentiation and provide a meshwork which is essential to maintain a proper intercellular milieu. The development of embryonic organs can be accompanied by alterations in the glycosaminoglycan pattern. In pregnancies with malformed fetuses, there are alterations in total glycosaminoglycans and their components (chondroitin 4–6 sulphate, dermatan sulphate, and hyaluronic acid) in amniotic fluid. We examined total glycosaminoglycans and the percentage variations of the single classes in both amniotic fluid and culture medium of fibroblasts from heart, lung, and skin obtained from five normal human fetuses and one with holoprosencephaly. In the amniotic fluid total glycosaminoglycans and their sulphate classes were increased, whereas hyaluronic acid was decreased, compared with controls. The extracellular glycosaminoglycans showed hyaluronic acid reduction in skin, while chondroitin 4–6 sulphate plus dermatan sulphate and heparan sulphate were higher in skin and heart. Our data demonstrate that variations in the glycosaminoglycan pattern are associated with alterations of the cellular environment, which can prevent normal organogenesis.

A new grid electrode for electrochemotherapy treatment of large skin tumors

Electrochemotherapy (ECT) is a local anticancer treatment for superficial tumors which involves the administration of a chemotherapeutic drug followed by short, high-voltage pulses. Incipient clinical experience with breast cancer patients are encouraging, with a local complete response rate ranging from 50 to 90%. However, since many patients present with multiple or widespread metastases, ECT, despite its high local antitumor efficacy, need to be applied several times in order to complete treatment delivery or maintain tumor control during the follow-up. In this paper, we propose the prototype of a new grid electrode aimed at the improvement of ECT application. The device is suitable for treating large, tumor-infiltrated skin surfaces as in breast cancer patients with chest wall metastases after mastectomy. According to our tests in different in vitro models, the new device allows to apply the voltage pulses more quickly and homogeneously when compared with standard pulse applicators. This technical advancement holds promise for improving ECT outcome and, hopefully, for sparing a number of patients from the need of multiple treatments.

The reduced sensitivity of the ProC Global test in protein S deficient subjects reflects a reduction in the associated thrombotic risk.

To investigate simultaneously a defect affecting the protein C/protein S (PC/PS) anticoagulant pathway is possible thanks to a methodological approach (ProC® Global; Dade Behring) based on the activation of endogenous plasma PC by a snake venom extract. Factor V (FV) Leiden, the most frequent cause of hereditary thrombosis, is well detected by the test with sensitivity of 100% irrespective of the presence/absence of thrombosis in the subjects investigated. The test is also suited to detect PC or PS defect, but in this case the in vitro impairment of the PC/PS pathway is less pronounced particularly for PS defects (sensitivity for PC and PS defect, 85–100 and 30–90%, respectively). In this study, we hypothesized that the lower sensitivity described for PS defect, compared with those of PC and FV Leiden defects, could also be related to the clinical condition of the subject investigated (symptomatic/asymptomatic) rather than solely to the PS plasma activity/level. Therefore, we analyzed 126 subjects with single congenital defects in the PC/PS pathway: 46 subjects with PS deficiency (26 thrombotic cases and 20 asymptomatic relatives), 40 subjects with PC deficiency (25 thrombotic cases and 15 asymptomatic relatives), and 40 heterozygous FV Leiden subjects (25 thrombotic cases and 15 asymptomatic relatives). By a cut-off of normalized Agkistrodon contortix snake venom ratio of 0.84, the sensitivity in the whole group of cases (sensitivity a) was 76.1, 95.0 and 100%, respectively, for PS, PC and FV Leiden defects. The test failed to detect 11 (23.9%) among the 46 PS-deficient subjects, and all these cases except two belonged to the asymptomatic subgroup (9/20; 45%). Excluding the 20 asymptomatic relatives, the new sensitivity (sensitivity b) for the PS defect was 92.3%. The comparison of the sensitivity in the symptomatic PS cases and in the asymptomatic ones was significantly different (P = 0.010). Among the 40 PC-deficient subjects, only two (5.0%) were not detected by the test and they belonged indifferently to the two subgroups. Finally, none of the 40 FV Leiden heterozygotes were misdiagnosed by the test. These results suggest that in symptomatic PS-deficient cases the test could reflect a post-thrombotic effect and/or reveal potential unidentified prothrombotic influences assessing a prothrombotic risk condition.