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We use the OMI-QA4ECV-v1.1 NO2 tropospheric columns over the 10-year 2008–2017 period to confront satellite-based trends in NO2 concentrations to those from the state-of-the-art regional chemistry-transport model CHIMERE and to evaluate the bottom-up anthropogenic and biogenic NOx emissions in Europe. A focus is made for the 30 most populated urban areas in Europe. Over urban areas in Western Europe, except for coastal cities, OMI confirms the drop in the simulated CHIMERE NO2 tropospheric columns based on the latest country emission official reporting. OMI hardly shows significant negative trends over Central and Eastern Europe urban areas. Increasing biogenic emissions helps reconciling CHIMERE and OMI trends over urban areas in Central Europe and over rural areas, confirming the importance of accounting for non-anthropogenic emissions to assess long-term trends. Over Eastern Europe, our results question emission reductions estimated for particular sectors and in particular the road transport, public power, and industrial emissions.

Study Design In vitro disk explant culture. Objective Notochordal cells (NCs) have been shown to upregulate matrix production by nucleus pulposus (NP) cells in coculture. To examine the translation of these in vitro results to a nativelike setting, the regenerative potential of NCs injected into NP tissue was assessed in this study. Methods NP explants were cultured after injection with NCs in phosphate-buffered saline (PBS) or with PBS alone (sham). At days 0 and 42, cell viability and morphology, water, DNA, sulfated glycosaminoglycan and hydroxyproline content, and gene expression of anabolic markers were analyzed. Results NCs remained viable during culture, but their morphology changed. The biochemical content remained unchanged, except for the DNA content in the NC group. Overall ACAN expression remained unchanged, whereas COL2A1 decreased during culture. Conclusions No overall anabolic response was observed when NCs were injected into NP explants. NCs were found to survive but did not display the typical NC morphology by the end of the culture period.

In intervertebral disc herniation with nucleus pulposus (NP) extrusion, the elicited inflammatory response is considered a key pain mechanism. However, inflammatory cytokines are reported in extruded herniated tissue, even before monocyte infiltration, suggesting that the tissue itself initiates the inflammation. Since herniated tissue swells, we investigated whether this simple mechanobiological stimulus alone could provoke an inflammatory response that could cause pain. Furthermore, we investigated whether sustained-release cyclooxygenase-2 (COX2) inhibitor would be beneficial in such conditions. Healthy bovine NP explants were allowed to swell freely or confined. The swelling explants were treated with Celecoxib, applied either as a bolus or in sustained-release. Swelling explants produced elevated levels of interleukin-6 (IL-6) and prostaglandin E2 (PGE2) for 28 days, while confined explants did not. Both a high concentration bolus and 10 times lower concentration in sustained release completely inhibited PGE2 production, but did not affect IL-6 production. Swelling of NP tissue, without the inflammatory system response, can trigger cytokine production and Celecoxib, even in bolus form, may be useful for pain control in extruded disc herniation. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1–8, 2015

Introduction Early degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs. Methods Canine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture. Results NCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa. Conclusions No regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×106 cells/mL) and NPCs+BMSCs (6×106 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10¿ng/mL transforming growth factor-ß1 (TGF-ß1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. I

Chemical processes occurring on leaf surface and in air inside vegetation canopies play significant and sometimes dominant roles on pollutant dry deposition budgets. Yet, these processes are seldom explicitly treated in dry deposition parameterizations/models. This report briefly summarizes the current knowledge, outstanding issues, and recommendations for pollutant leaf surface exchange including ammonia (NH3), ozone (O3) and nitrogen oxides (NOx = NO + NO2), acidifying pollutants, volatile organic compounds (VOCs), and atmospheric aerosols.

Volatile organic compounds (VOC) are a relatively minor component of the atmosphere and yet are widely recognized to have important roles in air quality and climate. With the exception of methane, an important greenhouse gas, atmospheric VOC are primarily of interest because of their impact on other atmospheric constituents, including oxidants and aerosol. Most of the global annual VOC emission is from biogenic sources but biomass burning, fossil fuel combustion and industrial activities dominate in some regions. Each of these major sources can be further categorized, e.g., biogenic sources include plant chloroplasts, plant specialized tissues, microbes, and animals. The processes for removing VOC from the atmosphere include VOC surface deposition, VOC deposition to particles, and surface deposition of their oxidation products including oxidized VOC, CO and CO2. Regional to global atmospheric chemistry and transport models (CTMs) routinely include at least some VOC emission and removal processes but in a highly simplified form. Climate models have previously included just methane but as they evolve into more comprehensive earth system models, other VOC are being included although the sources and sinks may be prescribed or highly simplified.

