Enhancement of Skeletal Cell Differentiation by Microsecond Pulsed Dielectric Barrier Discharge
Authors:   , Chernets Natalie , Freeman Theresa A. , Fridman Alexander A. , Friedman Gary G. 
Published: July 2021

Plasma medicine, specifically non-thermal plasma (NT-plasma) technology, has clinical potential in medicine and biology, which has yet to be realized. Current concepts being tested range from wound healing, cancer suppression and dental applications. To date clinical trials testing NT-plasma have mainly focused on its bacteriostatic and bacteriocidal properties in patients possessing chronic and acute wounds, or diverse skin and itching diseases. Further development of NT-plasma as a clinical tool depends on gaining a better understanding of the mechanisms underlying NT-plasma interactions with living organisms. Several strategies have been employed including; characterizing NT-plasmas, determining cellular response to specific NT-plasmas and identification of NT-plasma modifications to the environment surrounding cells and tissues. The successful accomplishment of these goals and to prove plasma medicine has true clinical potential requires a multiple disciplinary approach where physicists, chemists, biologists, and electrical, mechanical and biomedical engineers work together with clinicians to meet the needs of the medical community. The best-characterized mechanism underlying NT-plasma activity on both eukaryotic and prokaryotic cells is the generation of ROS and RNS at the cell/environment interface which elicits an immediate intracellular oxidative response. This thesis tests the hypothesis that NT-plasma promotes redox dependent changes to enhance developmental and cellular signaling. Initially, NT-plasma parameters including electrode size, plasma dose, filament formation and energy density were compared to determine both cellular response and treatment effectiveness. Once established, we were able to establish the mechanisms by which NT-plasma generated intracellular ROS and extra- and intracellular calcium flux promote murine limb autopod development and mesenchymal cell differentiation. Results of this study indicate that if NT-plasma is applied under the appropriate conditions it induces chondrocyte differentiation, osteoblast differentiation and promotes mouse limb autopod development. Effective NT-plasma dose was governed by both the biological model to which it was applied and to electrode geometry and size. Specifically, energy density was not an effective method for predicting cellular response nor to determine the resulting filament pattern created by the electrode. The cellular response at frequencies above 50 Hz filamentation pattern were instead strongly dependent on electrode size. Having established these conditions, we determined the biological responses to NT-plasma were dependent on increases in intracellular ROS production and the resulting activation of oxidative-stress responsive proteins and gene expression. Furthermore, both cellular differentiation and the enhancement of limb autopod elongation by NT-plasma were dependent on increases in intracellular calcium, through the activation of voltage gated calcium channels on the plasma membrane. In conclusion, this thesis identifies the potential of plasma medicine technology to induce and recapitulate normal cellular signaling and its potential in regenerative medical applications for skeletal tissue engineering, and if directly applied to improve healing after skeletal injury.