Passage-dependent relationship between mesenchymal stem cell mobilization and chondrogenic potential.

OBJECTIVE: Galvanotaxis, the migratory response of cells in response to electrical stimulation, has been implicated in development and wound healing. The use of mesenchymal stem cells (MSCs) from the synovium (synovium-derived stem cells, SDSCs) has been investigated for repair strategies. Expansion of SDSCs is necessary to achieve clinically relevant cell numbers; however, the effects of culture passage on their subsequent cartilaginous extracellular matrix production are not well understood. METHODS: Over four passages of SDSCs, we measured the expression of cell surface markers (CD31, CD34, CD49c, CD73) and assessed their migratory potential in response to applied direct current (DC) electric field. Cells from each passage were also used to form micropellets to assess the degree of cartilage-like tissue formation. RESULTS: Expression of CD31, CD34, and CD49c remained constant throughout cell expansion; CD73 showed a transient increase through the first two passages. Correspondingly, we observed that early passage SDSCs exhibit anodal migration when subjected to applied DC electric field strength of 6 V/cm. By passage 3, CD73 expression significantly decreased; these cells exhibited cell migration toward the cathode, as previously observed for terminally differentiated chondrocytes. Only late passage cells (P4) were capable of developing cartilage-like tissue in micropellet culture. CONCLUSIONS: Our results show cell priming protocols carried out for four passages selectively differentiate stem cells to behave like chondrocytes, both in their motility response to applied electric field and their production of cartilaginous tissue.

Time and dose-dependent effects of chondroitinase ABC on growth of engineered cartilage.

Tissue engineering techniques have been effective in developing cartilage-like tissues in vitro. However, many scaffold-based approaches to cultivating engineered cartilage have been limited by low collagen production, an impediment for attaining native functional load-bearing tensile mechanical properties. Enzymatic digestion of glycosaminoglycans (GAG) with chondroitinase ABC (chABC) temporarily suppresses the construct's GAG content and compressive modulus and increases collagen content. Based on the promising results of these early studies, the aim of this study was to further promote collagen deposition through more frequent chABC treatments. Weekly dosing of chABC at a concentration of 0.15 U/mL resulted in a significant cell death, which impacted the ability of the engineered cartilage to fully recover GAG and compressive mechanical properties. In light of these findings, the influence of lower chABC dosage on engineered tissue (0.004 and 0.015 U/mL) over a longer duration (one week) was investigated. Treatment with 0.004 U/mL reduced cell death, decreased the recovery time needed to achieve native compressive mechanical properties and GAG content, and resulted in a collagen content that was 65 % greater than the control. In conclusion, the results of this study demonstrate that longer chABC treatment (one week) at low concentrations can be used to improve collagen content in developing engineered cartilage more expediently than standard chABC treatments of higher chABC doses administered over brief durations.

Comparison of nanosecond and femtosecond pulsed laser deposition of silver nanoparticle films.

Nanoparticle (NP) films of silver were prepared using nanosecond (ns) and femtosecond (fs) pulsed laser deposition (PLD) in vacuum. The flux and energy distribution of the ions in the plasma part of the ablation plume were measured using a Langmuir ion probe. The deposition energy efficiencies of ns and fs silver PLD were also compared. For equivalent thickness up to ∼3 nm the NPs made by ns-PLD are well separated and roughly circular, but for higher thicknesses the NPs begin to coalesce. For equivalent thickness up to 7 nm the fs films are comprised of well separated NPs, though the mean NP size and the surface coverage increase with equivalent thickness. The mean Feret diameter for both ns- and fs-PLD films increases with increasing equivalent solid-density thickness. The surface plasmon resonance peak was observed to red shift for both ns- and fs-PLD films as the equivalent solid-density thickness was increased from 1 nm to 7 nm.

A comparative in vitro toxicity assessment of electronic vaping product e-liquids and aerosols with tobacco cigarette smoke.

