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.

Melanoma is one of the most aggressive metastatic cancers with resistance to radiation and most chemotherapy agents. This study highlights an alternative treatment for melanoma based on nanosecond pulsed dielectric barrier discharge (nsP DBD). We show that a single nsP DBD treatment, directly applied to a 5 mm orthotopic mouse melanoma tumor, completely eradicates it 66% (n = 6; p ≤ 0.05) of the time. It was determined that reactive oxygen and nitrogen species produced by nsP DBD are the main cause of tumor eradication, while nsP electric field and heat generated by the discharge are not sufficient to kill the tumor. However, we do not discount that potential synergy between each plasma generated component (temperature, electric field and reactive species) can enhance the killing efficacy.

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.

The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 ؎ 3% ␣-helix, 20 ؎ 3% ␤-pleated sheet, and 53 ؎ 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal. ABC3 transporters are required for transport of a wide variety of hydrophobic substances across cellular membranes, including drugs (1-3), lipids (4 -6), metabolites, peptides (7), and steroids (2). Typically, eukaryotic ABC proteins are composed of two tandem sets of six transmembrane helices followed by a Walker type A and a type B nucleotide-binding motif (8). To date, 48 members of the human ABC transporter family have been identified, which, based on sequence homology, have been divided into seven subfamilies, ABCA through ABCG (2-3, 9).A retina-specific variant of the ABCA subfamily, the ABCA4 gene product (ABCR), was first described as the bovine and Xenopus Rim proteins identified in the rims of the rod outer segment discs (10 -11). Mutations in the ABCA4 gene appear to lead to defects in the energy-dependent transport of all-transretinal and lead to the accumulation of cytotoxic lipofuscin fluorophores in the retinal pigment epithelium characteristic of the diseases such as Stargardt disease, fundus flavimaculatus, and autosomal recessive cone-rod dystrophy, as well as increased susceptibility to age-related macular degeneration (12)(13)(14)(15)(16)(17)(18)(19)(20). Although ultimately these diseases await the promise of a cure through gene therapy (21-22), current treatments have been directed toward slowing the progression of the disease. Consequently, understanding how genetic mutations lead to retinal degeneration is critical for the development of novel therapies.Studies in several laboratories and the establishment of ABCA4 ...