Ultra-Low Intensity Post-Pulse Affects Cellular Responses Caused by Nanosecond Pulsed Electric Fields.

High-intensity nanosecond pulse electric fields (nsPEF) can preferentially induce various effects, most notably regulated cell death and tumor elimination. These effects have almost exclusively been shown to be associated with nsPEF waveforms defined by pulse duration, rise time, amplitude (electric field), and pulse number. Other factors, such as low-intensity post-pulse waveform, have been completely overlooked. In this study, we show that post-pulse waveforms can alter the cell responses produced by the primary pulse waveform and can even elicit unique cellular responses, despite the primary pulse waveform being nearly identical. We employed two commonly used pulse generator designs, namely the Blumlein line (BL) and the pulse forming line (PFL), both featuring nearly identical 100 ns pulse durations, to investigate various cellular effects. Although the primary pulse waveforms were nearly identical in electric field and frequency distribution, the post-pulses differed between the two designs. The BL's post-pulse was relatively long-lasting (~50 µs) and had an opposite polarity to the main pulse, whereas the PFL's post-pulse was much shorter (~2 µs) and had the same polarity as the main pulse. Both post-pulse amplitudes were less than 5% of the main pulse, but the different post-pulses caused distinctly different cellular responses. The thresholds for dissipation of the mitochondrial membrane potential, loss of viability, and increase in plasma membrane PI permeability all occurred at lower pulsing numbers for the PFL than the BL, while mitochondrial reactive oxygen species generation occurred at similar pulsing numbers for both pulser designs. The PFL decreased spare respiratory capacity (SRC), whereas the BL increased SRC. Only the PFL caused a biphasic effect on trans-plasma membrane electron transport (tPMET). These studies demonstrate, for the first time, that conditions resulting from low post-pulse intensity charging have a significant impact on cell responses and should be considered when comparing the results from similar pulse waveforms.

Gene electrotransfer of FGF2 enhances collagen scaffold biocompatibility.

Tendon injuries are a common athletic injury that have been increasing in prevalence. While there are current clinical treatments for tendon injuries, they have relatively long recovery times and often do not restore native function of the tendon. In the current study, gene electrotransfer (GET) parameters for delivery to the skin were optimized with monophasic and biphasic pulses with reporter and effector genes towards optimizing underlying tendon healing. Tissue twitching and damage, as well as gene expression and distribution were evaluated. Bioprinted collagen scaffolds, mimicking healthy tendon structure were then implanted subcutaneously for biocompatibility and angiogenesis analyses when combined with GET to accelerate healing. GET of human fibroblast FGF2 significantly increased angiogenesis and biocompatibility of the bioprinted implants when compared to implant only sites. The combination of bioprinted collagen fibers and angiogenic GET therapy may lead to better graft biocompatibility in tendon repair.

Characterization and biological properties of a novel synthesized silicon-substituted hydroxyapatite derived from eggshell.

In the present study, the effect of adding different concentrations of silicon on physical, mechanical and biological properties of a synthesized aqueous precipitated eggshell-derived hydroxyapatite (e-HA) was evaluated. No secondary phases were detected by X-ray diffraction for the specimens e-HA and e-HA containing silicon (Si-e-HAs) before and after heating at 1200°C. A reduction in the crystallite size and a-axis as well as an increase in c-axis was occurred when silicon replacement was happened in the structure of e-HA. The presence of Si-O vibrations and carbonate modes for Si-e-HAs was confirmed by Fourier transform infrared spectroscopy analysis. The range of porosity and density was varied from 25% and 2.4 g cm-3 to 7% and 2.8 g cm-3 for e-HA and Si-e-HAs. The values of Young's modulus ( E) and compressive strength were varied for e-HA and Si-e-HAs. The porous structure of the samples was reduced when they were heated as e-HA kept the porous microstructure containing some dense areas and Si-e-HAs possessed a rough surface including slight levels of microporosity. The acellular in vitro bioactivity represented different apatite morphologies for e-HA and Si-e-HAs. The G-292 osteoblastic cells were stretched well on the surface with polygon-shaped morphology for 0.8Si-e-HA after 7 days of culture. According to MTT assay and alkaline phosphatase test, the maximum cell activity was related to 0.8Si-e-HA. The minimum inhibitory concentration for 0.8Si-e-HA and e-HA was estimated to be about 3.2 and 4.4 mg/mL, respectively. In overall, the sample 0.8Si-e-HA exhibited a higher bacteriostatic effect than e-HA against gram-negative bacterial strain Escherichia coli.

Regeneration of full-thickness skin defects using umbilical cord blood stem cells loaded into modified porous scaffolds.

In this study, we have demonstrated the ability of cord blood (CB)-derived unrestricted somatic stem cells (USSCs) and chitosan-modified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffold to promote skin regeneration. Afterward, the scaffolds were evaluated by structural, microscopic, physical, and mechanical assays and cell culture analyses. Results of structural, physical, and mechanical analyses also showed a good resilience and compliance with movement as a skin graft. Cellular experiments showed a better cell adhesion, growth, and proliferation inside the modified scaffolds compared with unmodified ones. In animal models with histological examinations, all groups, excluding the control group especially the groups treated with stem cells, exhibited the most pronounced effect on wound closure, with the statistically significant improvement in wound healing being seen at postoperative day 21. These data suggest that chitosan-modified PHBV scaffold loaded with CB-derived USSCs could significantly contribute to wound repair and be potentially used in the tissue engineering.