Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation.

The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (μsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with μsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with μsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on μsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd

Nonthermal atmospheric pressure plasma enhances mouse limb bud survival, growth, and elongation.

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. Real-time 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

Skeletal cell differentiation is enhanced by atmospheric dielectric barrier discharge plasma treatment.

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

NT-plasma induced osteogenic differentiation using qPCR markers.

<p>(<b>A</b>) Fold increases in the expression of alkaline phosphatase (ALKP), bone morphogenetic protein 2 (BMP2), bone sialoprotein (BSP) and fibroblast growth factor-2 (FGF-2) in response to NT-plasma treatment, H₂O₂ or β-glycerol phosphate (βGP) normalized to sham treated control cells after 24 hr. ALKP was upregulated 3- (NS) and 11-fold (p<0.01), respectively, and BMP2 and BSP were increased 5 fold (p<0.05). βGP increased expression 14–23 fold (p<0.01). FGF-2 expression was not affected by NT-plasma, H₂O₂ or βGP. (<b>B</b>) After a 24 hr incubation in βGP NT-plasma was applied. 24 hr later there was a 2–7 fold increase in the induction of BMP2, ALKP (p<0.05) and Osterix was increased 15 fold (p<0.01) compared to βGP treatment alone. At 56 hr, BSP and osteocalcin (OSTCN) were increased 17–24 fold above βGP-treated control (p<0.05). The results are expressed as the mean ± standard deviation (n = 2). Statistical significance was determined by the Wilcoxin Mann-Whitney test for non-parametric data; * (p<0.05), ** (p<0.01).</p

Direct effects of NT-plasma on cytotoxicty and cell proliferation.

<p>Osteoblast-like, MLO-A5 cells were treated with NT-plasma at frequencies of 50, 1000 and 3500 Hz for 10 seconds. (<b>A</b>) Cell death measured by nuclear PI incorporation at 24 hr showed increased cell death in response to frequency 50 Hz, 3500 Hz (p<0.01) and H₂O₂ (p<0.001) (n = 3). There was no significant cell death in response to frequency 1000 Hz. (<b>B</b>) Cell detachment was observed at 3500 Hz as shown by toluidine staining of cells after NT-plasma treatment. (<b>C</b>) Cell viability after NT-plasma was assessed by a lactate dehydrogenase (LDH) release assay. 1000 Hz and H₂O₂ both showed an increase in LDH release (p<0.01 and p<0.001). LDH release was reduced in the presence of TEMPOL for both H₂O₂ (p<0.01) and NT-plasma treatments (ns, n = 3). (<b>D</b>) No difference in cell cycle profile was observed between NT-plasma at 50 Hz or 1000 Hz as compared to sham control. (<b>E</b>) Western blots show no H2Ax or cytochrome c release in cells treated at 1000 Hz for 10 s as compared to the 30 and 60 sec NT-plasma treatments. Statistical significance was determined by the Mann-Whitney U test for non-parametric data; * or # (p<0.01) ** or ## (p<0.001.</p

PCR primer information.

<p>PCR primer information.</p

Direct effects of NT-plasma on intracellular ROS production.

<p>(<b>A and B</b>) Intracellular O₂^−<b>.</b> and H₂O₂ levels were measured using fluorescent indicators MitoSox (n = 3) and dihydrorhodamine (DHR), (n = 3) respectively. Immediately post-treatment (POST-PL) and at 1 hr, 1000 Hz NT-plasma treated cells generated significantly increased amounts of O₂^−<b>.</b> and H₂O₂ (p<0.001) as compared to sham control or pre-treatment levels (PRE-PL). Amounts of both ROS were decreased by 24 hr. NAC and TEMPOL quenched the ROS increase POST-PL and at 1 hr (p<0.01–0.001). However, H₂O₂ levels in the TEMPOL (p<0.01) inhibitor group were significantly increased above control at 24 hr, presumably due to removal of baseline NO<b>^.</b> The results are expressed as the mean ± standard deviation. Statistical significance was determined by the Mann-Whitney U test for non-parametric data; * or # (p<0.01) ** or ## (p<0.001.</p