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

Abstract

The enhanced differentiation of mesenchymal cells into chondrocytes or osteoblasts is of paramount importance in tissue engineering and regenerative therapies. A newly emerging body of evidence demonstrates that appendage regeneration is dependent on reactive oxygen species (ROS) production and signaling. Thus, we hypothesized that mesenchymal cell stimulation by nonthermal (NT)-plasma, which produces and induces ROS, would (1) promote skeletal cell differentiation and (2) limb autopod development. Stimulation with a single treatment of NT-plasma enhanced survival, growth, and elongation of mouse limb autopods in an in vitro organ culture system. Noticeable changes included enhanced development of digit length and definition of digit separation. These changes were coordinated with enhanced Wnt signaling in the distal apical epidermal ridge (AER) and presumptive joint regions. Autopod development continued to advance for approximately 144 h in culture, seemingly overcoming the negative culture environment usually observed in this in vitro system. Realtime quantitative polymerase chain reaction analysis confirmed the up-regulation of chondrogenic transcripts. Mechanistically, NT-plasma increased the number of ROS positive cells in the dorsal epithelium, mesenchyme, and the distal tip of each phalange behind the AER, determined using dihydrorhodamine. The importance of ROS production/signaling during development was further demonstrated by the stunting of digital outgrowth when anti-oxidants were applied. Results of this study show NT-plasma initiated and amplified ROS intracellular signaling to enhance development of the autopod. Parallels between development and regeneration suggest that the potential use of NT-plasma could extend to both tissue engineering and clinical applications to enhance fracture healing, trauma repair, and bone fusion.

Non‐Equilibrium Dielectric Barrier Discharge Treatment of Mesenchymal Stem Cells: Charges and Reactive Oxygen Species Play the Major Role in Cell Death

“…Computational modeling and optical emission spectroscopy of atmospheric plasma discharges in air have shown the production of various oxygen species . ROS, such as hydrogen peroxide, can function as a signaling molecule to regulate cell processes, but at high concentrations, they can become agents that lead to cell death .…”

Skin Wound Healing: An Update on the Current Knowledge and Concepts

“…Concomitantly, longer exposures might lead to overdosing of the plasma application inducing apoptotic cell death with a further disturbed wound healing process [93,94]. Chernets et al [95] examined appendage regeneration stimulated by NTP dependent on reactive oxidative species. Single treatment of in vitro organ culture systems with NTP was already able to enhance the survival, growth, and elongation of mouse limb autopods.…”

“…Single treatment of in vitro organ culture systems with NTP was already able to enhance the survival, growth, and elongation of mouse limb autopods. Perceptible transformations comprised an improved development of the digit length as well as the definition of digit separation [95]. In our own group, we have treated cultivated fibrocytes with NTP for different periods [unpublished data].…”

Review of Major Directions in Non-Equilibrium Atmospheric Plasma Treatments in Medical, Biological, and Bioengineering Applications

“…Discussed are the biological and biochemical processes involved in initiation of differentiation processes as well as the cell's specialization into appropriate daughter cell. 353 Discussed are the key findings on the plasma-cell interaction mechanisms and the reported increase in the MCS differentiation rate using microsecond-pulsed floating electrode-dielectric barrier discharge in in vitro cell studies and in vivo mouse model with injections of MSC-loaded Matrigel ® .…”