title: 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.
authors.affiliation: Department of Biomedical Engineering, Drexel University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Marla J
authors.initials: MJ
authors.lastname: Steinbeck
authors.affiliation: Department of Electrical Engineering, Drexel University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Natalie
authors.initials: N
authors.lastname: Chernets
authors.affiliation: Department of Orthopaedics, The Second Hospital of Jilin University, Chang Chun, Jilin, China ; Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Deepa S
authors.initials: DS
authors.lastname: Kurpad
authors.affiliation: Department of Biomedical Engineering, Drexel University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Gregory
authors.initials: G
authors.lastname: Fridman
authors.affiliation: Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Alexander
authors.initials: A
authors.lastname: Fridman
authors.affiliation: Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Theresa A
authors.initials: TA
authors.lastname: Freeman
mesh_terms: D000818:Animals; D001272:Atmosphere / Q000737:chemistry*; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004560:Electricity; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D058626:Plasma Gases / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D058626:Plasma Gases; D017382:Reactive Oxygen Species