The use of electronic vaping products (EVPs) continues to increase worldwide among adult smokers in parallel with accumulating information on their potential toxicity and relative safety compared to tobacco smoke. At this time, in vitro assessments of many widely available EVPs are limited. In this study, an in vitro battery of established assays was used to examine the cytotoxic (Neutral red uptake), genotoxic (In vitro micronucleus) and mutagenic (Bacterial reverse mutation) responses of two commercial EVPs (blu GO™ disposable and blu PLUS+™ rechargeable) when compared to smoke from a reference cigarette (3R4F). In total, 12 commercial products were tested as e-liquids and as aerosols. In addition, two experimental base liquids containing 1.2% and 2.4% nicotine were also assessed to determine the effect of flavour and nicotine on all three assays. In the bacterial reverse mutation (Ames) and in vitro micronucleus (IVM) assays, exposures to e-liquids and EVP aerosols, with and without nicotine and in a range of flavourings, showed no mutagenic or genotoxic effects compared to tobacco smoke. The neutral red uptake (NRU) assay showed significantly reduced cytotoxicity (P < .05) for whole undiluted EVP aerosols compared to tobacco smoke, which by contrast was markedly cytotoxic even when diluted. The reduced in vitro toxicological responses of the EVPs add to the increasing body of scientific weight-of-evidence supporting the role of high-quality EVPs as a harm reduction tool for adult smokers.

Two phase I studies of carboplatin dose escalation in chemotherapy-naive ovarian cancer patients supported with granulocyte-macrophage colony-stimulating factor.

Recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) may reduce myelosuppression and, thus, allow dose escalation of certain chemotherapeutic agents. We conducted two sequential phase I trials of escalating doses of carboplatin and a fixed dose and schedule of rHuGM-CSF in ovarian cancer patients who had not previously had chemotherapy, i.e., chemotherapy-naive patients. In the first trial, patients were assigned to regimens of increasing dose levels of carboplatin (starting at 400 mg/m2) and fixed doses and schedules of cyclophosphamide (600 mg/m2) and rHuGM-CSF (10 micrograms/kg given subcutaneously once daily on days 2-11). Chemotherapy was given every 3 weeks (regimen A). In the subsequent trial, the design was the same except that cyclophosphamide was omitted (regimen B). Fifteen patients received regimen A, and seven patients received regimen B. In regimen A, all three patients treated at the first dose level tolerated five cycles at full doses. Hematologic toxicity was dose limiting at the 600-mg/m2 dose level. When 500 mg/m2 carboplatin was given, six of eight patients tolerated three or four cycles at full doses before requiring dose reductions or treatment delays. In regimen B, doses could not be escalated above the first dose level (600 mg/m2) because of severe hematological toxicity. Nonhematological toxicity was tolerable and managed with acetaminophen, antihistamines, and/or nonsteroidal, anti-inflammatory medication. Compliance was excellent. We conclude that (a) rHuGM-CSF can be given safely and reliably to chemotherapy-naive ovarian cancer patients receiving these treatment regimens, (b) early and severe thrombocytopenia was a major problem with or without cyclophosphamide with doses of carboplatin at or above 600 mg/m2, and (c) 500 mg/m2 carboplatin administered every 3 weeks is the highest dose in regimen A that can be given safely in the outpatient setting.

Effects of ionizing radiation and pretreatment with [D-Leu6,des-Gly10] luteinizing hormone-releasing hormone ethylamide on developing rat ovarian follicles.

To assess the effects of a gonadotropin-releasing hormone agonist, [D-Leu6,des-Gly10] luteinizing hormone-releasing hormone ethylamide, in ameliorating the damage caused by ionizing radiation, gonadotropin-releasing hormone agonist was administered to rats from day 22 to 37 of age in doses of 0.1, 0.4, and 1.0 microgram/day or vehicle and the rats were sacrificed on day 44 of age. There were no effects on estradiol, progesterone, luteinizing, or follicle-stimulating hormone, nor an effect on ovarian follicle numbers or development. In separate experiments, rats treated with gonadotropin-releasing hormone agonist in doses of 0.04, 0.1, 0.4, or 1.0 microgram/day were either irradiated or sham irradiated on day 30 and all groups sacrificed on day 44 of age. Irradiation produced a reduction in ovarian weight and an increase in ovarian follicular atresia. Pretreatment with the agonist prevented the reduction in ovarian weight and numbers of primordial and preantral follicles but not healthy or atretic antral follicles. Such putative radioprotection should be tested on actual reproductive performance.