Study Design Preclinical in vitro culture of human degenerated nucleus pulposus (NP) tissue. Objective Cyclooxygenase 2 inhibitors (e.g., celecoxib) inhibit prostaglandin E2 (PGE2) production, and they have been shown to upregulate regeneration of articular cartilage. In this study, we developed an explant culture system for use with human tissue and tested the potential of celecoxib. Methods NP explants were cultured with or without 1 µM of celecoxib and were analyzed at days 0 and 7 for biochemical content (water, sulfated glycosaminoglycans, hydroxyproline, and DNA), gene expression (for disk matrix anabolic and catabolic markers), and PGE2 content. Results Water and biochemical contents as well as gene expression remained close to native values after 1¿week of culture. PGE2 levels were not increased in freshly harvested human NP tissue and thus were not reduced in treated tissues. Although no anabolic effects were observed at the dosage and culture duration used, no detrimental effects were observed and some specimens did respond by lowering PGE2. Conclusions Human degenerated NP explants were successfully cultured in a close to in vivo environment for 1 week. Further research, especially dosage-response studies, is needed to understand the role of PGE2 in low back pain and the potential of celecoxib to treat painful disks.

Bone marrow stromal cells (BMSCs) have shown promising potential to stop intervertebral disc degeneration in several animal models. In order to restore a healthy state, though, this potential should be further stimulated. Notochordal cells (NCs), influential in disc development, have been shown to stimulate BMSC differentiation, but it is unclear how this effect will translate in an environment where resident disc cells (nucleus pulposus cells [NPCs]) could also influence BMSCs. The goal of this study was, therefore, to evaluate the effects of NCs on BMSCs when cocultured with NPCs, in a simplified 3D in vitro system. Bovine BMSCs and NPCs were mixed (Mix) and seeded into alginate beads. Using culture inserts, the Mix was then cocultured with porcine NCs (alginate beads) and compared to coculture with empty beads or porcine skin fibroblasts (SFs, alginate beads). NPCs alone were also cocultured with NCs, and BMSCs alone cultured under chondrogenic conditions. The effects of coculture conditions on cell viability, matrix production (proteoglycan and collagen), and gene expression of disc markers (aggrecan, type II collagen, and SOX9) were assessed after 4 weeks of culture. The NC phenotype and gene expression profile were also analyzed. Coculture with NCs did not significantly influence cell viability, proteoglycan production, or disc marker gene expression of the Mix. When compared to NPCs, the Mix produced the same amount of proteoglycan and displayed a higher expression of disc marker, indicating a stimulation of the BMSCs (and/or NPCs) in the Mix. Additionally, during the 4 weeks of culture, the NC phenotype changed drastically (morphology, gene expression profile). These results show that NCs might not be as stimulatory for BMSCs in an NPC-rich environment, as believed from individual cultures. This absence of effects could be explained by a mild stimulation provided by (de)differentiating NCs and the costimulation of BMSCs and NPCs by each other.

Recent studies suggest that notochordal cells (NCs) might be involved in intervertebral disc homeostasis, a role exploitable to counteract matrix degradation as observed during degeneration. This study aimed to evaluate the potential of NCs to promote matrix production by nucleus pulposus cells (NPCs) and to compare it to the currently proposed addition of bone marrow stromal cells (BMSCs). Using alginate beads, bovine NPCs were exposed for 28 d to porcine NC conditioned medium (NCCM); direct co-culture with porcine NCs or bovine BMSCs; or the combination of BMSCs and NCCM. Effects on cell proliferation, disc matrix production (proteoglycans, collagens) and disc matrix protein expression (aggrecan, collagen 1 and 2, SOX9) were determined and compared to TGFß stimulation. NCCM strongly promoted NPC proliferation (× 2.2) and matrix production (× 3.9) to levels similar to that with TGFß, whereas the direct addition of NCs had no effect. Co-culture of NPCs and BMSCs led to proteoglycan synthesis similar to NPCs alone, which was slightly improved by NCCM (× 1.5). Histological analysis confirmed biochemical data. Gene expression of analysed proteins remained stable for all groups and unaffected by medium conditions. NCs could substantially stimulate NPCs through factors secreted into conditioned medium and in levels similar to the addition of BMSCs. This study showed that molecular agents secreted by NCs constitute a promising alternative to the proposed "standard" injection of BMSCs for disc repair: their effects are similar, do not require the injection of a large number of cells and can be further amplified when the factors are identified.

Transplantation of mesenchymal stem cells (MSCs) with electrotransferred bone morphogenetic protein-2 (BMP-2) transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1 alpha, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications