Skeletal Cell Differentiation Is Enhanced by Atmospheric Dielectric Barrier Discharge Plasma Treatment

Abstract

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury.

Nonthermal Atmospheric Pressure Plasma Enhances Mouse Limb Bud Survival, Growth, and Elongation

“…The treatment voltage, frequency, pulse width, and time were based on our previous publication. 1 Briefly, NT-plasma was generated using a microsecond pulsed voltage of 20 kV (peak to peak) with a pulse width of 10 ms, a typical rise rate of 5 V/ns, and a pulse repetition rate of 1000 Hz applied through a high-voltage electrode for 10 s. The high-voltage electrode was composed of an inner copper core surrounded by an outer insulating shell of acrylic, also detailed by Fridman et al 29 ( Fig. 1A).…”

“…A pie chart was created to display the results between each pair of contralateral limbs by tallying which treatment received the higher score. Then, the scoring results [1][2][3][4] from all contralateral experiments were tabulated to provide an overall assessment of growth and development independent of the contralateral limb. A histogram was generated by tallying the scores for all limbs for each treatment NT-plasma or sham.…”

“…Recently, we reported that nonthermal (NT) dielectric barrier discharge (DBD) plasma treatment stimulated reactive oxygen species (ROS)-associated cell signaling to enhance both osteoblast and chondrocyte differentiation. 1 Results from this study led us to investigate the effects of NT-plasma treatment on the more complex in vitro tissue model of mouse limb autopod development. Enhancement of ROS signaling during development is supported by studies showing tail regeneration in Xenopus, and zebrafish is dependent on ROS production.…”

Extremely low frequency electromagnetic field sensitizes cisplatin-resistant human ovarian adenocarcinoma cells via P53 activation

“…Free radicals are known to directly activate multiple proteins involved in signaling pathways that regulate cell function. Free radicals responsive MAP kinases are known to control a wide range of cellular processes including: cellular differentiation, cell cycle control, cytokine levels and apoptosis (Steinbeck et al 2013). These effects may be enhanced when EL-EMFs and cisplatin are applied in combination and in this way also intensify the effects of cisplatin to cell death induction.…”

Analysis of Cytotoxic Effects of Medical Gas-Discharge Devices

“…In this connection, gas-discharge plasma technologies applied both in Russia and abroad are of great interest [1][2][3]. Such medical gas-discharge devices as quartz lamp (discharge of mercury vapors) [4] and apparatus for darsonvalization (silent corona discharge) [5] are widely being used in Russia.…”